Biomedical subjects

B Kleuser

Publications and source records attributed to B Kleuser.

At least 19 recordsLinked to original sources

PubMed2001

Depolarisation induces rapid and transient formation of intracellular sphingosine-1-phosphate.

Formation of sphingosine-1-phosphate (SPP) by sphingosine kinase serves as a signalling pathway for various membrane receptors. Here, we show that membrane depolarisation is another mechanism by which this pathway can be activated. Formation of [(3)H]SPP as well as levels of endogenous SPP were rapidly and transiently increased in PC12 pheochromocytoma cells depolarised with high KCl. Time course and maximum were similar to those induced by bradykinin. Depolarisation-induced SPP production was also observed in RINm5F insulinoma cells, dependent on extracellular Ca(2+) and fully suppressed by verapamil, thus apparently caused by Ca(2+) influx via voltage-gated Ca(2+) channels. Studies with sphingosine kinase inhibitors and overexpression of sphingosine kinase revealed a partial contribution of this pathway to depolarisation-induced noradrenaline release and Ca(2+) increase.

Animals↗

PubMed2001

Stimulation of nuclear sphingosine kinase activity by platelet-derived growth factor.

Subcellular fractionation revealed that a significant fraction of total sphingosine kinase, the enzyme that phosphorylates sphingosine to form the bioactive lipid metabolite sphingosine-1-phosphate, resides in the nuclei of Swiss 3T3 cells, localized to both the nuclear envelope and the nucleoplasm. Platelet-derived growth factor, in addition to rapidly stimulating cytosolic sphingosine kinase, also induced a large increase in nucleoplasm-associated activity after 12-24 h that correlated with progression of cells to the S-phase of the cell cycle and translocation of sphingosine kinase-green fluorescent protein fusion protein to the nuclear envelope. Our results add sphingosine kinase to the growing list of lipid-metabolizing enzymes associated with the nucleus, and suggest that sphingosine-1-phosphate may also play a role in signal transduction in the nucleus.

3T3 Cells↗

PubMed2001

An improved high-performance liquid chromatographic method for the determination of sphingosine-1-phosphate in complex biological materials.

Sphingosine-1-phosphate (SPP) has been proposed to act both as an intracellular second messenger and as an extracellular mediator via specific cell surface receptors. Based on the increasing diverse cellular roles methods to quantify endogenous and exogenous SPP are highly required. Here, we report a rapid HPLC method that allows quantification of SPP in the picomolar range even in complex biological systems. A two-step lipid extraction serves to separate SPP from most interfering phospholipids and sphingolipids. Importantly, dihydrosphingosine-1-phosphate (dihydro-SPP), not detectable in all cultured cells and biological samples in considerable amounts, possesses equal extraction properties and therefore is an ideal internal standard. Following extraction SPP and dihydro-SPP are converted to fluorescent isoindol derivatives by ortho-phthaldialdehyde (OPA) and separated by HPLC using a gradient program with methanol and 0.07 M K2HPO4 as eluents. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.5 pM to 0.2 nM. The identity of SPP and dihydro-SPP was confirmed by the use of the ion pair reagent tetraammoniumsulfate, which induced a shift of both peaks but did not alter peak areas. Moreover, enzymatic conversions to sphingosine and sphinganine by bovine intestinal mucosa alkaline phosphatase (AP) excluded the existence of overlapping compounds. Levels of SPP were determined in a variety of biological samples like serum, thrombocytes, primary keratinocytes and several cell lines. Furthermore, we were able to detect increases of intracellular SPP levels in human keratinocytes after exposure to 1alpha,25-dihydroxyvitamin D3(1,25-(OH)2D3) for which a stimulation of sphingosine kinase activity has been recognized.

Blood Platelets↗

PubMed2001

1Alpha,25-dihydroxyvitamin D3 protects human keratinocytes from apoptosis by the formation of sphingosine-1-phosphate.

Owing to its ability to induce growth arrest and differentiation of keratinocytes, 1alpha,25-dihydroxyvitamin D3 and its analogs are useful for the treatment of hyperproliferative skin diseases, such as psoriasis vulgaris. It has been implicated that the 1alpha,25-dihydroxyvitamin D3-induced differentiation of keratinocytes is mediated, at least in part, by the formation of ceramides; however, ceramides have also been identified to induce apoptosis in many cells, including keratinocytes. Therefore, it was of interest to investigate the influence of 1alpha,25-dihydroxyvitamin D3 on apoptosis in keratinocytes. Most interestingly, physiological concentrations of 1alpha,25-dihydroxyvitamin D3 did not induce apoptosis in keratinocytes, despite the formation of ceramides. Moreover, 1alpha,25-dihydroxyvitamin D3 appeared cytoprotective and made keratinocytes resistant to apoptosis induced by ceramides, ultraviolet irradiation, or tumor necrosis factor-alpha. The cytoprotective effect was accompanied by the formation of the sphingolipid breakdown product sphingosine-1-phosphate, which prevented apoptosis in analogy to 1alpha,25-dihydroxyvitamin D3. The effect of 1alpha,25-dihydroxyvitamin D3 was specific as the almost inactive precursor cholecalciferol neither induced sphingosine-1-phosphate formation nor prevented cells from apoptosis. Besides this, the cytoprotective aptitude of 1alpha,25-dihydroxyvitamin D3 was completely abolished by the sphingosine kinase inhibitor N,N-dimethylsphingosine, which blocked sphingosine-1-phosphate formation. Moreover, sphingosine-1-phosphate was able to restore the cytoprotective effect of 1alpha,25-dihydroxyvitamin D3 in the presence of N,N-dimethylsphingosine. Taken together, here we report for the first time that 1alpha,25-dihydroxyvitamin D3 protects keratinocytes from apoptosis and additionally this cytoprotection is mediated via the formation of sphingosine-1-phosphate.

Apoptosis↗

PubMed1999

Skin penetration and metabolism of topical glucocorticoids in reconstructed epidermis and in excised human skin.

PURPOSE: To investigate pharmacokinetic differences between the nonhalogenated double ester prednicarbate (PC) and the fluorinated monoester betamethasone 17-valerate (BM17V) their metabolism in human keratinocytes and fibroblasts as well as their permeation and biotransformation in reconstructed epidermis and excised human skin was compared. Special attention was given to the 17-monoesters because of their high receptor affinity and antiproliferative effects. METHODS: Glucocorticoid penetration was determined using Franz diffusion cells, quantifying metabolite concentrations by HPLC. Chemical stability and reactivity of the monoesters was determined by molecular modeling analysis. RESULTS: PC accumulated in the stratum corneum. A considerable amount of penetrating PC was hydrolyzed by viable keratinocytes to prednisolone 17-ethylcarbonate (PI7EC), P17EC permeated the skin very rapidly when compared to BM17V. Overall P17EC concentrations in viable tissue were low. Inside of the acceptor fluid, but not within the tissue, P17EC was converted to the more stable prednisolone 21-ethylcarbonate (P21EC). CONCLUSIONS: The inactivation of highly potent, but also cell toxic, 17-monoesters to almost inactive 21-congeners seen with isolated cell monolayers appears less important in the skin. In vitro determination of the dermal 17-monoesters concentrations may allow the prediction of the atrophogenic risk in man. BM17V levels exceeding P17EC concentration about 6-fold may contribute to its lower tolerance when compared to PC.

Administration, Topical↗

PubMed1999

Fusion of the binding domain of Raf-1 kinase with green fluorescent protein for activated Ras detection by fluorescence correlation spectroscopy.

Ras proto-oncogenes play a central role in cell proliferation by the regulation of signal transduction pathways from receptors of the outer cell membrane to the nucleus via the activation of transcription factors. Wild-type Ras cycles between the activated GTP-bound and the inactivated GDP-bound state, and the GTPase reaction is a timer for the interaction between Ras-GTP and effector molecules such as Raf-1 protein kinase. Mutations of ras resulting in the loss of the intrinsic GTPase activity result in autonomous proliferation. Mutated Ras is found in a variety of human tumors. Therefore, monitoring of GTP-loaded conformation of Ras related proteins could be utilised in cancer diagnosis. To develop a fluorescence based bioassay we have coupled the gene for the N-terminal Ras binding domain (RBD) of Raf-1 protein kinase with the gene for the green fluorescent protein (GFP). The chimeric fusion protein RBDGFP was identified by immunoblotting and subsequently investigated by fluorescence correlation spectroscopy (FCS), a new analytical technology allowing the measurement of characteristic diffusion times of fluorescently labeled molecules. Molecular interactions increase the molecular weight and influence the diffusion time of RBDGFP. FCS diffusion value of the recombinant protein was in coincidence with the molecular weight of the construct. Fluorimetric measurements of RBDGFP versus GFP showed clearly that the recombinant protein contains functional GFP. Increased FCS transition times indicated the interaction of RBDGFP with its corresponding antibody. Suboptimal binding of the fusion protein to activated Ras, how ever, resulted in a modest influence on the diffusion value. Taken together our rational design and construct shows the way for a ready characterisation of novel GFP-connected fusion proteins employing FCS.

Blotting, Western↗

PubMed1998

N,N-Dimethylsphingosine is a potent competitive inhibitor of sphingosine kinase but not of protein kinase C: modulation of cellular levels of sphingosine 1-phosphate and ceramide.

Sphingosine 1-phosphate (SPP), a lipid second messenger formed by the action of sphingosine kinase, has been implicated in regulating diverse biological processes, including growth, survival, and differentiation. N,N-Dimethylsphingosine (DMS) inhibits sphingosine kinase and has been used to investigate the biological roles of SPP; however, little is known of the mechanism of inhibition of sphingosine kinase by DMS. In addition, DMS has been shown to inhibit protein kinase C in vitro. Here we report that DMS is a competitive inhibitor of sphingosine kinase from U937 monoblastic leukemia cells, Swiss 3T3 fibroblasts, and PC12 pheochromocytoma cells. DMS decreases basal levels of SPP and prevents increases in SPP in response to physiological stimuli known to activate sphingosine kinase. DMS also effectively increases cellular levels of ceramide in a variety of cell types, and resetting of the ceramide/SPP rheostat may account for the pro-apoptotic effects of DMS. Moreover, DMS, at concentrations which effectively inhibit sphingosine kinase, has no effect on protein kinase C activity or its membrane translocation. Thus, DMS acts as a specific competitive inhibitor of sphingosine kinase in diverse cell types and is a useful tool to elucidate the role of SPP as an intracellular second messenger.

3T3 Cells↗

PubMed1998

1Alpha,25-dihydroxyvitamin D3 inhibits programmed cell death in HL-60 cells by activation of sphingosine kinase.

Sphingolipid breakdown products [ceramide, sphingosine, and sphingosine-1-phosphate (SPP)] are emerging as a new class of bioactive molecules. In agreement with previous studies, treatment of human promyelocytic leukemia HL-60 cells with 1-alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induced a transient increase of ceramide levels within 2 h, which then returned to basal levels within 8 h. In contrast, sphingosine kinase activity increased more slowly and reached maximal levels only after 20 h of exposure, leading to a concomitant increase in SPP level. Unlike treatments with cell-permeable ceramide analogues or sphingomyelinase, which induce apoptosis, 1,25-(OH)2D3 did not induce apoptosis, despite the early formation of ceramide. Moreover, prolonged treatment of HL-60 cells with 1,25-(OH)2D3 suppressed ceramide-induced apoptosis. There was a correlation between the time course and dose response of the activation of sphingosine kinase by 1,25-(OH)2D3 and the protection against apoptosis. In contrast, treatment with all-trans-retinoic acid neither stimulated sphingosine kinase activity nor protected cells from ceramide-induced apoptosis. Treatment with SPP protected HL-60 cells from ceramide-induced apoptosis, and N,N-dimethylsphingosine (DMS), a competitive inhibitor of sphingosine kinase, prevented the survival effect of 1,25-(OH)2D3. The effect of DMS was counteracted by SPP, suggesting that SPP is a critical component of the cytoprotective effect of 1,25-(OH)2D3. Chelerythrine chloride, an inhibitor of protein kinase C, markedly reduced sphingosine kinase activity and the apoptosis-sparing effect of 1,25-(OH)2D3, and conversely, the tumor promoter 12-O-tetradecanoylphorhol-13-acetate not only suppressed ceramide-induced apoptosis but also stimulated sphingosine kinase activity. Moreover, the protective effect of 12-O-tetradecanoylphorbol-13-acetate was blocked by DMS. Collectively, our observations indicate that the cytoprotective effect of 1,25-(OH)2D3 is mediated by SPP, which is formed as a consequence of activation of sphingosine kinase.

Alkaloids↗

PubMed1997

Prednicarbate versus conventional topical glucocorticoids: pharmacodynamic characterization in vitro.

PURPOSE: Pharmacodynamic characterization of topical glucocorticoids as prednicarbate (PC), its metabolites prednisolone 17-ethylcarbonate (PEC) and prednisolone (PD), betamethasone 17-valerate (BMV), betamethasone (BM) and desoximetasone (DM) by evaluating their effects on epidermal and dermal cells. Synopsis of pharmacokinetic and pharmacodynamic studies, possibly explaining the improved benefit-risk ratio of prednicarbate. METHODS: Isolated foreskin keratinocytes were used to investigate the influence on epidermal inflammatory processes, dermal fibroblasts of the same origin to study antiproliferative activities of glucocorticoids. Interleukins were measured by ELISA-assay, the influence on II-1 alpha-production also on mRNA-level by RNAse protection assay. Proliferation was assessed by 3H thymidine incorporation and biodegradation by HPLC/UV-absorption. Cell viability was controlled by MTT assay. RESULTS: In keratinocytes, inflammation was induced by TNF alpha, resulting in an increased II-1 alpha synthesis. This cytokine was particularly suppressed by PC and BMV, whereas PEC, PD, DM and BM were less potent (p < or = 0.05). Since, however, the double ester PC is rapidly degraded in keratinocytes, a RNAse-protection assay of II-1 alpha mRNA was performed allowing short incubation times and thus minimizing biodegradation effects. In agreement with the previous experiment, the antiinflammatory potency of native PC was confirmed. In fibroblasts, II-1 alpha and II-6 synthesis indicate proliferation and inflammation respectively. Whereas PC inhibited II-1 alpha and II-6 production in fibroblasts to a minor extent only, it was strongly reduced by the conventional glucocorticoids and PEC (p < or = 0.05). The minor unwanted effect of PC on fibroblasts was also reflected by its low influence on cell proliferation as assayed by 3H thymidine incorporation. More pronounced antiproliferative features were observed with BM, PEC and especially BMV. CONCLUSIONS: Correlating antiphlogistic effects in keratinocytes (suppression of II-1 alpha) with antiproliferative effects in fibroblasts (suppression of II-1 alpha and II-6), the improved benefit-risk ratio of PC compared to conventional glucocorticoids does not result only from distinct drug metabolism in the skin but also from a specific influence on the cytokine network.

Administration, Topical↗

PubMed1996

Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.

Ceramide is an important regulatory participant of programmed cell death (apoptosis) induced by tumour-necrosis factor (TNF)-alpha and Fas ligand, members of the TNF superfamily. Conversely, sphingosine and sphingosine-1-phosphate, which are metabolites of ceramide, induce mitogenesis and have been implicated as second messengers in cellular proliferation induced by platelet-derived growth factor and serum. Here we report that sphingosine-1-phosphate prevents the appearance of the key features of apoptosis, namely intranucleosomal DNA fragmentation and morphological changes, which result from increased concentrations of ceramide. Furthermore, inhibition of ceramide-mediated apoptosis by activation of protein kinase C results from stimulation of sphingosine kinase and the concomitant increase in intracellular sphingosine-1-phosphate. Finally sphingosine-1-phosphate not only stimulates the extracellular signal-regulated kinase (ERK) pathway, it counteracts the ceramide-induced activation of stress-activated protein kinase (SAPK/JNK). Thus, the balance between the intracellular levels of ceramide and sphingosine-1-phosphate and their regulatory effects on different family members of mitogen-activated protein kinases determines the fate of the cell.

3T3 Cells↗

PubMed1996

Measurement of phospholipase A2 and 1-alkylglycerophosphocholine acetyltransferase activities in stimulated alveolar macrophages by HPLC analysis of NBD-labeled ether lipids.

The importance of phospholipases in cellular signaling and 1-alkylglycerophosphocholine acetyltransferase in the formation of platelet-activating factor (PAF) has stimulated demand for methods to measure these enzyme activities in inflammatory cells. Most of the assays currently used rely on radiolabeled substrates. We have synthesized NBD-labeled ether lipids as substrates for measuring enzyme activities of the PAF cycle and of lysosomal phospholipase A2 (PLA2). The fluorescent lipids were incubated with homogenates of stimulated bovine alveolar macrophages. The generated products were separated from the substrates by HPLC on a normal phase and monitored with a fluorescence detector. NBD-lyso-PAF was well accepted by acetyl- and acyltransferases of the cell-free preparations, which metabolized the substrate into NBD-PAF and NBD-alkyl-acylglycerophosphocholines. Homogenates of stimulated cells showed an enhanced production of NBD-PAF. The increased formation of the biological mediator was dependent on the nature of the stimuli and the time of stimulation. Lysosomal PLA2 was measured with 1-O-(12-NBD-aminododecyl)-2-acyl-sn-glycero-3-phosphocholine as substrate. By varying the pH and the calcium concentration, it was possible to distinguish between the cytosolic PLA2 and the lysosomal PLA2 activity. Optimal conditions for the determination of the lysosomal PLA2 were obtained at pH 4.5 and in the presence of EDTA. Stimulation with particulate agonists induced an enhancement of the lysosomal PLA2 activity in macrophages.

4-Chloro-7-nitrobenzofurazan↗

PubMed1996

Eukaryotic expression systems: a comparison.

Eukaryotic expression systems are frequently employed for the production of recombinant proteins as therapeutics as well as research tools. Most commonly used expression systems are based on stably transfected adherent CHO cells or nonadherent lymphoid cell lines. An efficient alternative is the infection of insect cells by recombinant baculoviruses. Transient expression in mammalian cells, e.g., COS cells, is often used for the production of smaller quantities of proteins. The choice of a suitable expression system depends largely on the biochemical and biological properties of the protein of interest, as well as on the nature of the planned experiments and the amount of recombinant protein required. We summarize here the expression of the cytokine human Leukemia Inhibitory Factor (hu-LIF) in five of the most commonly used systems, namely in CHO, Sp2/0, MEL, COS, and insect cells, in conjunction with an outline of the principles and characteristics of each of these expression systems. In result, the stably transfected cell lines, CHO, Sp2/0, and MEL cells, gave rise to production of fully glycosylated hu-LIF at variable product titers; incompletely glycosylated, albeit biological action hu-LIF could be rapidly produced by transient expression in COS cells or by baculovirus-mediated infection of insect cells.

Animals↗

PubMed1995

A COS-cell-based system for rapid production and quantification of scFv::IgC kappa antibody fragments.

Single-chain Fv (scFv) antibody (Ab) fragments were transiently produced in COS-1 cells utilizing a mammalian expression vector featuring a murine immunoglobulin (Ig) light-chain leader sequence for efficient secretion and a murine Ig kappa constant domain (IgC kappa) for detection. Several hundred milliliters of supernatants from large-scale COS cell transfections were sufficient to purify the scFv::IgC kappa fusion proteins by one-step affinity chromatography utilizing an immobilized rat anti-mouse IgC kappa monoclonal Ab. Furthermore, the murine IgC kappa domain allowed for accurate quantification of the scFv::IgC kappa fusion protein secreted into the COS cell supernatant by a sandwich enzyme-linked immunosorbent assay (S-ELISA).

Animals↗

PubMed1995

The E-selectin-ligand ESL-1 is a variant of a receptor for fibroblast growth factor.

E-SELECTIN is an inducible cell-adhesion molecule on endothelial cells, which mediates the binding of neutrophils and functions as a Ca(2+)-dependent lectin. We have recently identified a 150K glycoprotein as the major ligand for E-selectin on myeloid cells, using a recombinant antibody-like form of mouse E-selectin as an affinity probe. Here we report the isolation of a mouse complementary DNA for this E-selectin ligand (ESL-1). The predicted amino-acid sequence of ESL-1 is 94% identical (over 1,078 amino acids) to the recently identified chicken cysteine-rich fibroblast growth-factor receptor, except for a unique 70-amino-acid aminoterminal domain of mature ESL-1. Fucosylation of ESL-1 is imperative for affinity isolation with E-selectin-IgG. A fucosylated, recombinant antibody-like form of ESL-1, but not of L-selectin, supports adhesion of E-selectin-transfected Chinese hamster ovary cells. Antibodies against ESL-1 block the binding of mouse myeloid cells to E-selectin. ESL-1, with a structure essentially identical to that of a receptor, thus functions as a cell adhesion ligand of E-selectin.

Amino Acid Sequence↗

PubMed1992

High-level expression of a human immunoglobulin gamma 1 transgene depends on switch region sequences.

We describe that chimeric mouse-human immunoglobulin heavy chain (IgH) genes lacking a switch region and controlled by an IgH promoter and the intronic enhancer are only weakly expressed in transgenic mice. Insertion of part of the human C gamma 1 or murine Cmu switch region into the major intron of the chimeric IgH gene results in a 10(2)-to 10(3)-fold increase in transgene expression. Analysis of B cell hybridoma clones from transgenic mice suggests that switch sequences influence IgH transgene expression at the cellular level. However, the effect of switch region sequences on IgH gene expression observed in vivo is not apparent in transfected B cell lines. These results indicate that switch region sequences which are located proximal to the constant part of the gene and which are normally present in a rearranged IgH gene after class switching represent a novel type of regulatory element that plays a critical role in IgH gene expression in vivo.

Animals↗

PubMed1985

Interrelation between cellular rRNA content and regulation of the cell cycle of normal and transformed mouse cell lines.

The relation between cellular rRNA content, as a measure of cell size, and the regulation of the cell cycle has been investigated for swiss 3T3 and the spontaneously transformed swiss 3T6 cell line. It is shown that the characteristic of percent of quiescent cells stimulated into the cell cycle versus cellular rRNA content is basically different for 3T3 and 3T6 cells: 3T3 cells do not enter the cell cycle below a certain threshold of cellular rRNA content, whereas 3T6 cells start proliferation without a substantial increase of rRNA. These data are interpreted as consistent with transformation of 3T6 cells being in essence their uncoupling from the requirement of normal cells of passing over a threshold of cellular rRNA content (cell size) before initiating DNA-replication.

Animals↗

PubMed1985

Selective effects by valinomycin on cytotoxicity and cell cycle arrest of transformed versus nontransformed rodent fibroblasts in vitro.

The effect of submicromolar concentrations of the K+ ionophore valinomycin on proliferation, viability, distribution of cell population over phases of the cell cycle, and cellular adenosine triphosphate content of different permanent rodent cell lines in vitro was investigated. Valinomycin inhibits proliferation of all cell lines tested with a saturating effect at about 20 to 100 nM. The effect of valinomycin on nontransformed 3T3 mouse and Rat-1 cells is nontoxic, whereas it acts with increasing toxicity on the transformed cells in the order 3T6 mouse, polyoma-3T3 mouse, temperature-sensitively Rous sarcoma virus-transformed Rat-1 at permissive temperature, and SV40-3T3 cells. According to these and some other criteria, the essential action of valinomycin appears to be to impose on the cells at low growth densities a state of limiting growth condition which normally is encountered only at high cell densities and/or low serum concentration. Nontransformed cells are proliferation arrested by valinomycin essentially in the G1 phase of the cell cycle, whereas all transformed cells under this condition are not arrested selectively in G1. In all cell lines tested (3T3, 3T6, and SV40-3T3), cellular adenosine triphosphate content is decreased by about 33% upon treatment with 20 nM valinomycin. Evidence is presented for a mitochondrial site of action of valinomycin.

Adenosine Triphosphate↗

PubMedDate not supplied

Cutaneous inflammation and proliferation in vitro: differential effects and mode of action of topical glucocorticoids.

The nonhalogenated double ester of prednisolone, prednicarbate (PC), is the first topical glucocorticoid with an improved benefit/risk ratio verified clinically and in vitro. To evaluate if this is due to unique characteristics of this steroid, a new compound created according to an identical concept, prednisolone 17-ethylcarbonate, 21-phenylacetate (PEP), and the new halogenated monoester desoximetasone 21-cinnamate (DCE) were tested and compared to PC, desoximetasone (DM) and betamethasone 17-valerate (BMV). Isolated foreskin keratinocytes served for in vitro investigations of anti-inflammatory processes in the epidermis, fibroblasts of the same origin were used to investigate the atrophogenic potential. Inflammation was induced by TNFalpha, resulting in an increased interleukin 1alpha (Il-1alpha) synthesis. As quantified by ELISA, all drugs significantly reduced Il-1alpha production. But PC and BMV appeared particularly potent, followed by DM and the two new congeners, which revealed minor anti-inflammatory activity. Glucocorticoid esters including PEP are rapidly degraded in keratinocytes (85% within 12 h). Hence, a ribonuclease protection assay of Il-1alpha mRNA was performed allowing short incubation times and thus minimizing biodegradation. This assay confirmed the anti-inflammatory potency of native PC and BMV. In contrary DCE and PEP did not reduce Il-1alpha mRNA to a significant extent. Therefore PEP acts as a prodrug only. In fibroblasts, Il-1alpha and Il-6 syntheses indicate proliferation and inflammation, respectively. Whereas PC and PEP inhibited Il-1alpha and Il-6 production in fibroblasts only to a minor extent, cytokine synthesis was strongly affected by the conventional glucocorticoids BMV and DM, but also by DCE. The minor unwanted effect of PC and PEP on fibroblasts was also reflected by their low influence on cell proliferation as derived from (3)H-thymidine incorporation. Again, more pronounced antiproliferative features were seen with the halogenated glucocorticoids. In the following, the correlation between antiphlogistic effects in keratinocytes (suppression of Il-1alpha) and antiproliferative effects in fibroblasts (suppression of Il-1alpha and Il-6; (3)H-thymidine incorporation) was analyzed. Here, PC is revealed as the only glucocorticoid with an improved benefit/risk ratio. Native PEP is shown to be almost ineffective and DCE presents exactly the opposite features of PC. It is tempting to speculate if this is due to different glucocorticoid receptor subtypes or different signaling pathways in keratinocytes and fibroblasts.

Administration, Topical↗