The spatial gene-gene interaction networks and the analysis of its properties.

<p>The results were based on the contact data with reads that were mapped to multiple locations on the genome removed. (<b>A</b>) The gene-gene interaction network of genes residing in chromosome 14 for the CALL-4 cell line. (<b>B</b>) The distribution of node degrees. (<b>C</b>) The histogram of shortest path lengths. (<b>D</b>) The plot of average clustering coefficient against of the degree (the number of neighbors) of a node. (<b>E</b>) The plot of closeness centralities against the degree of a node. (<b>F</b>) The stress distribution. (<b>G</b>) The plot of topological coefficients against the degree of a node. The network and its properties were visualized and analyzed by Cytoscape <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058793#pone.0058793-Shannon1" target="_blank">[32]</a>.</p

The contact matrices, correlation matrices, and difference matrices of chromosome 14 after applying normalization.

<p>(<b>A–D</b>) denote the contact matrices after removing reads mapped to multiple locations on the genome, (<b>E-H</b>) the contact matrices after applying SCN normalization method to the maps in (<b>A–D</b>), (<b>I–L</b>) the contact matrices after applying both genomic sequential distance based method and SCN to maps in (<b>A–D</b>), (<b>M–P</b>) the correlation matrices based on maps in E-H, and (<b>Q–T</b>) the difference matrices of between correlation matrices in M-P of normal and malignant samples.</p

The contact matrices, correlation matrices, and difference matrices of chromosome 14.

<p>This figure illustrates the original contact matrices, correlation matrices, and difference matrices of chromosome 14 for the normal human B-cell, human acute lymphoblastic leukemia B-cell, human MHH-CALL-4 B-ALL cell line, and human lymphoma RL cell-line. Heat maps (A-D) visualize the original number of contacts within the chromosome, (<b>E</b>-<b>H</b>) the Pearson’s correlation matrices generated from the original contact matrices, and (<b>I</b>-<b>L</b>) the absolute difference matrices generated from the correlation matrices.</p

Role of PMNs in the Pathogenesis of LPS-Induced Lung Injury

<div><p>(A) Depletion of granulocytes attenuates the endotoxin induced rise in alveolocapillary permeability. Pretreatment of mice with anti-Gr-1 was followed by a significant decrease in the number of granulocytes (left graph) and a significant reduction of the total amount of protein (right graph) recovered by BAL 48 h after IT LPS injection.</p> <p>(B) The more granulocytes immigrated into the alveolar spaces, the higher the alveolocapillary permeability rose. Bivariate analysis according to Pearson revealed a statistically significant correlation (<i>p</i> < 0,001) between the number of PMNs and the amount of protein in the BAL fluid 48 h after IT LPS injection, suggesting that inflammatory lung injury after IT injection of LPS is mostly mediated by granulocytes.</p></div

IT Administration of A_2AR Agonist Protects from Increased Death Rate upon Oxygenation of Mice with Acute Lung Injury

<p>Compensation for the oxygenation-associated loss of the hypoxia → adenosine → A_2AR signaling pathway by IT injection of CGS21680 significantly decreased the oxygen-exacerbated death rate in mice with acute lung injury induced by IT injection of SEB and LPS. For further explanation, see legend for <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030174#pbio-0030174-g001" target="_blank">Figure 1</a>.</p

Intratracheal Injection of A_2AR Selective Agonist Mimics Protective Effects of Hypoxia

<div><p>(A) IT injection of the A_2AR agonist CGS21680 into endotoxin-inflamed lungs provides protection similar to that observed in hypoxia-treated mice. Number of PMNs recovered after 48 h by BAL from endotoxin-injected animals that were kept at normal 21% oxygen atmosphere was significantly diminished by IT injections of CGS21680 compared to placebo-treated mice. Lung PMNs (left graph) from A_2AR agonist-treated animals also produced lower levels of reactive oxygen metabolites (H₂O₂; right graph).</p> <p>(B) Significantly decreased lung vascular permeability (protein in BAL; left graph) and improved lung gas exchange (p_aO₂; right graph) in endotoxin-injected mice after treatment with the A_2AR agonist CGS21680.</p> <p>(C) Histologic evidence for the lung tissue-protecting effects of A_2AR agonist during endotoxin- and oxygenation-induced lung damage. Quantitative analysis of lung histopathology by a pathologist blinded to the experimental design revealed inhibition of PMN sequestration in IT LPS-injected mice after treatment with the A_2AR-selective agonist CGS21680 for 48 h. The lung tissue damage was also significantly decreased as assessed by the LIS (<i>n</i> = 9, mean ± standard deviation). Representative H&E-stained slices in the right two photomicrographs show less intracapillary PMN sequestration and almost no intraalveolar accumulation of PMNs in CGS21680-treated mice. These CGS21680-induced changes are similar to those observed for the effects of hypoxia on endotoxin- injected animals (compare with <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030174#pbio-0030174-g006" target="_blank">Figure 6</a>C).</p></div

Evidence for the Critical Role of Immunosuppressive A_2AR in Lung Protection from Inflammatory Damage

<div><p>(A) In <i>A_2AR</i> gene-deficient mice, number of PMNs (left graph) and amount of protein recovered (center graph) 48 h after IT LPS injection by BAL was significantly higher than in similarly treated wild-type control mice, reflecting increased lung damage in the absence of A_2AR. The arterial oxygen tension (right graph) was lower in <i>A_2AR</i> gene-deficient mice as compared with wild-type mice.</p> <p>(B) Pharmacologic inactivation of A_2AR leads to exacerbated inflammatory lung tissue damage and decreased lung funciton. After estimation of biologically relevant half-life of A_2AR antagonist ZM241385 (ZM) in vivo (unpublished data), the IT LPS-injected mice were administered ZM241385 at a dose of 10 mg/kg body weight every 6 h subcutaneously to ensure sufficient levels of the antagonist. This dosing regimen of the A_2AR antagonist caused significant more lung tissue damage, as reflected by increased number of PMNs (left graph) and protein levels (center graph) in the BAL fluid obtained after 48 h. In parallel experiments, the A_2AR antagonist decreased lung function (right graph) as compared to untreated wild-type mice, in agreement with results of experiments with <i>A_2AR</i> gene-deficient mice.</p></div

Increased Death Rate upon Oxygenation of Mice with Acute Lung Injury

<p>Mice were IT injected with SEB and LPS to model polymicrobial infection and were exposed to 21% or 100% oxygen for 48–60 h. Determination of time-dependent survival curves was prohibited by considerations of unrelieved severe respiratory distress in NIH-approved animal care protocol, which required termination of experiments immediately after differences between groups became apparent. Major differences between groups occurred within 48–60 hours after IT injection of toxins, when the majority of oxygenated animals with inflamed lungs had died, while the nonoxygenated, obviously sick control mice with inflamed lungs were still alive.</p

Exacerbation of Inflammatory Lung Injury after Exposure of Mice to Different Concentrations of Oxygen

<div><p>(A) Enhanced lung vascular permeability (left graph) and impairment of lung gas exchange (right graph) in mice breathing 100% O₂ upon induction of acute lung injury. Following IT injection of mice with SEB and LPS, animals breathed 21% or 100% oxygen. After 48 h, lung vascular permeability and lung gas exchange were determined by the amount of protein recovered by BAL or by measuring pO₂ values in arterial blood drawn, respectively, 15 min after return of mice to normal atmosphere. During this equilibration period, three out of seven mice previously exposed to 100% oxygen died, so that no arterial blood gas analyses could be performed, but BAL protein concentrations were determined immediately thereafter.</p> <p>(B) Increased lung vascular permeability (left graph) and impairment of lung gas exchange (right graph) in mice with acute lung injury even upon exposure to lower levels of oxygen (60%), which are considered clinically safe in humans. Experimental conditions were the same as in (A), except oxygen concentration was 60% instead of 100%.</p></div

Hypoxia Down-Regulates Neutrophils and Protects Lung Tissue from Inflammatory Damage

<div><p>(A) Exposure of IT LPS-injected mice to hypoxic (10%) oxygen levels for 48 h atmosphere leads to a significantly decreased accumulation of PMNs (left graph), production of LPS-triggered oxygen reactive metabolites in lungs (center graph), and improved lung gas exchange (right graph) compared to a control group of endotoxin-treated mice that were kept at ambient (21%) oxygen. To standardize conditions, the arterial blood samples were taken 15 min after return of the previously hypoxia-exposed animals to normal atmosphere.</p> <p>(B) Treatment by a shorter period of hypoxia attenuates PMN sequestration (left graph) and lung vascular permeability (right graph). Hypoxic treatment of mice even for only 24 h was sufficient to delay PMN sequestration and to diminish the increase in lung vascular permeability.</p> <p>(C) Histologic evidence for the hypoxic inhibition of pulmonary PMN sequestration. Quantitative analysis of lung slices by a pathologist blinded to the experimental design revealed inhibition of PMN sequestration in IT LPS-injected mice following 4-h exposure to hypoxia. Hypoxia not only attenuated PMN accumulation, but the lung tissue damage was also significantly decreased as assessed by the LIS (<i>n</i> = 9, mean ± standard deviation). The representative H&E-stained slices in the right two photomicrographs show less intravascular granulocyte sequestration, less thickening of the alveolocapillary membrane, and almost no granulocytes in the alveolar spaces as compared to IT endotoxin-injected animals breathing 21% O₂. These observations demonstrate that hypoxia also inhibited the transmigration of granulocytes from capillaries into the alveolar spaces.</p></div