Glucocorticoids induced down-regulation of glucocorticoid receptor mRNA expression in asthma.

Although their precise mechanism of action remains to be elucidated, glucocorticoids represent the most effective therapy in the treatment of asthma. Interactions between the glucocorticoid receptor and the AP-1 complex have been shown to regulate the transcription of some genes, including glucocorticoid receptor itself. The aim of the present study was to compare the expression of mRNA for glucocorticoid receptor in human blood monocytes obtained from seven unstable untreated asthmatic patients who were subsequently treated with high doses of parenteral corticosteroid (methyl prednisolone 120 mg/day) for 10 days. mRNA expression was identified after RNA extraction using RNAzol and analysed after reverse transcriptase, by polymerase chain reaction using a semiquantitative competitive hybridization assay. All asthmatic patients showed an improvement in their FEV1 values after corticosteroid treatment (per cent of predicted value 68.28 +/- 4.93 versus 95.57 +/- 6.41, P < 0.02), and a significant decrease for glucocorticoid receptor mRNA expression (P < 0.02) was observed in their monocytes. This is the first report of an ex vivo down-regulation for the glucocorticoid receptor mRNA expression, following corticosteroid treatment.

Nonradioactive quantification of glucocorticoid receptor expression during differentiation of human monocytic cells (U937).

We describe a method for relative quantification of specific mRNA using a nonradioactive assay based on DNA strand competition between identical sequences of biotin- and fluorescein-labeled amplicon (probe) and unlabeled amplicon (target) during hybridization. As the target quantity increased, that of the double-labeled probe decreased in accordance with the mass action law. This technique was successfully applied to evaluate differences in glucocorticoid receptor expression in U937 cells before and after the addition of potent differentiation inducers: 12-O-tetradecanoylphorbol 13-acetate (TPA) and a combination of all-trans retinoic acid (RA) and 1,25-dihydroxyvitamin D2 (VD). We observed that TPA treatment was associated with an increase in specific binding of [3H]dexamethasone and up-regulation of GR mRNA while no enhanced GR expression was perceived with RA/VD treatment.

Imaging reactive oxygen species in asthma.

Inflammatory processes in asthma are characterized by an infiltration of inflammatory cells including mononuclear phagocytes. It has been observed that mononuclear phagocytes, alveolar macrophages and blood monocytes, release higher quantities of reactive oxygen species in asthmatic patients than in healthy subjects. Chemiluminescence assays were developed to measure the superoxide anion and the other reactive oxygen species. The chemiluminescence response was first analysed with a luminometer, which made it possible to study cells in suspension before and after PMA-stimulation. Secondly a video-imaging camera was used in experiments on adherent cells before and after stimulation with PMA and/or specific stimulus IgE/anti-IgE. Both techniques showed that human alveolar macrophages, blood monocytes, PMN and lymphocytes were spontaneously primed in vivo and were more easily stimulated in asthma. Analysis of adherent cells in vitro may provide give information on the physiological condition of adherent cells in vivo.

Reciprocal effects between opioid peptides and human polymorphonuclear leukocytes--II. Enhancement of phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocyte by opioid peptides previously exposed to activated oxygen species.

Activated oxygen species (AOS) have often been shown to promote strong modifications in peptide structures and thus in their biological functions. In the present study, the immunomodulatory effects of Leu-enkephalin, beta-endorphin, dynorphin and some fragments are evaluated, before and after exposure of peptides to AOS, by studying their influence on human polymorphonuclear leukocyte (PMN) respiratory burst. None of the tested opioid peptides (modified or not) were shown to affect resting oxidative metabolism in the PMNs. The effects of peptides on phorbol myristate acetate (PMA)-stimulated production of AOS were measured in a lucigenin-enhanced chemiluminescence assay. Before AOS exposure, the opioid peptides suppressed the PMA-stimulated respiratory burst in human PMNs and a U-shaped dose-response relationship was observed. Conversely, after AOS exposure the opioid peptides enhanced the PMA-stimulated respiratory burst in human PMNs and an inverted U-shaped dose-response relationship was observed. In both cases, the maximal effect was reached at peptide concentrations of 10(-10)M-10(-12) M.

Correlation in inflammatory fibroblasts between the number of glucocorticoid receptors and PGE2 release.

The anti-inflammatory effects of glucocorticoids are mediated through steroid receptor occupancy and there is a significant correlation between the extent of receptor saturation and the extent of the biological effects. In a previously published study, we found that the number of these receptors was higher in inflammatory fibroblasts than in quiescent ones. PGE2 release, measured at the same time as the number of steroid receptors, was higher when the cells were from inflammatory tissue. Our aim, in the present study, was to determine whether the PGE2 released by cells during inflammatory processes could participate in increasing the number of steroid receptors. Fibroblasts obtained from rat quiescent subcutaneous connective tissue and granulomas were subcultured in monolayers. The specific binding of [3H]dexamethasone was assessed and analyzed by a method described by Kalimi et al. After a freeze-thaw cycle, we observed a decrease in the number of receptors in inflammatory fibroblasts. When the frozen and thawed fibroblasts were subcultured in the presence of PGE2 (10(-8) M), the number of receptors was enhanced in fibroblasts from inflammatory tissue. Cycloheximide (3 X 10(-7) M) prevented this increase. The release of PGE2 decreased after freezing and then increased simultaneously with the number of receptors in inflammatory cells. These findings suggest that PGE2 may play a role in regulating steroid effects on fibroblast function.

Glucocorticoid receptors in fibroblasts from synovial tissue. Changes during the inflammatory process. Preliminary results.

There is known to be a significant correlation between the number of glucocorticoid receptors in tissues and their anti-inflammatory effect. In this work, the specific binding of glucocorticoids was studied in inflammatory fibroblasts. Human fibroblasts were obtained from the knee joint of a rheumatoid patient undergoing surgery; experimental fibroblasts were from rat granulomas. The same study was carried out in quiescent synovial fibroblasts from a healthy subject (post-traumatic amputation) and from rat subcutaneous conjunctive tissue. Fibroblasts were obtained by explant cultures and subcultures in monolayers. The stimulation state of cells was evaluated by the amounts of PGE2 and PGF2 alpha released into the culture media. Analysis of the proportions of steroid bound to whole cells showed evidence of specific glucocorticoid receptors in all fibroblasts. Their number was three times higher in cells from inflammatory tissues than from controls. This increased number of receptors in inflammatory cells could be the result of the action of one or more mediators that promote their biosynthesis.

[New derivatives of benzopyran-4-one with analgesic, anti-inflammatory and antiplatelet clumping activity]. / Nouveaux dérivés de la benzopyrannone-4, à activités analgésique, anti-inflammatoire et antiagrégante plaquettaire.

A series of 35 new derivatives of 3-benzylidene benzopyran-4-one carboxylic acid was synthesized; their antiinflammatory and analgesic activities were investigated and compared with those of acetylsalicylic acid, phenylbutazone, ketoprofene and glafenine. Among these compounds, four were found to be more potent than the references compounds in tests of antiinflammatory and analgesic activity. Seven compounds were tested on rat blood platelet aggregation induced by ADP. From these tests, HD-039, HD-041, HD-044 and HD-049 appear to be promising antiinflammatory, analgesic and anticlotting agents, although this original series shows little structural analogy with classical non-steroidal antiinflammatory agents.