Also there was a higher percentage of apop totic neutrophils detected by the Tunel assay compared with those cells that were Av PI. There was a significant correlation between Av PI neutrophils and the percentage of neutrophils displaying morphological features of apoptosis. Using the Annexin V PI method for the assessment of selleck bio apoptosis, granulocytes were gated using the forward and side scatter dot plot and assessed for staining of Annexin V and or PI. Subjects with COPD and healthy smokers had a statistically significant reduc tion in early stage apoptosis compared to non smokers, p 0. 001. There was a trend for an increase in late apoptosis sec ondary Inhibitors,Modulators,Libraries necrosis in subjects with COPD and healthy smokers compared to non smokers.
Despite differences in methodologies, similar findings to those with the Annexin V PI assay were seen using both the Tunel assay and the morphological Inhibitors,Modulators,Libraries identification of apoptosis. A significant reduction in the per centage of Tunel positive cells was observed in COPD sub jects compared to non smokers and also in the percentage of cells that were morphologically identified as apoptotic using light microscopy. This study also examined the plasma levels of soluble Fas APO I receptor, an inhibitor of apoptosis, and sol uble Fas ligand, an inducer of apoptosis, in all three subject groups. Although plasma sFas lev els were slightly higher in COPD subjects compared to non smokers there was no statistical significance between the groups. Similarly assessment of sFasL showed no sig nificant differences between the subject groups.
I B phosphorylation and NF B activation in induced sputum Localisation of single granulocyte nuclei allowed Inhibitors,Modulators,Libraries for the quantification of NF B activation in sputum from COPD subjects, healthy smoking controls and non smoking con trols. p50 and p65 subunit expression Inhibitors,Modulators,Libraries in sputum neutrophil nuclei showed a significant increase in the expression of both NF B subunits in COPD sub jects compared to non smokers. Although there was a higher expression of p50 and p65 positive neutrophil nuclei in healthy smokers compared to non smokers, this did not reach significance. An association was observed between sFasL in plasma from non smokers and activation of p65 in sputum. Inhibitors,Modulators,Libraries To further assess the role of NF B in controlling sputum neutrophil apoptosis in COPD subjects, the phosphoryla tion of I B in sputum was investigated.
Although there was a trend towards a higher level of phosphorylated I B in healthy smokers compared to non smokers, this did not reach significance. Discussion In this study, flow cytometric analysis demonstrated not that apoptosis is abnormally regulated in COPD subjects that this appears to be, at least in part, regulated by the activa tion of NF B. It is postulated that oxidative stress and sur vival factors contribute to the longevity of the neutrophil that perhaps overwhelms the macrophage clearance mechanism leading to persistent airway inflammation in COPD subjects.