Differences in smoking attitudes of adolescents and young adults

This study employed the Theory of Planned Behavior to examine the differences between adolescent (n = 182) and young adults (n = 209) in their intention to smoke and examined possible differences. Analysis showed that young adults had more positive self-reported attitudes toward smoking than adolescents, had higher intentions to smoke, lower perceived behavioral control over smoking and perceived they were more informed about smoking. The Theory of Planned Behavior provided good prediction of intention for both young adults (R-2 = .70, attitudes, information, and past behavior significant) and adolescents (R-2 = .68, attitudes, past behavior significant). For both samples attitudes were the strongest predictor of intentions to smoke. Implications for understanding intention toward smoking between adolescents and young adults are discussed

Isolation and phenotypic characterization of large vein endothelial cells.

<p>Cells isolated from surgical specimens were characterized <i>in-vitro</i> by morphological assessment (<b>A</b>) and multiparametric flow cytometry analysis (<b>B</b>). In <b>A</b>, cell morphology was examined by using phase-contrast microscopy. VEC cultures were characterized by regular polygonal shape and dimensions and uniform monolayer. On the other hand, non-VEC cultures appeared with a fibroblast-like morphology characterized by elongated shapes and growing in an uneven manner. Representative images of VEC and non-VEC cultures, at two different <i>in vitro</i> passages (p = 0 and p = 3), are shown. Left panels: 10X, original magnification; right panels: 20X original magnifications. In <b>B</b>, multiparametric flow cytometry analyses were performed with a specific panel of endothelial cells defining antibodies. VEC were defined as CD146⁺/CD144⁺/CD31⁺/CD105⁺/CD34⁺/CD45⁻/CD14⁻ while non-VEC displayed a more variable and random pattern of antigens expression. Two representative multiparametric flow cytometry analysis panels of a non-VEC and pure VEC cultures are shown as two-colors dot plots.</p

Analysis of ICAM-1 and VCAM expression in control and pathological VEC.

<p>In <b>A–C</b>, surface expression of ICAM-1 antigen was evaluated by flow cytometry in either pathological and control-VEC. In <b>A</b>, colored histograms represent cells stained with monoclonal antibodies specific for the indicated antigens and white histograms represent background fluorescence obtained by staining the same cells with isotype-matched control antibodies. A representative panel for control-, C2- and C3-VEC is shown. In <b>B</b>, the expression levels of ICAM-1 were determined for all VEC samples (8 C2-VEC, 13 C3-VEC and 5 control VEC) by flow cytometry analysis and expressed as mean fluorescence intensity (MFI). *<i>P</i><0.05 compared to control VEC. In <b>C</b>, comparative analysis of ICAM-1 surface expression, reported as MFI, at different VEC passages. In <b>D–E</b>, pathological and control-VEC cultures were exposed to TNF-α for 18 hours before ICAM-1 and VCAM surface expression analysis by flow cytometry (<b>D</b>), and mRNA levels analysis by quantitative RT-PCR (<b>E</b>). In <b>D</b>, two representative panels are shown: dotted histograms represent background fluorescence obtained by staining the same cells with isotype-matched control antibodies. The expression levels of ICAM-1 and VCAM, determined for all VEC samples by flow cytometry analysis upon TNF-α stimulation, are expressed as mean fluorescence intensity (MFI). In <b>E</b>, mRNA expression levels of ICAM-1 and VCAM were determined both in unstimulated and TNF-α-stimulated VEC cultures. Results from amplifications, done in duplicate, are expressed as arbitrary units, after normalization for the housekeeping gene. Horizontal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles.</p

Analysis of the NF-kB, OPG and VEGF levels in VEC culture supernatants and serum samples.

<p>In <b>A</b>, NF-kB-p65 DNA binding activity was assessed in duplicate using the TransAM assay. Results are reported as absorbance values (O.D.) per 20 µg of cell lysate protein. In <b>B</b>, OPG and VEGF levels were determined by ELISA in VEC culture supernatants. In <b>C</b>, OPG and VEGF levels were determined by ELISA in sera of C2 and C3 patients as well as in sex and age-matched normal controls. In <b>A–C</b>, horizontal bars are median, upper, and lower edges of box are 75th and 25th percentiles; lines extending from box are 10th and 90th percentiles. *, <i>P</i><0.05 compared to either control VEC (<b>A–B</b>) or control sera (<b>C</b>).</p

Establishment of airway inflammation mouse model.

<p>Mice were exposed to an OVA “sensitization plus challenge” protocol. In <b>A</b>, the schedule and the timeline of OVA treatments of BALB/c mice are shown. In <b>B</b>, the circulating levels of total IgE were determined in serum samples of controls (n = 16) and OVA-treated mice (n = 16), harvested at the indicated experimental time points. Results are expressed as mean ± SD. In <b>C</b>, representative hematoxylin and eosin stained sections of lung tissue isolated (at day 32) from either healthy control or OVA-treated mice. Lungs of OVA-treated mice exhibit airway remodeling and thickness of epithelial cells (original magnification 40X).</p

Effect of recombinant TRAIL on OVA-induced CXCL-1/KC expression.

<p>In <b>A</b>, the levels of CXCL-1/KC in the BAL fluids of Controls, OVA- and TRAIL+OVA-mice were measured by Multiplex assay. In <b>B</b>, the expression levels of CXCL-1/KC mRNA in lung tissue of Controls, OVA- and TRAIL+OVA-mice were determined by quantitative RT-PCR. Results from amplifications, done in duplicate, are expressed as arbitrary units after normalization for the housekeeping gene. In <b>A–B</b>, horizontal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles; *, P<0.05 (Mann-Whitney rank-sum test).</p

<i>In vivo</i> biodistribution of recombinant TRAIL.

<p>Mice were i.n. instilled with recombinant TRAIL (20 µg). In <b>A</b>, the distribution of the labeled Cy5.5-TRAIL was recorded at the indicated time intervals by whole body scans in six mice. Results of one representative mouse before (control) and after i.n. instillation are shown. In <b>B</b>, <i>ex vivo</i> representative images of lungs isolated from mice sacrificed at the indicated time points after i.n. instillation of Cy5.5-TRAIL compared to a representative lung isolated from a control mouse (i.n. instillation with control vehicle). In <b>C</b>, after i.n. instillation a time course detection of circulating recombinant human TRAIL was assessed by ELISA performed on murine sera samples. Results are reported as mean ± SD of 6 mice.</p

Baseline characteristics of subjects.

<p>Baseline characteristics of subjects.</p

Effect of recombinant TRAIL on OVA-induced airway inflammation.

<p>Mice were treated with recombinant TRAIL before OVA challenges and the cellular influx in the BAL was characterized. In <b>A</b>, the schedule and the timeline of recombinant TRAIL treatments with regard to OVA sensitizations/challenges of BALB/c mice are shown. At the time of sacrifice (day 32), the circulating levels of total IgE were determined in serum samples of controls, OVA- and TRAIL+OVA-mice and expressed as fold of modulation with respect to controls (<b>insert</b>). In <b>B</b>, total cell count of cells present in BAL fluids of controls, OVA- and TRAIL+OVA-mice (n = 16 mice/group) is shown. Horizontal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles; *, P<0.01. Representative images of May-Grünwald Giemsa stained cytospins of BAL cells from each experimental group are shown (original magnification, 20X). In <b>C</b>, gating strategy utilized in multiparametric flow cytometry analysis to identify the major BAL infiltrating cell types. In <b>D</b>, the percentage of eosinophils, neutrophils, lymphocytes, alveolar macrophages (AM) and dendritic cells (DC) in the BAL of OVA-mice and of TRAIL+OVA-mice is expressed as fold of modulation with respect to the percentages of control healthy mice (set at 1). Results are mean ± SD. *, P<0.05 compared to healthy control mice; ^§, P<0.05 compared to OVA-mice (Mann-Whitney rank-sum test). In <b>E</b>, blinded semi-quantitative analysis of histology from Controls, OVA- and TRAIL+OVA-mice was done to evaluate airway remodeling (<b>left panel</b>) and inflammation (<b>right panel</b>), scored as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115387#s2" target="_blank">Material and Methods</a> section. Results are derived from at least 3 microscopy fields for each mouse (n = 8 for each experimental group). Data are expressed as mean ± SD. *, P<0.05 compared to healthy control mice (Mann-Whitney rank-sum test).</p

Differential phenotype between control and pathological VEC.

<p>Surface expression of CD31 and CD146 was evaluated by flow cytometry in either pathological and control-VEC. In <b>A</b>, colored histograms represent cells stained with monoclonal antibodies specific for the indicated antigens and white histograms represent background fluorescence obtained by staining the same cells with isotype-matched control antibodies. Representative panels for control, C2- and C3-VEC are shown. In <b>B</b>, the expression levels of the indicated antigens were determined for all VEC samples (8 C2-VEC, 13 C3-VEC and 5 control-VEC) by flow cytometry analysis and expressed as mean fluorescence intensity (MFI). Horizontal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles. *<i>P</i><0.05 compared to control VEC.</p