Evaluation of serum levels of TGFβ1 and VEGF during colorectal carcinogenesis.

<p>The levels of TGFβ1 (A) and VEGF (B) were measured in controls and patient groups (N: control; TA: tubular adenoma; TVA: tubulovillous adenoma; I-IV AC: malignant stages of adenocarcinoma). TGFβ1 and VEGF were detected by ELISA and values expressed as ng/ml and pg/ml serum, respectively (see materials and methods). A: dots correspond to single TGFβ1 values and black lines represent the mean values within the experimental groups. Mean values ± SEM: N 6.3±0.4; TA 7.2±0.45; TVA 6.3±0.6; IAC 6.9±0.3; IIAC 4.9±0.3; IIIAC 4.4±0.3; IVAC 5.7±0.5. *Significantly different versus control group (p<0.05). B: dots correspond to single VEGF values and black lines represent the mean values within the experimental groups. Mean values ± SEM: N 227.0±18.7; TA 215.2±31.3; TVA 221.1±36.4; IAC 216.3±34.4; IIAC 205.0±26.6; IIIAC 266.5±38.6; IVAC 295.2±70.2.</p

Distribution and expression of MMP-9 on paraffin embedded CRC tissue sections.

<p>A: a representative image of carcinoma tissue at stage III showed strong staining for MMP-9, localized in the cytoplasm of both tumor cells and inflammatory cells (10x/0.22 objective). For experimental details see materials and methods. B: higher magnification of a MMP-9 positive area of the same cancer section (40x/0.65 objective). The images were obtained using Digital Microscope DMD Leica, Leica Microsystems, Milan, Italy.</p

Evaluation of serum levels of IL-8 and IL-6 during colorectal carcinogenesis.

<p>The levels of IL-8 (A) and IL-6 (B) were measured in controls and patient groups (N: control; TA: tubular adenoma; TVA: tubulovillous adenoma; I-IV AC: malignant stages of adenocarcinoma). IL-8 and IL-6 were detected by ELISA and values expressed as pg/ml serum (see materials and methods). A: dots correspond to IL-8 single values and black lines represent the mean values within the experimental groups. Mean values ± SEM: N 17.0±2.5; TA 15.9±3.7; TVA 18.4±2.2; IAC 25.4±4.8; IIAC 38.7±5.1; IIIAC 37.2±5.7; IVAC 30.5±5.7. *Significantly different versus control group (p<0.05). B: dots correspond to single IL-6 values and black lines represent the mean values within experimental groups. Mean values ± SEM: N 247.1±55; TA 375.9±54; TVA 407.9±80; IAC 237.9±68; IIAC 472.3±89; IIIAC 521.3±152; IVAC 436.3±118. *Significantly different versus control group (p<0.05).</p

Evaluation of serum MMP activity and levels during colorectal carcinogenesis.

<p>A: active forms of MMP-2 and MMP-9 are shown in a representative gelatin zymography. B: MMP-9 activation in the serum was evaluated by densitometric analysis as percentage of active MMP-9 compared to controls (taken as 100%). Dots correspond to the activated MMP-9 value for each subject and black lines represent mean values within the experimental groups. Mean values ± SEM: TA 120±3.9; TVA 121±8.5; IAC 151.63±17.2; IIAC 163.81±16.6; IIIAC 164.2±10.6; IVAC 129.0±13.0. *Significantly different versus control group (p<0.01). C: serum levels of MMP-9 protein were detected by ELISA and expressed as ng/ml serum. Dots correspond to the MMP-9 value for each subject and black lines represent the mean values within the experimental groups. Mean values ± SEM: N 1.5±0.2; TA 1.9±0.3; TVA 1.9±0.3; I AC 2.7±0.3; IIAC 2.8±0.3; IIIAC 3.0±0.5; IVAC 2.6±0.4. *Significantly different versus control group (p<0.05). N: control; TA: tubular adenoma; TVA: tubulovillous adenoma; I-IV AC: malignant stages of adenocarcinoma.</p

Evaluation of serum levels of CRP during colorectal carcinogenesis.

<p>The levels of CRP were measured in controls and patient groups (N: control; TA: tubular adenoma; TVA: tubulovillous adenoma; I-IV AC: malignant stages of adenocarcinoma). CRP was detected by latex immunoassay analysis and expressed as mg/L serum (see materials and methods). Dots correspond to single CRP values and black lines represent the mean values within the experimental groups. Mean values ± SEM: N 3.5±0.1; TA 3.7±0.7; TVA 5.7±1.1; IAC 7.5±1.8; IIAC 24.3±8.5; IIIAC 27.9±7.5; IVAC 17.2±4.1.</p

Average diameter, polydispersity index and zeta potential of the nanoparticle formulations.

<p>Average diameter, polydispersity index and zeta potential of the nanoparticle formulations.</p

Effect of oxysterols on expression of CD36, β1-integrin, IL-8, MCP-1, and MMP-9.

<p>Gene expression was quantified by real-time RT-PCR in SH-SY5Y cells treated for 6 h with 5 µM 7β-hydroxycholesterol (7β-OH), 24-hydroxycholesterol (24-OH), 27-hydroxycholesterol (27-OH) or with a 15 µM mixture of these three oxysterols. Untreated cells (Control) were taken as controls. Data, normalized to β₂-microglobulin, are expressed as mean values ± SD of three different experiments. *P<0.05, **P<0.01, and ***P<0.001 vs. control.</p

Protection exerted by quercetin-loaded nanoparticles on CD36 and β1-integrin, IL-8, MCP-1, and MMP-9 expression induced by oxysterols.

<p>Gene expression was quantified by real-time RT-PCR in SH-SY5Y cells treated for 6 h with 5 µM 7β-hydroxycholesterol (7β-OH), 24-hydroxycholesterol (24-OH), 27-hydroxycholesterol (27-OH). Some cells were pretreated for 1 h with 5 µM free quercetin (Q_F) or with 5 µM quercetin loaded into nanoparticles (Q_N) before oxysterol treatment. Untreated cells (Control) were taken as controls, and cells treated with 31.2 mM ethanol (Et-OH) as solvent controls. Cells supplemented with blank nanoparticles (NPs) or with blank nanoparticles plus oxysterols, were taken as internal controls. Data, normalized to β₂-microglobulin, are expressed as mean values ± SD of five different experiments. *P<0.05, **P<0.01, and ***P<0.001 vs. control; #P<0.05, ##P<0.01, and ###P<0.001 vs. the specific oxysterol; §P<0.05, §§P<0.01, and §§§P<0.001 vs. Q_F+ specific oxysterol.</p

Cell viability and cell uptake of β-CD-dodecylcarbonate nanoparticles.

<p>A) SH-SY5Y cells were incubated with β-CD-dodecylcarbonate nanoparticles with (Q_N) or without (NPs) being loaded with quercetin (5 µM). Some cells were treated with 5 µM quercetin alone (Q_F). Untreated cells (Control) were taken as controls. Cell viability was measured in terms of release of the enzyme lactate dehydrogenase (LDH), as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096795#s2" target="_blank">Materials and Methods</a> section. Data represent the mean values ± SD of three different experiments. B) SH-SY5Y cells were incubated with fluorescent coumarin 6-β-CD-dodecylcarbonate nanoparticles for the times indicated and then analyzed by confocal laser scanning microscopy (40X/0.75).</p

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<p>Francisella tularensis is a highly infectious Gram-negative bacterium that is the etiologic agent of tularemia in animals and humans and a Tier 1 select agent. The natural incidence of pneumonic tularemia worldwide is very low; therefore, it is not feasible to conduct clinical efficacy testing of tularemia medical countermeasures (MCM) in human populations. Development and licensure of tularemia therapeutics and vaccines need to occur under the Food and Drug Administration's (FDA's) Animal Rule under which efficacy studies are conducted in well-characterized animal models that reflect the pathophysiology of human disease. The Tularemia Animal Model Qualification (AMQ) Working Group is seeking qualification of the cynomolgus macaque (Macaca fascicularis) model of pneumonic tularemia under Drug Development Tools Qualification Programs with the FDA based upon the results of studies described in this manuscript. Analysis of data on survival, average time to death, average time to fever onset, average interval between fever and death, and bacteremia; together with summaries of clinical signs, necropsy findings, and histopathology from the animals exposed to aerosolized F. tularensis Schu S4 in five natural history studies and one antibiotic efficacy study form the basis for the proposed cynomolgus macaque model. Results support the conclusion that signs of pneumonic tularemia in cynomolgus macaques exposed to 300–3,000 colony forming units (cfu) aerosolized F. tularensis Schu S4, under the conditions described herein, and human pneumonic tularemia cases are highly similar. Animal age, weight, and sex of animals challenged with 300–3,000 cfu Schu S4 did not impact fever onset in studies described herein. This study summarizes critical parameters and endpoints of a well-characterized cynomolgus macaque model of pneumonic tularemia and demonstrates this model is appropriate for qualification, and for testing efficacy of tularemia therapeutics under Animal Rule.</p