Description of the 3 asthmatic mouse models.

<p>Data are means ± standard error of the mean. P-values were obtained using Wilcoxon-Mann-Whitney rank sum test. BAL: bronchoalveolar lavage.</p

Comparison of Penh and lung resistance.

<p>A) Bronchial hyperresponsiveness (BHR) to methacholine was determined at Day 75 in unrestrained conscious mice by single-chamber plethysmography. The results were expressed as a ratio of Penh measured in response to 8 mg/ml methacholine to that with normal saline. B) Bronchial hyperresponsiveness (BHR) to methacholine was also determined at Day 77 in anaesthetised and intubated animals by invasive plethysmography. The results were expressed as a ratio of LR measured in response to 8 mg/ml methacholine to that with normal saline. Results from control (white bars) and OVA-sensitized mice (black bars) are presented.</p

Correlation matrix between micro-CT bronchial parameters and plethysmographic, BAL and histological data.

<p>Data are Spearman rank correlation coefficients. Data in parentheses are P-values.</p><p>BAL: bronchoalveolar lavage. PBA: peribronchial mean attenuation, Normalized PBA corresponds to 1– PBA/total lung mean attenuation.</p

Bland-Altman analysis of manual and semi-automatic methods for peribronchial attenuation (PBA) measurements.

<p>A) Correlation of peribronchial mean attenuation (PBA) between the two methods. Dashed line represents the line of equality. Solid line corresponds to the regression line. B) Means of measurement between the two methods are plotted against their differences. Solid line corresponds to the mean difference. Dashed lines correspond to the mean difference ±2 standard deviations. C) Means of measurement between the two methods are plotted against their standard deviations.</p

Comparison of micro-CT parameters.

<p>A) Total lung attenuation, B) peribronchial mean attenuation (PBA), and C) normalized PBA are presented for control (white box plots) and OVA-sensitized (grey box plots) mice at each endpoint. Box plots summarise medians with 25% and 75% interquartiles. Error bars represent 5th and 95th percentiles. *p<0.05 using Wilcoxon’s signed-rank tests between control and OVA.</p

Semi-automatic 3D method for assessing peribronchial attenuation.

<p>A) Native axial (top) and coronal thin-section reformatted (bottom) micro-CT images of the bronchial tree. B) Automatic segmentation of the bronchial lumen (pink). C) Automatic 8-voxels dilatation of the lumen volume. D) Second automatic segmentation of the bronchial lumen volume (green) overwriting bronchial lumen from the previous volume of interest. E) After subtraction of the bronchial lumen, the resultant volume of interest includes only the peribronchial area of the whole bronchial tree. From the created peribronchial volume, the software provides the peribronchial mean attenuation (PBA) value.</p

Typical axial native micro-CT images of control (left) and OVA-sensitized mice (right) at different endpoints: A) Day 36, B) Day 76 and C) Day 111.

<p>The insert at the right bottom of each panel corresponds to a selected part of a new image generated by normalizing each pixel attenuation value by the total lung attenuation value. The green circles delineating the lumen and the 8-voxels peribronchial atmosphere help to demonstrate the differences in normalized peribronchial attenuation between control and OVA-sensitized mice at day 76 and day 111.</p

Typical coronal curved reformatted micro-CT images of the bronchial tree with numerical values of peribronchial mean attenuation (PBA) and normalized PBA.

<p>Images were obtained from control mice (left) and OVA-sensitized (right) at different endpoints: A) Day 36, B) Day 76 and C) Day 111.</p

Protease Activated Receptor-2 Expression and Function in Asthmatic Bronchial Smooth Muscle

<div><p>Asthmatic bronchial smooth muscle (BSM) is characterized by structural remodeling associated with mast cell infiltration displaying features of chronic degranulation. Mast cell-derived tryptase can activate protease activated receptor type-2 (PAR-2) of BSM cells. The aims of the present study were (i) to evaluate the expression of PAR-2 in both asthmatic and non asthmatic BSM cells and, (ii) to analyze the effect of prolonged stimulation of PAR-2 in asthmatic BSM cells on cell signaling and proliferation.</p><p>BSM cells were obtained from both 33 control subjects and 22 asthmatic patients. PAR-2 expression was assessed by flow cytometry, western blot and quantitative RT-PCR. Calcium response, transduction pathways and proliferation were evaluated before and following PAR-2 stimulation by SLIGKV-NH₂ or trypsin for 1 to 3 days.</p><p>Asthmatic BSM cells expressed higher basal levels of functional PAR-2 compared to controls in terms of mRNA, protein expression and calcium response. When PAR-2 expression was increased by means of lentivirus in control BSM cells to a level similar to that of asthmatic cells, PAR-2-induced calcium response was then similar in both types of cell. However, repeated PAR-2 stimulations increased the proliferation of asthmatic BSM cells but not that of control BSM cells even following lentiviral over-expression of PAR-2. Such an increased proliferation was related to an increased phosphorylation of ERK in asthmatic BSM cells.</p><p>In conclusion, we have demonstrated that asthmatic BSM cells express increased baseline levels of functional PAR-2. This higher basal level of PAR-2 accounts for the increased calcium response to PAR-2 stimulation, whereas the increased proliferation to repeated PAR-2 stimulation is related to increased ERK phosphorylation.</p></div

Increased PAR-2 expression in asthmatic bronchial smooth muscle cells.

<p>PAR-2 levels were assessed by flow cytometry (A), western blot (B) and quantitative RT-PCR (C). Normalized median fluorescence intensities were calculated by dividing median fluorescence intensity of PAR-2 by that of isotype control (A). Representative blots stained with anti–PAR-2 or anti–β-actin antibodies are shown (B). The RT-PCR expression of PAR-2 was presented as an arbitrary unit and normalized to endogenous references (geometric averaging of three internal control genes; <i>i.e.</i> YWHAZ, HPRT-1, and PO) according to GeNorm (C). Bronchial smooth muscle cells were obtained from asthmatic (black bars, n = 7) and control subjects (white bars, n = 7). Results are expressed as mean ± SD. *<i>P</i><0.05 using Mann & Whitney test.</p