MOESM1 of Westernization of lifestyle affects quantitative and qualitative changes in adiponectin

Additional file 1: Table A. Relationships of Matsuda Index by regression analysis with total APN (A) and C1q-APN/total-APN ratio (B) as the dependent variables in native Japanese and Japanese-Americans

Sputum Leucine-Rich Alpha-2 Glycoprotein as a Marker of Airway Inflammation in Asthma

<div><p>Background</p><p>Asthma is a chronic inflammatory disease of airways, but an ideal biomarker that accurately reflects ongoing airway inflammation has not yet been established. The aim of this study was to examine the potential of sputum leucine-rich alpha-2 glycoprotein (LRG) as a new biomarker for airway inflammation in asthma.</p><p>Methods</p><p>We obtained induced sputum samples from patients with asthma (N = 64) and healthy volunteers (N = 22) and measured LRG concentration by sandwich enzyme-linked immunosorbent assay (ELISA). Ovalbumin (OVA)-induced asthma model mice were used to investigate the mechanism of LRG production during airway inflammation. The LRG concentrations in the bronchoalveolar lavage fluid (BALF) obtained from mice were determined by ELISA and mouse lung sections were stained with anti-LRG antibody and periodic acid-Schiff (PAS) reagent.</p><p>Results</p><p>Sputum LRG concentrations were significantly higher in patients with asthma than in healthy volunteers (p = 0.00686). Consistent with patients’ data, BALF LRG levels in asthma model mice were significantly higher than in control mice (p = 0.00013). Immunohistochemistry of lung sections from asthma model mice revealed that LRG was intensely expressed in a subpopulation of bronchial epithelial cells, which corresponded with PAS-positive mucus producing cells.</p><p>Conclusion</p><p>These findings suggest that sputum LRG is a promising biomarker of local inflammation in asthma.</p></div

Effect of SK-216 on fibroblast to myofibroblast differentiation induced by TGF-β1 in human primary lung fibroblasts.

<p>After pre-incubation in serum free medium for 24 hours, ILD-derived and normal human primary lung fibroblasts were pretreated in the presence or absence of SK-216 (50, 150 μM) for 1 hour followed by stimulation with TGF-β1 (5 ng/ml). Real-time quantitative RT-PCR analysis and western blotting analysis were performed after incubation for 24 and 48 hours, respectively. (A) mRNA expression of α-SMA, fibronectin, and COL1A1 in ILD-derived human primary lung fibroblasts evaluated using real-time quantitative RT-PCR. Results are expressed as fold change from the control and reflect the mean ± SEM of 4 experiments. (B-C) Western blot analysis of α-SMA, fibronectin, and type I collagen in (B) ILD-derived and (C) normal human primary lung fibroblasts. Data normalized to β-actin levels are shown as fold change from the control and reflect the mean ± SEM of 3 independent experiments. *p <0.05, **p <0.01 vs untreated cells, ^#p <0.05, ^##p <0.01 vs cells treated with TGF-β1.</p

Detection of LRG in BALF, serum and lung section in a murine model of asthma.

<p>A) Levels of LRG in BALF and serum in a mouse model of asthma. Concentrations of BALF and serum LRG were determined by ELISA. The Student’s t-test was used for statistical analysis. The individual values are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162672#pone.0162672.s005" target="_blank">S3B–S3E File</a>) Localization of LRG in mouse lung. Paraffin sections of the lung from control (B and D) and OVA-treated (C and E) mouse were stained with anti-LRG antibody (B and C) and PAS (D and E). Scale bar, 100 μm. F and G) Immunohistochemisry of MUC5AC (F) and LRG (G) of the lung from OVA-treated mouse. Arrows show MUC5AC (F) or LRG (G) positive cells. Scale bar, 20 μm</p

Effect of SK-216 on morphological transformation and the expression of epithelial or mesenchymal markers in A549 cells undergoing TGF-β-induced EMT.

<p>Cells were pre-incubated in the presence or absence of SK-216 (50, 100 μM) for 1 hour and then co-incubated with TGF-β1 (5 ng/ml). All assays were performed after 48 hours of incubation. (A) Phase contrast images of A549 cells. A549 cells untreated with TGF-β1 maintained epithelial cobblestone-shape with cell-cell contacts. The treatment with TGF-β1 transformed the cells into mesenchymal spindle-shape cells with loss of cell-cell adhesion. The co-incubation with SK-216 reversed these TGF-β-induced morphologic alterations. Magnification: ×200. Scale bar = 100 μm. (B) Proliferation of A549 cells after treatment with SK-216 and TGF-β1. (C) A549 cells stained for E-cadherin (green), vimentin (red) and nuclei (blue). Magnification: ×200. Scale bar = 100 μm. (D) Fluorescence intensities for E-cadherin and vimentin. Results are expressed as Box plot of at least 6 fields. (E) Western blot analysis of E-cadherin, vimentin, and N-cadherin. Data normalized to β-actin levels are shown as fold change from the control, and reflect the mean ± SEM of 3 independent experiments. (F) Measurement of total soluble collagen in the culture medium. Data are normalized to the amount of total protein in the cell lysate. Results are expressed as the mean ± SEM of 4 independent experiments. *p <0.05, **p <0.01 vs untreated cells, ^#p <0.05, ^##p <0.01 vs cells treated with TGF-β1.</p

Effect of SK-216 on BLM-induced pulmonary fibrosis in mice.

<p>Mice were allocated into three groups: PBS+DW group, BLM+DW group, BLM+SK-216 group. Mice were intratracheally instilled with PBS or PBS containing BLM (1.5 mg/kg body weight) on day 0. PBS+DW group; PBS intratracheally instilled and orally administered with distilled water, BLM+DW group; BLM intratracheally instilled and orally administered with distilled water, BLM+SK-216 group; BLM intratracheally instilled and orally administered with distilled water on days from 0 to 8 and distilled water containing SK-216 (1000ppm) on days from 9 to 21. (A) mRNA expression levels of PAI-1, α-SMA, and COL1A1 in lung. Left lung harvested on day 11 was analyzed using real-time quantitative RT-PCR. Results are expressed as fold change from the control. (B) Levels of active PAI-1 and TGF-β1 in BALF. BALF was collected on day 11. (C) The degrees of pulmonary fibrosis were analyzed by measuring hydroxyproline contents in the whole lungs on day 21. (D) Histological and immunohistochemical analysis of bleomycin-injured lung. Right lungs were excised and sectioned on day 21. Scale bar = 200 μm. Results are presented as the mean ± SEM of 6 mice per group. *p <0.05, **p <0.01 vs PBS+DW group, ^#p <0.05, ^##p <0.01 vs BLM+DW group.</p

Additional file 2: of MUC1 in lung adenocarcinoma: cross-sectional genetic and serological study

The dataset supporting the conclusions of this article is available in this datasheet. (XLS 133 kb

Induction of LRG in primary bronchial epithelial cells.

<p>A) LRG secretion in culture supernatant by primary bronchial epithelial cells. Cells were incubated with IL-13 for 5 days and then treated with indicated cytokines for 24 h. Control cells were incubated without IL-13 for 5 days and further stimulated by cytokines. LRG protein in culture supernatant was detected by western blot. B) LRG gene expression in primary bronchial epithelial cells treated with IL-13 for 5 days. Cells were stimulated by indicated cytokines for 6 h. LRG mRNA expression was analyzed by quantitative PCR. Dunnett’s test was used for statistical analysis. The individual values are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162672#pone.0162672.s006" target="_blank">S4 File</a>.</p

Clinical characteristics of the study subjects.

<p>Clinical characteristics of the study subjects.</p

Up-regulation of LRG in asthmatic patients.

<p>Levels of LRG in sputum obtained from healthy volunteers and patients with asthma. Concentrations of sputum LRG were determined by ELISA. The Mann-Whitney U-test was used for statistical analysis. The individual values are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162672#pone.0162672.s004" target="_blank">S2 File</a>.</p