Publications

Chronic obstructive pulmonary disease (COPD) is an inflammatory disease associated with increased levels of IFNγ which may subsequently contribute to the underlying pathophysiology. IFNγ activates the JAK/STAT pathway via STAT1 phosphorylation leading to increased release of inflammatory mediators; therefore inhibiting this response may provide therapeutic benefit. This pilot study established an intracellular flow cytometry staining protocol to measure STAT1 phosphorylation in sputum cells from COPD patients and healthy smokers. Induced sputum was collected from COPD patients (n=6) and healthy smokers (n=3). Sputum was solubilised with DTT and cells were stimulated with 100ng/ml IFNγ for 20min, fixed with 4% (w/v) paraformaldehyde and permeabilised with ice-cold methanol. A JAK inhibitor (PF956980, 10 -5 M, 1h) was used to inhibit the JAK/STAT pathway ex-vivo . Flow cytometry was performed and cell populations were determined by their forward scatter/side scatter profile. STAT1 phosphorylation was detected in macrophages from 8/9 samples but not in any neutrophil population. Macrophages from both COPD patients and healthy smokers had similar baseline phosphorylated STAT1 signals (MFI: 250.3±17.9 and 214.4±6.7 respectively). This was increased to a greater extent from COPD patients after IFNγ stimulation compared to those from healthy smokers (MFI 447.9±36.1and 385.4±25.0 respectively). STAT1 phosphorylation in macrophages was completely blocked by PF956980. Sputum is a difficult matrix to perform intracellular flow cytometry staining with and this is the first report of STAT1 phosphorylation being detected in sputum cells from COPD patients and healthy smokers.

No abstract is provided for this article.

No abstract is provided for this article.

No abstract is provided for this article.

At present most systems used for three-dimensional reconstruction (3-D) of two-dimensional intracoronary ultrasound (ICUS) images are based on an image acquisition with a pull-back device which withdraws the catheter with a constant speed, not taking account of cardiac motion and coronary dynamic/pulsation. Cyclic changes of the vessel dimensions and the movement of the catheter inside the vessel result in artifacts and inaccuracies of quantitative measurements. This phenomenon limits accuracy and resolution when an attempt of 3-D reconstruction is performed, since images obtained in different phases of the cardiac cycle are compiled. To overcome these limitations the authors developed a custom-designed pull-back device driven by a stepping motor, which is controlled by a steering logic ensuring an ECG-gated image acquisition.

Epithelial-Mesenchymal transition is an important process in the repair and maintenance of the epithelial barrier in the lungs. During EMT epithelial cells gain increased motility and increased production of proteins, such as collagen and actin. EMT is important in a number of lung diseases, and is associated with fibrosis in asthma, remodeling in COPD and increased metastasis and invasiveness in lung cancer. Epigenetic regulation including post-translational modifications of histones is thought to play an important role in gene regulation and cell differentiation. Histone modifications such as acetylation and methylation are recognized by transcriptional co-factors such as Brd4 and JMJD3. We used type 1, alpha 1, collagen, encoded by the gene COL1A1 , as a marker of the EMT process normal human bronchial epithelial cells. Stimulation with TGFβ1 strongly induced COL1A1 transcription. Pretreating epithelial cells with the H3K27 demethylase JMJD3 inhibitors (GSKJ1 and GSKJ4) and the Brd4 inhibitor (JQ1) significantly inhibited TGFβ1 induced collagen expression. Our results indicate that following stimulation of epithelial cells with TGFβ1 the histones in the COL1A1 promoter region are demethylated by JMJD3 allowing transcriptional activation. As Brd4 recognizes changes to histone acetylation our results indicate that during EMT the histones of the COL1A1 promoter become acetylated. Therefore epigenetic modification of histones plays a key role during the process of TGFβ1 induced EMT.

We studied the effects of ovalbumin (OA) sensitization and challenge on inducible nitric oxide synthase (iNOS) gene and protein expression in Brown‐Norway rats in vivo . By use of Northern analysis, a 4.4‐kb iNOS mRNA transcript was weakly observed in control rat lung but there was a 3 fold increase in lungs sensitized to OA alone ( P <0.05). In sensitized rats, four hours after exposure to OA aerosol, there was a 6 fold increase in iNOS mRNA transcript ( P <0.05), which returned to baseline at 24 h. Immunostaining with an anti‐mouse iNOS antibody revealed some patchy staining of airway epithelium in naïve rats. There were no changes in sensitized rats exposed to saline, but sensitized and OA‐exposed rats showed increased expression in iNOS staining in macrophages. Electrophoretic mobility shift assays of lung nuclear extracts showed a marked increase in nuclear factor‐κB (NF‐κB)‐binding activity at 2 h after allergen exposure with return to baseline at 6, 12 and 24 h. We concluded that there is increased iNOS gene and protein expression associated with increased NF‐κB DNA‐binding in lungs of sensitized and challenged rats. The increase in iNOS expression may underlie the increase in exhaled NO found after allergen challenge and may contribute to the development of allergen‐induced airway hyperresponsiveness.

No abstract is provided for this article.

No abstract is provided for this article.

Background The prognostic impact of high-sensitivity C-reactive protein (CRP) levels in patients with left main coronary artery disease (LMCAD) treated with percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) is unknown. We sought to determine the effect of elevated baseline CRP levels on the 3-year outcomes after LMCAD revascularization and to examine whether CRP influenced the relative outcomes of PCI versus CABG. Methods In the EXCEL trial, patients with LMCAD and Synergy between PCI with Taxus and Cardiac Surgery (SYNTAX) scores ≤32 were randomized to PCI versus CABG. The primary composite outcome of death, myocardial infarction (MI), or stroke was analyzed according to baseline CRP levels. Results Among 999 patients with available CRP levels, median CRP was 3.10 mg/L (interquartile range 1.12-6.40 mg/L). The rate of the primary composite end point of death, MI, or stroke at 3 years steadily increased with greater baseline CRP levels. The adjusted relationship between the 3-year composite rate of death, MI, or stroke and baseline CRP modeled as a continuous log-transformed variable demonstrated steadily increasing event rates with greater CRP levels (adjusted hazard ratio, 1.26, 95% CI 1.10-1.44, P = .0008). Similarly, patients with CRP ≥10 mg/L had a 3-fold higher risk of the 3-year primary end point compared to patients with lower CRP levels (adjusted hazard ratio 2.92, 95% CI 1.88-4.54, P < .0001). The association between an elevated CRP level and the adjusted 3-year risk of the primary composite end point did not differ according to revascularization strategy (P interaction = .75). Conclusions In patients with LMCAD undergoing revascularization, elevated baseline CRP levels were strongly associated with subsequent death, MI, and stroke at 3 years, irrespective of the mode of revascularization. Further studies are warranted to determine whether anti-inflammatory therapies may improve the prognosis of high-risk patients with LMCAD following revascularization.