Anti-inflammatory activity of beta2-agonists in primary lung epithelial cells is independent of glucocorticoid receptor.

In patients with asthma and chronic obstructive pulmonary disease, the addition of long-acting beta(2)-agonists (LABA) to glucocorticosteroids (GCS) results in better control than increasing the dose of GCS alone. In smooth muscle cells and fibroblasts, one apparent underlying mechanism involves the ability of LABAs to activate the glucocorticoid receptor (GR). The present study investigates the effects of formoterol (FORM), salmeterol (SALM) and budesonide (BUD) on GR activation in bronchial epithelial cells via tumour necrosis factor-alpha-stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) release, GR nuclear translocation and GR-regulated reporter gene activity. Both BUD and FORM inhibited GM-CSF release by < or = 50%. The combination of these two drugs, in clinically relevant concentrations, inhibited GM-CSF release by 85% down to unstimulated levels. A similar inhibition was obtained when combining BUD and SALM. The ability of FORM to inhibit GM-CSF synthesis was not altered by small interfering RNA-mediated depletion of GR and FORM nor SALM-induced GR translocation into the cell nucleus. In addition, FORM did not activate GR-regulated reporter gene activity (SALM was not tested), in contrast to the clear effect of BUD. It was concluded that in bronchial epithelial cells, inhibition of granulocyte-macrophage colony-stimulating factor synthesis by formoterol and salmeterol does not act via previously demonstrated glucocorticoid receptor-related mechanisms, suggesting an alternative pathway in these cells.

ROS in the local and systemic pathogenesis of COPD.

An imbalance between oxidants and antioxidants is proposed in the pathogenesis of COPD. Potential alterations responsible for an imbalance in oxidant production and intra- and extracellular antioxidant defense systems are discussed with respect to COPD-related changes in the pulmonary compartment. In line with the current view of COPD as a disease with multiple systemic consequences, there is increasing evidence that imbalances in the redox milieu extend beyond the diseased lung in COPD patients. Skeletal muscle dysfunction is often observed in COPD and may result from imbalances in the redox environment of skeletal muscle. Potential triggers of oxidative stress in the muscle compartment include inflammation and hypoxia, and local sources of reactive oxygen and nitrogen species are discussed, as well the mechanisms by which skeletal muscle trophical state, contractility and fatigability may be affected by oxidative stress, resulting in skeletal muscle dysfunction.

Effects of formoterol and budesonide on GM-CSF and IL-8 secretion by triggered human bronchial epithelial cells.

The effect of formoterol, alone and in combination with budesonide, upon tumour necrosis factor-alpha stimulated (10 ng x mL(-1)) human bronchial epithelial cells was investigated. Addition of formoterol (> or = 10(-10) M) reduced granulocyte macrophage-colony stimulating factor (GM-CSF) levels, as assessed by enzyme-linked immunosorbent assay, by 40-50% and increased interleukin (IL)-8 levels by approximately 50%. The effects of formoterol were long lasting (23 h). Budesonide (10(-8) M) reduced the amounts of both cytokines (GM-CSF and IL-8) by 40%. Simultaneous addition of formoterol and budesonide reduced GM-CSF levels approximately 75%, while IL-8 levels were decreased approximately 40%, similar to the reduction obtained with budesonide alone. The glucocorticoid receptor (GR) antagonist RU486 did not influence the effect of formoterol, suggesting no involvement of the GR. Formoterol rapidly induced an elevation in intracellular cyclic adenosine monophosphate, which was reduced in the presence of propranolol. In addition, the alterations in cytokine secretion induced by formoterol could be fully blocked by propranolol, demonstrating that these effects are beta2-receptor mediated. In conclusion, the combination of budesonide and formoterol reduces the secretion of granulocyte macrophage-colony stimulating factor to basal levels and counteracts the capacity of formoterol alone to induce interleukin-8 production, modulations which may facilitate improved asthma control.

Cytokine-induced activation of nuclear factor-kappa B is inhibited by hydrogen peroxide through oxidative inactivation of IkappaB kinase.

Rapid activation of the IkappaB kinase (IKK) complex is considered an obligatory step in the activation of nuclear factor-kappaB (NF-kappaB) in response to diverse stimuli. Since oxidants have been implicated in the regulation of NF-kappaB, the focus of the present study was the activation of IKK by tumor necrosis factor alpha (TNFalpha) in the presence or absence of hydrogen peroxide (H(2)O(2)). Exposure of mouse alveolar epithelial cells to H(2)O(2) was not sufficient to activate IKK, degrade IkappaBalpha, or activate NF-kappaB. In contrast, TNFalpha induced IKK activity rapidly and transiently resulting in IkappaBalpha degradation and NF-kappaB activation. Importantly, in the presence of H(2)O(2), the ability of TNFalpha to induce IKK activity was markedly decreased and resulted in prevention of IkappaBalpha degradation and NF-kappaB activation. Neither tyrosine kinases nor phosphatidylinositol 3-kinases, known regulators of NF-kappaB by oxidants, were involved in IKK inhibition by H(2)O(2). Direct addition of H(2)O(2) to the immunoprecipitated IKK complex inhibited enzyme activity. Inhibition of IKK activity by H(2)O(2) was associated with direct oxidation of cysteine residues present in the IKK complex and occurred only in enzymatically active IKK. In contrast to previously published observations, our findings demonstrate that the oxidant H(2)O(2) reduces NF-kappaB activation by inhibiting activated IKK activity.

Alpha and beta glucocorticoid receptor mRNA expression in skeletal muscle.

The aim of the present study was to investigate the occurrence and autoregulation of both glucocorticoid receptor mRNAs in rat gastrocnemius muscle. The expression of both receptor forms was studied 1, 4 or 12 hours after intra-tracheal instillation of a high dose (100 micrograms) of budesonide; muscular expression was compared with glucocorticoid receptor expression in lung tissue. After Northern blot analysis, hybridization was performed with glucocorticoid receptor, glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase probes. In the gastrocnemius muscle, both the alpha and beta glucocorticoid receptor mRNA forms were detected and found to be downregulated four hours after the budesonide instillation. alpha/beta glucocorticoid receptor ratios were lower in the gastrocnemius (1.1 +/- 0.2) than in the lungs (2.6 +/- 0.6). In the lungs, at all time points, the average alpha glucocorticoid receptor mRNA levels did not differ from controls, although glutamine synthetase mRNA levels were upregulated. The beta glucocorticoid receptor mRNA was slightly reduced at 1 and 4 hours. In conclusion, after intra-tracheal instillation of budesonide, both alpha and beta glucocorticoid receptor forms were downregulated in muscle tissue. The difference in alpha/beta glucocorticoid receptor mRNA ratios and concentrations between lung and gastrocnemius muscle supports the hypothesis of differential gene regulation by glucocorticoids in different cell types.

Glucocorticoid receptor mRNA levels in bronchial epithelial cells of patients with COPD: influence of glucocorticoids.

Glucocorticoids (gcs) are known to be effective in the treatment of asthma. In chronic obstructive pulmonary disease (COPD), however, no beneficial effects are demonstrated in most patients. Hypothetically, this may be explained by an overexpressed beta-glucocorticoid receptor (GR) compared to the alpha-GR. The aim of this study was to investigate alpha- and beta-GR mRNA levels and ratios in patients with COPD with or without glucocorticoid treatment. GR and, as a control, metallothionein (MT) 2 mRNA levels were compared between patients with COPD receiving glucocorticoids (COPD + gcs), glucocorticoid naive COPD-patients (COPD - gcs) and non-COPD control patients not using gcs. Bronchoscopy was performed and bronchial epithelial cells were sampled with brushing. Smoking did not influence alpha- and beta-GR levels and ratios, nor the MT2 mRNA expression level. The alpha-GR mRNA expression was lower in the COPD - gcs group than in controls. Both GR forms were higher in the COPD + gcs patients than in the COPD - gcs patients, but not different from the levels measured in the controls. alpha 1/beta-GR mRNA ratios did not differ between the groups and averaged 1.7, suggesting no inhibitory effect of the beta-GR on the alpha 1 form. MT2 levels were upregulated in the COPD + gcs patients as compared to the COPD - gcs group, indicating a pharmacological glucocorticoid effect. In the present study it is demonstrated that basal GR mRNA levels are lower in patients with COPD. Although this needs to be investigated further, this might explain, in part, the non-responsiveness of patients with COPD to gcs.

Interaction between glucocorticoids and beta2-agonists: alpha and beta glucocorticoid-receptor mRNA expression in human bronchial epithelial cells.

Recent studies have suggested that regular use of beta2-agonists has adverse effects on asthma control, due to the cross-talk between cAMP responsive element binding proteins (CREB) and glucocorticoid receptors (GR). The aim of this study was to investigate the interaction between GR and CREB on cytoplasmic protein level with a gel mobility shift assay and to determine the effect of this interaction on mRNA levels by Northern blot analysis. After exposing human bronchial epithelial cells for 1 hr to either 1 microM terbutaline or budesonide, more binding of CREB and GR, respectively, was observed to their responsive elements in DNA. Simultaneous exposure to terbutaline and budesonide also increased the binding of CREB and GR to DNA. After 4 hr, both alpha and beta GR mRNAs were down-regulated by 1 microM budesonide. Simultaneous addition of 1 microM terbutaline prevented this down-regulation. Adding 100 times more budesonide compared to terbutaline again down-regulated both GR forms, although significantly less compared to the down-regulation induced by 1 microM budesonide alone. Addition of terbutaline to cells already exposed to budesonide did not reverse the GR mRNA expression within 44 hr. Similar results were obtained with metallothionein-2 (MT2) mRNA levels. In conclusion, beta2-agonists interfere with the GR function in human bronchial epithelial cells when given simultaneously, with this being overcome by sequential exposure of the cells to first glucocorticoids and later beta2-agonists.

In vitro and in vivo modulation of alpha- and beta-glucocorticoid-receptor mRNA in human bronchial epithelium.

Despite the central role bronchial epithelial cells play in asthmatic reactions, and the widespread use of inhaled corticosteroids in asthma, no information is available on the effect of glucocorticoids on glucocorticoid- receptor (GR) gene expression. In this study, the effect of budesonide on alpha- and beta-GR gene expression in human bronchial epithelial cells was investigated in vitro and in vivo. A bronchial epithelial cell line was exposed in vitro to budesonide, and a dose- and time-dependent synchronous downregulation of alpha- and beta-GR mRNA was observed. A 1-h exposure resulted in a reversible and reduced downregulation as compared with continuous exposure. In healthy volunteers (n = 10), no difference on average was present in GR mRNA expression before or after 4 wk of budesonide inhalation in either bronchial epithelial cells or alveolar macrophages. The time between the last inhalation and sampling of cells ranged from 0.5 to 8 h. However, a significant downregulation of alpha-GR mRNA was observed when the time between the last inhalation and sampling of cells was more than 2 h. Normalization of the downregulation of GR mRNA expression in bronchial epithelial cells is explained by the pharmacokinetics of inhaled budesonide in the human lung.

Comparative study of ibuprofen lysine and acetaminophen in patients with postoperative dental pain.

This single-dose, double-blind, parallel-group, single-site study compared ibuprofen lysine 400 mg with acetaminophen 1000 mg and placebo in 240 patients with moderate-to-severe postoperative dental pain. The relative onset of analgesic response, overall analgesic efficacy, duration of effect, and safety were assessed over a 6-hour postdose period. Analgesic efficacy was assessed by patient self-rating of pain intensity, pain relief, time to meaningful pain relief, need for additional analgesic medication, and patient global evaluation. Both ibuprofen lysine 400 mg and acetaminophen 1000 mg were significantly (P < or = 0.05) more effective than placebo. Ibuprofen lysine had a significantly (P < or = 0.05) faster onset of action with greater peak and overall analgesic effect than did effect than did acetaminophen. All treatments were generally well tolerated.

Comparison of single-dose ibuprofen lysine, acetylsalicylic acid, and placebo for moderate-to-severe postoperative dental pain.

In a single-dose, double-blind, parallel-group, single-site study, ibuprofen lysine 200 mg (IBL 200) was compared with acetylsalicylic acid 500 mg (ASA 500) and placebo in 183 patients with moderate-to-severe postoperative dental pain. The relative onset of analgesic response, duration and degree of analgesia, and safety were assessed over a 6-hour postdose period. Analgesic efficacy was assessed by patient self-rating of pain intensity, pain relief, time to meaningful pain relief, global evaluation, and requirement for additional analgesic medication; both IBL 200 and ASA 500 were significantly more effective than placebo. IBL 200 also had a significantly faster onset of action, greater peak and overall analgesic effect, and longer duration of analgesia than ASA 500. All treatments were generally well tolerated.

K-ras point mutations in routinely processed tissues: non-radioactive screening by single strand conformational polymorphism analysis.

AIMS: To develop a non-radioactive method to screen routinely fixed, paraffin wax embedded specimens for the occurrence of point mutations; to evaluate the single strand conformational polymorphism (SSCP) analysis technique for the detection of K-ras point mutations as a result of electrophoretic mobility shifts. METHODS: DNA was extracted from archival specimens of colon cancer and from established colon cancer cell lines with known point mutations. A K-ras gene fragment containing codons 12 and 13 of exon 1 was amplified with the polymerase chain reaction (PCR). Denatured DNA fragments were run on 10% polyacrylamide gels under non-denaturing conditions. After electrophoresis DNA was blotted and the single stranded DNA was detected using a digoxigenin labelled ras probe. The nature of the detected point mutations was identified and confirmed by sequencing and hybridisation with oligonucleotides using 32P labelling. RESULTS: Wild type and aberrant alleles were detected caused by mobility shifts after electrophoresis of the PCR products. Commonly occurring mutations in the K-ras gene--in the first two positions of codon 12--could easily be detected in DNA from archival paraffin wax embedded colon cancer tissue. In all the colon tumour samples studied wild type gene alleles were also found, presumably derived from normal cells in the specimen. CONCLUSIONS: The SSCP method permits rapid non-radioactive screening of adenomas or carcinomas for the occurrence of point mutations in the K-ras gene. But if a mutation is detected by an electrophoretic mobility shift, its identification requires confirmation by sequencing or oligonucleotide hybridisation.