Airway responsiveness testing in an excised rat lung.

<p>Slice selective VFA FLASH images of positively responding <i>ex vivo</i> rat lungs after intravenous challenges of 60 µg methacholine with subsequent reversal produced by flushes of intravenous 5% glucose and 1000 µg salbutamol. Images were performed using a constant inhalation syringe (suction) volume of <i>V_S</i> = 5 mL. Imaging parameters: 4 mm central slice, matrix 128×64, FOV = 46.9×30.0 mm². Positioning of the lung as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073468#pone-0073468-g002" target="_blank">Fig. 2</a>.</p

Normalization of hyperpolarized ¹²⁹Xe distribution by total signal intensity and position along the anterior-posterior axis.

<p>(A) Integrated signal intensity (taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073468#pone-0073468-g003" target="_blank">Fig. 3</a>) in arbitrary units (a.u.) as a function of the image row number <i>m</i> (in z-direction); (B) Integrated signal intensity after normalization by the total signal intensity (i.e. the integrated intensity of all voxels, , of the respective MR image); (C) Normalized integrated signal intensity as in (B) but as a function of position along the lung posterior-anterior axis (z-axis) from base to apices. Independent of inhalation volume and actual lung expansion, the 0.0 point refers the base of the lung, whereas 1.0 refers to the apices. The 50% signal intensity position in the lungs is indicated by grey vertical line (C) i.e. 50% of the total signal intensity lies to both sides of the grey line.</p

Relationship between syringe suction volume and inhaled gas volume^¹.

1<p>Applied syringe suction volumes, <i>V_s</i>, with corresponding values for inhaled volume, <i>V_i</i>, determined by the water bell method for three Sprague-Dawley rats (weight 250–300 g). Errors listed are experimental relative errors. The omitted values were not determined.</p

Outline of the hyperpolarized ¹²⁹Xe gas delivery to the <i>ex vivo</i> lung.

<p>(A) Experimental <i>ex vivo</i> setup with hp ¹²⁹Xe administered from a balloon reservoir chamber into the storage volume (<i>V_B</i>) before being inhaled by the lung. The lung is caused to inhale (exhale) by the negative (positive) external ‘pleural’ pressure applied via the suction volume (<i>V_s</i>) from the ventilation syringe upon the artificial pleural cavity; (B) <i>Ex vivo</i> lung submerged with its orifice down (sutured to a cannula) in 5% glucose solution within the ventilation chamber with its posterior-anterior axis aligned in z-direction. In this sketch, a negative pleural pressure caused by <i>V_s</i> leads to a partial inflation of the <i>ex vivo</i> lung, inhaling a selected gas (hp ¹²⁹Xe, or N₂ or O₂) from the storage volume <i>V_B</i>. Drugs are administered via a cannula sited in the right ventricle with the excess fluid outlet located below the fluid level in the chamber. All resulting MR images shown in subsequent figures are depicted with the lung orifice pointing upwards.</p

Non-slice selective coronal VFA FLASH MR images used for calculation of residual volume (<i>RV</i>).

<p>(A) Acquired after inhalation to <i>V_s</i> = 5 mL (actual inhalation, <i>V_i</i> = 3.09 mL); (B) Inhalation to <i>V_s</i> = 5 mL followed by full exhalation to <i>V_s</i> = 0 mL (<i>V_i</i> = 0 mL) before the MR image is acquired. Image resolution is 128×64 with FOV = 46.9 mm in the longitudinal and FOV = 30.0 mm in the axial dimensions, respectively. In this presentation, the orifice of the lung is pointing up with the posterior-anterior axis aligned with the z-direction.</p

Hyperpolarized ¹²⁹Xe gas distribution on increasing inhalation volumes.

<p>Non-slice selective coronal VFA FLASH images as a function of increasing suction volume (<i>V_s</i>) (and inhaled volume (<i>V_i</i>)). The corresponding histograms displaying integrated intensities, , for each row, <i>m</i>, are shown to the right of the images. The vertical axis of the image is parallel to the direction of the <i>B_o</i> field (z-direction) and corresponds to the posterior-anterior axis (base to apex) of the lung in the magnet. Phase encoding is applied transverse to the <i>B_o</i> field direction. As the suction volume increases from 0.5 mL to 6.0 mL the image contrast is greatly enhanced. The effect is caused by the increasing quantities of inhaled hp gas contained in the lung as the suction volume rises. Matrix 128×64 with FOV = 46.9×30.0 mm².</p

Airway responsiveness testing in an excised guinea pig lung.

<p>Slice-selective VFA FLASH images of <i>ex vivo</i> guinea pig lungs after intravenous challenges with 5% glucose solution alone and 10 µg methacholine. Subsequent reversal was produced by flushes of intravenous 5% glucose and 200 µg salbutamol. Images were performed with a constant inhalation syringe (suction) volume of <i>V_S</i> = 5 mL. Imaging parameters: 4 mm central slice, matrix 128×64, FOV = 46.9×30.0 mm². Positioning of the lung as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073468#pone-0073468-g002" target="_blank">Fig. 2</a>.</p

Timed release of hyperpolarized ¹²⁹Xe during constant inhalation volumes.

<p>Coronal slice selective VFA FLASH images for directed ventilation schemes with a histogram that displays the integrated intensities in each row are shown to the right of the images. Scheme 1 (A–C)- initial inhalation consists of a known volume of hp gas, <i>V_s(hp)</i>, followed by dark gas, <i>V_s(Dark)</i>. Scheme 2 (D–E)- the reversal with the inhalation of <i>V_s(Dark)</i> followed by <i>V_s(hp)</i>. Full 5.0 mL inhalation of hp gas with edge detection using Kirsch operator <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073468#pone.0073468-Kirsch1" target="_blank">[71]</a> with window level adjusted to show lower signal intensities (F). Z-axis along <i>B_o</i> in posterior-anterior axis (base to apex) of the lung in the magnet and x-axis along indirect (phase encoding) dimension. Imaging parameters: 4 mm central slice, matrix 128×64, FOV = 46.9×30.0 mm². Positioning of the lung as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073468#pone-0073468-g002" target="_blank">Fig. 2</a>.</p

Large-Scale Label-Free Quantitative Mapping of the Sputum Proteome

Analysis of induced sputum supernatant is a minimally invasive approach to study the epithelial lining fluid and, thereby, provide insight into normal lung biology and the pathobiology of lung diseases. We present here a novel proteomics approach to sputum analysis developed within the U-BIOPRED (unbiased biomarkers predictive of respiratory disease outcomes) international project. We present practical and analytical techniques to optimize the detection of robust biomarkers in proteomic studies. The normal sputum proteome was derived using data-independent HDMS^E applied to 40 healthy nonsmoking participants, which provides an essential baseline from which to compare modulation of protein expression in respiratory diseases. The “core” sputum proteome (proteins detected in ≥40% of participants) was composed of 284 proteins, and the extended proteome (proteins detected in ≥3 participants) contained 1666 proteins. Quality control procedures were developed to optimize the accuracy and consistency of measurement of sputum proteins and analyze the distribution of sputum proteins in the healthy population. The analysis showed that quantitation of proteins by HDMS^E is influenced by several factors, with some proteins being measured in all participants’ samples and with low measurement variance between samples from the same patient. The measurement of some proteins is highly variable between repeat analyses, susceptible to sample processing effects, or difficult to accurately quantify by mass spectrometry. Other proteins show high interindividual variance. We also highlight that the sputum proteome of healthy individuals is related to sputum neutrophil levels, but not gender or allergic sensitization. We illustrate the importance of design and interpretation of disease biomarker studies considering such protein population and technical measurement variance

Large-Scale Label-Free Quantitative Mapping of the Sputum Proteome

Analysis of induced sputum supernatant is a minimally invasive approach to study the epithelial lining fluid and, thereby, provide insight into normal lung biology and the pathobiology of lung diseases. We present here a novel proteomics approach to sputum analysis developed within the U-BIOPRED (unbiased biomarkers predictive of respiratory disease outcomes) international project. We present practical and analytical techniques to optimize the detection of robust biomarkers in proteomic studies. The normal sputum proteome was derived using data-independent HDMS^E applied to 40 healthy nonsmoking participants, which provides an essential baseline from which to compare modulation of protein expression in respiratory diseases. The “core” sputum proteome (proteins detected in ≥40% of participants) was composed of 284 proteins, and the extended proteome (proteins detected in ≥3 participants) contained 1666 proteins. Quality control procedures were developed to optimize the accuracy and consistency of measurement of sputum proteins and analyze the distribution of sputum proteins in the healthy population. The analysis showed that quantitation of proteins by HDMS^E is influenced by several factors, with some proteins being measured in all participants’ samples and with low measurement variance between samples from the same patient. The measurement of some proteins is highly variable between repeat analyses, susceptible to sample processing effects, or difficult to accurately quantify by mass spectrometry. Other proteins show high interindividual variance. We also highlight that the sputum proteome of healthy individuals is related to sputum neutrophil levels, but not gender or allergic sensitization. We illustrate the importance of design and interpretation of disease biomarker studies considering such protein population and technical measurement variance