Tregs and HLA-DR expression in sputum cells of COPD patients treated with tiotropium and formoterol.

Immune cells expressing the activation markers HLA-DR and regulatory T cells (Tregs) may be involved in the regulation of chronic inflammation in chronic obstructive pulmonary disease (COPD). In this study we analyzed native and activated cell profiles in sputum of 22 stable COPD patients receiving formoterol (F) or formoterol + tiotropium (F + T) for 3 months. Cells were isolated from induced sputum and were examined on Coulter flow cytometer using fluorescent antibodies specific for CD3, CD4, CD8, CD14, CD19, CD25, CD127, and HLA-DR antigens. Cell profiles and cell activation were assessed by analysis of HLA-DR, CD25, and CD127 co-expression in double-stained samples. Tregs were defined as CD4⁺CD25(high) CD127(low) cells. We found that the combined therapy significantly decreased the CD8⁺ cell number (p < 0.01). At baseline, HLA-DR was expressed in about 10 % of sputum T or B cells and a higher expression was found on monocytes. The HLA-DR expression on lymphocytes, but not monocytes, was significantly lower (p < 0.01) in patients treated with F + T. Fractions of activated [CD4⁺ CD25⁺] cells were also significantly lower in the combined therapy group, except for the subpopulation of CD4⁺CD25(high) CD127(low) cells which was not altered. We conclude that tiotropium in add-on therapy to formoterol affects Treg cell profiles and decreases HLA-DR expression in airway lymphocytes.

Histone acetylation and arachidonic acid cytotoxicity in HepG2 cells overexpressing CYP2E1.

The aim of this work was to assess the role of ethanol-derived acetate and acetate-mediated histone acetylation in arachidonic acid-induced stress in HepG2 cells and cells overexpressing CYP2E1. Cells were grown for 7 days with 1 mM sodium acetate or 100 mM ethanol; their acetylated histone proteins and histone deacetylase 2 expression was quantified using Western blot. Ethanol- or acetate-pretreated cells were also treated for 24 h with 60 µM arachidonic acid to induce oxidative stress. Cytotoxicity was estimated by lactate dehydrogenase release, 3-[4,5-dimethylthiazolyl-2] 2,5-diphenyltetrazolium bromide test, and by DNA damage, while oxidative stress was quantified using dichlorofluorescein diacetate. Cells grown with ethanol or acetate had increased acetylated histone H3 levels in both cell types and elevated acetylated histone H4 levels in cells overexpressing CYP2E1 but not in naïve cells. In cells overexpressing CYP2E1 grown with ethanol, expression of histone deacetylase 2 was reduced by about 40 %. Arachidonic acid altered cell proliferation and was cytotoxic mostly to cells engineered to overexpress CYP2E1 but both effects were significantly lower in cells pretreated with ethanol or acetate. Cytotoxicity was also significantly decreased by 4-methylpyrazole--a CYP2E1 inhibitor and by trichostatin--an inhibitor of histone deacetylases. In cells pretreated with acetate or ethanol, the oxidative stress induced by arachidonic acid was also significantly lower. Our data indicate that histone hyperacetylation may in some extent protect the cells against oxidative stress. It is possible that acetate may act as an antioxidant at histone level. This mechanism may be relevant to alcohol-induced liver injury.

Propofol protects rat astroglial cells against tert-butyl hydroperoxide-induced cytotoxicity; the effect on histone and cAMP-response-element-binding protein (CREB) signalling.

Propofol can be potentially beneficial in oxidative stress related malignancies as neurodegenerative diseases and traumatic brain injury but its signalling pathways are poorly understood. In this study effect of propofol on astroglial signalling in oxidative stress was evaluated. Ten days old cultures of rat astroglial cells were treated for 1 hour with t-butyl hydroperoxide (tBHP) to induce oxidative stress following by 1 hour propofol. We measured cytotoxicity, changes in cell growth and apoptosis as well as alterations in expression and acetylation of chromatin core H3 and H4 histone proteins and changes in native and phosphorylated cAMP-response-element-binding protein (CREB). tBHP induced limited cytotoxicity, increased apoptosis, decreased glutamine synthetase and enolase activities, decreased nuclear CREB, CREB-P and histone proteins but unchanged cytosolic CREB and histone acetyltransferase (HDAC) expression. Propofol clearly protected the cells against tBPH-induced toxicity, normalized alterations in cell growth, restored to some extent glial enzyme activities and reduced apoptotic cell numbers. Also, propofol restored H3 but not H4 expression/activation, but was without effect on decreased nuclear CREB expression/activation. These data show that oxidative stress in cultured astroglia significantly affects nuclear CREB and histone proteins and point to the protective role of propofol.