Frederik B. J. M. Thunnissen

Interaction between glucocorticoids and β2-agonists: α and β 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.

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.

Exonuclease enhances hybridization efficiency: Improved direct cycle sequencing and point mutation detection

Solution hybridization is an essential step in sequencing and some point mutation detection methods. In practice, this hybridization is hampered resulting in the need of additional purification of the amplification products. The use of T7 gene 6 exonuclease may lead to efficient production of single-stranded DNA. In this study, the effect of pretreatment with exonuclease on direct cycle sequencing and point mutation detection was analyzed. Exonuclease-treated products were directly cycle sequenced without further purification. This resulted in highly efficient quality improvement for sequencing allowing detection of heterozygotes. Point mutation detection by Point-EXACCT (exonuclease-amplification coupled capture technique) demonstrated detection of one cell containing a mutation in an excess of 75000 wild type cells. Exonuclease-enhanced detection methods offer simple, rapid detection strategies that are easily adaptable for widespread clinical laboratory use. With the use of exonuclease, the detection of heterozygosity using fluorescent cycle sequencing is becoming more reliable. The high sensitivity of Point-EXACCT due to the use of exonuclease makes it a highly promising method for large-scale screening of (pre)malignant changes in patients with a high risk for developing cancer.

Clinical value of DNA image cytometry in effusions with atypia

Malignant cells in serosal effusions provide essential information about the extent of malignant disease. The main aim of this study was to examine the additional diagnostic value of DNA image cytometry for cases with uncertainty in the cytological diagnosis. In addition, the feasibility of automated nuclei detection was investigated. Out of 457 cases, 33 samples in 32 patients were diagnosed with “atypia” (probably benign) and 21 as “suspicious for malignancy.” DNA image cytometry was performed on these 54 cases and on an additional group of 14 cytologically malignant cases.

α and β glucocorticoid receptor mRNA expressionin 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 12hours after intra-tracheal instillation of a high dose (100μg) 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 α and β glucocorticoid receptor mRNA forms were detected and found to be downregulated four hours after the budesonide instillation. α/β 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 α glucocorticoid receptor mRNA levels did not differ from controls, although glutamine synthetase mRNA levels were upregulated. The β glucocorticoid receptor mRNA was slightly reduced at 1 and 4hours. In conclusion, after intra-tracheal instillation of budesonide, both α and β glucocorticoid receptor forms were downregulated in muscle tissue. The difference in α/β glucocorticoid receptor mRNA ratios and concentrations between lung and gastrocnemius muscle supports the hypothesis of differential gene regulation by glucocorticoids in different cell types.