A randomized, double-blind, vehicle-controlled, half-side comparison with a herbal ointment containing Mahonia aquifolium, Viola tricolor and Centella asiatica for the treatment of mild-to-moderate atopic dermatitis.

OBJECTIVE: Only a few clinical trials have been published on the topical treatment of atopic dermatitis with herbal ointments. An ointment containing extracts from Mahonia aquifolium, Viola tricolor and Centella asiatica has previously been studied in open uncontrolled trials with children. However, no data exist on adult patients in a randomized controlled trial. METHODS: A total of 88 patients with mild-to-moderate atopic dermatitis were enrolled in a double-blind, vehicle-controlled, randomized, half-side comparison. Patients between 18 and 65 years of age were treated for 4 weeks with an ointment containing Mahonia aquifolium, Viola tricolor and Centella asiatica. The primary endpoint was a summary score for erythema, edema/papulation, oozing/crust, excoriation and lichenification according to a 4-point scale. Secondary efficacy variables were assessment of pruritus severity (10 cm VAS) and a global assessment of effectiveness as well as tolerability. RESULTS: The study ointment reduced the primary and secondary endpoints slightly more than the base cream which was used as vehicle; the differences were not statistically significant. Since the climatic conditions during the study duration varied from very mild and sunny to very cold and dry, a post-hoc subanalysis was performed with a subset of 64 patients whose treatment was at a mean outside temperature of 10 degrees C or less. Under these conditions the primary endpoint showed high statistical significance. CONCLUSION: In this trial, an ointment containing Mahonia aquifolium, Viola tricolor and Centella asiatica could not be proven to be superior to a base cream for patients with mild-to-moderate atopic dermatitis. However, a subanalysis indicated that the cream might be effective under conditions of cold and dry weather.

Cooperative effects of interferon-gamma on the induction of NADPH oxidase by retinoic acid or 1,25(OH)2-vitamin D3 in monocytic U937 cells.

The effect of retinoic acid (RA), 1,25-dihydroxyvitamin D3 (1,25-D3) or human recombinant interferon-gamma (IFN-gamma) on the induction of NADPH oxidase was studied in premonocytic U937 cells. Differentiation with the combination of either RA (1 microM) or 1,25-D3 (10 nM) with IFN-gamma (100 IU/ml) induced NADPH oxidase activity as demonstrated by increased superoxide anion (O2-) generation in response to stimulation with phorbol myristate acetate (PMA, 100 nM). Induction of NADPH oxidase activity was preceded by increases in mRNA levels of p47-phox, p67-phox and gp91-phox, which encode three subunits of the enzyme, and immunoblot analysis of the p47-phox and p67-phox proteins revealed that the increases in mRNA levels were equally reflected by increases in protein levels. In contrast, RA, 1,25-D3 or IFN-gamma alone did not induce NADPH oxidase activity which correlated with their failure to increase p67-phox and gp91-phox mRNA levels. The mRNA of p21 rac1, a GTP-binding protein that regulates NADPH oxidase activity in macrophages, was constitutively expressed in undifferentiated cells and was not affected by differentiation. These data indicate that induction of a functional NADPH oxidase in premonocytic U937 cells requires the cooperative actions of IFN-gamma plus RA or 1,25-D3 and is reflected in the increased expression of p67-phox and gp91-phox.

Physical state, expression and regulation of two glucocorticoid-controlled genes on bovine papilloma virus vectors.

We have studied the extrachromosomal maintenance and the transcription regulation of two glucocorticoid-inducible genes on bovine papilloma virus (BPV) vectors in c127 mouse fibroblasts. These genetic elements were the rat tryptophan oxygenase (TOase) gene promoter, which is active in vivo only in hepatocytes, and the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR). From both genes, fusions of the 5'-flanking region of the transcription unit to the bacterial gene for chloramphenicol acetyltransferase (CATase) were constructed. These fusion genes were inserted either into pCGBPV9, a BPV vector encoding G418 resistance, into pBPV-BV1, a vector containing "stabilizing" segments of the human beta-globin gene, or into a BPV construct, whose bacterial plasmid sequences could be removed before transfection. Five constructs of the two latter groups, selectable in c127 cells only as foci, were normally maintained in the extrachromosomal state. In contrast, three out of five constructs based on pCGBPV9 and selectable for resistance against G418 were maintained in a high molecular weight form, most probably of intrachromosomal concatemeric nature, while the remaining two G418-resistant constructs appeared alternatively in this or the extrachromosomal monomeric form. In contrast to its absence of expression in fibroblasts in vivo, the TOase gene element present on BPV vectors was found to be active in fibroblasts in these transfection experiments. As judged by CATase activities and for TOase also by mapping of the transcription start sites, transcription of both genes was under hormonal regulation. All BPV vectors proved to be useful tools in the study of these regulated genes, and in only one out of ten constructs was regulation atypical, possibly due to effects from flanking vector sequences.

Arachidonic acid increases activation of NADPH oxidase in monocytic U937 cells by accelerated translocation of p47-phox and co-stimulation of protein kinase C.

Arachidonic acid (AA) has been implicated as an important amphiphilic co-factor in the activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in neutrophils and reconstituted cell-free systems. To assess the role of AA in the activation of O2- generation in monocytic cells, we studied pre-monocytic U937 cells differentiated with 1,25-(OH)2-vitamin D3 plus interferon-gamma (IFN-gamma). AA dose-dependently enhanced phorbol myristate acetate (PMA)-stimulated O2- generation, with a maximum increase of 4,5-fold, through: (1) a more than 50% reduction of the lag-phase, defined as the time between addition of PMA and detection of O2-; and (2) a more than 60% increase in the constant rate of O2- generation. Reduction of the lag phase was associated with increased protein kinase C (PKC)-independent translocation of the cytosolic subunit of NADPH oxidase p47-phox to the cell membrane, whereas increased generation of O2- correlated with enhanced activation of PKC. The data indicate that AA increases activation of NADPH oxidase by accelerating its assembly and by co-stimulating PKC in monocytic U937 cells.

CCAAT/enhancer binding protein expression is rapidly extinguished in TA1 adipocyte cells treated with tumor necrosis factor.

Tumor necrosis factor (TNF) has been shown to have diverse effects on a wide variety of cell types. In the mouse adipogenic TA1 cell line, TNF completely abolishes differentiation and reverts fully differentiated fat cells into fibroblasts. This block in differentiation and its reversal is due to the rapid reduction in the expression of adipose-specific genes. This study reports that the transcription factor, CCAAT/enhancer binding protein (C/EBP), previously reported to promote the differentiation of 3T3-L1 adipocytes, is expressed in TA1 cells. During their growth in culture, the levels of C/EBP, as evidenced by its cellular levels of specific mRNA, protein, and DNA binding activity, increase dramatically when cells reach confluence and proceed to differentiate. Addition of TNF to cultured preadipocytes or fully differentiated adipocytes rapidly reduces C/EBP levels and is accompanied by the decrease in expression of adipose-specific genes. C/EBP binding sites occur in several adipose-specific genes, and here it is demonstrated that its presence in a novel adipose-specific gene, Clone 47, also referred to as FSP27, may be responsible for the strong down-regulation of the expression of the Clone 47 (FSP27) promoter-linked chloramphenicol acetyl transferase gene by TNF. This study proposes that the loss of C/EBP in response to TNF treatment may in part explain the loss of the adipocyte differentiated state.

Modulation of the expression of early genes by polyunsaturated fatty acids.

Expression of early genes is a characteristic immediate cellular response to mitogenic or inflammatory stimulation. Various second messenger systems have been found to transduce the signal from the plasma membrane to the nucleus. Recent observations indicate that in addition to well characterized second messenger systems, polyunsaturated fatty acids, especially the n-6 fatty acid arachidonic acid and its endogenously produced metabolites affect the expression of early genes in different cell types. At least in fibroblasts, the stimulatory effect of arachidonic acid can be antagonized by n-3 polyunsaturated fatty acids. Further identification of the mechanisms through which polyunsaturated fatty acids modulate early gene expression and regulate subsequent cellular responses, like cell growth, may help to define novel concepts in the management of cardiovascular diseases.

Effects of different polyunsaturated fatty acids on growth-related early gene expression and cell growth.

Effects of polyunsaturated fatty acids on expression of early-response genes c-fos and Egr-1 and induction of cell growth were assessed in Swiss 3T3 fibroblasts. Stimulation with arachidonic acid increased mRNA levels of c-fos and Egr-1. This effect was inhibited by preincubation with cyclooxygenase inhibitors and restored by addition of prostaglandin E2 (PGE2), the predominant eicosanoid produced in Swiss 3T3 fibroblasts. Further signaling of PGE2, was mediated by a protein kinase C-dependent pathway, since downregulation, or inhibition, of protein kinase C reduced increases in mRNA levels. Parallel to the stimulatory effects on mRNA levels, AA and PGE2 also increased cell growth, as determined by uptake of [3H]-thymidine. In contrast to arachidonic acid, n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) did not increase c-fos and Egr-1 mRNA levels or cell growth. Furthermore, preliminary data indicate that EPA and DHA even reduce the stimulatory effect of AA, which is associated with reduced formation of PGE2. In conclusion, our data indicate that AA increases expression of growth-related early genes c-fos and Egr-1 Swiss 3T3 fibroblasts by its conversion to PGE2 and subsequent activation of protein kinase C, whereas n-3 fatty acids do not activate this signaling cascade.

Petasites hybridus extracts in vitro inhibit COX-2 and PGE2 release by direct interaction with the enzyme and by preventing p42/44 MAP kinase activation in rat primary microglial cells.

Rhizomes of butterbur, Petasites hybridus L. (Asteraceae), have been used since ancient times for the treatment of inflammatory diseases. In the present study, the effects of lipophilic extracts from rhizomes of Petasites hybridus on the formation and release of prostaglandin E2 were investigated. The extracts had different contents of petasin and isopetasin: A: 2.1 % and 0.4 %, B: 0.2 % and 0.1 %, C: 12.1 % and 6.1 % and D: 21.9 % and 9.4 %, respectively. Direct inhibition of cyclooxygenase (COX) -1 and -2 isoenzymes and inhibition of the expression of COX-2 and p42/44 MAP kinase in rat primary microglial cells were tested. All extracts were found to be only weak direct inhibitors of COX-1 (IC50> 400 microg/mL). However, most extracts revealed a strong inhibitory activity against the inducible isoform COX-2 ( A: IC50=30.4 microg/mL; B: IC50=60.6 microg/mL; C: IC50=22.6 microg/mL; D: IC50=20.0 microg/mL). This activity was not correlated to the content of petasin and isopetasin. Pure petasin and isopetasin neither inhibited COX-1 nor COX-2 (IC50 > 400 microM for both compounds and enzymes). Petasites extracts dose-dependently inhibited LPS-induced and thus COX-2-mediated PGE2 release in primary rat microglial cells (A: IC50= 2.4 microg/mL; C: IC50=5.8 microg/mL and D: IC50=4.6 microg/mL). Also this effect was independent from the petasin and isopetasin content. COX-2 synthesis in microglia was totally blocked with 5 microg/mL of C whereas COX-1 synthesis was not influenced. C and D did not affect the LPS-induced activation of p38 MAPK and IkappaBalpha, but they prevented the LPS-induced activation of p42/44 MAPK. Therefore, these Petasites hybridus extracts can be regarded as natural selective inhibitors of COX-2 and its expression, an effect which is independent from the petasin content.

Steroid controlled expression of the chicken lysozyme and the rat tryptophan oxygenase gene after transfer into eukaryotic cells.

To study the mechanism of steroid induced transcriptional control we have introduced recombinants of the chicken lysozyme gene and the rat tryptophan oxygenase (TO) gene into heterologous and homologous cells. To monitor the activity of the TO-promoter, 1.9 kb of the TO 5'-flanking sequences were fused with sequences coding for the bacterial enzyme chloramphenicol acetyltransferase (CAT). Upon transfer into mouse L-cells the transient expression of the TO-CAT recombinant was found to be inducible by dexamethasone. Transient expression of chicken lysozyme gene recombinants after introduction into various cell types could only be detected in chicken oviduct cells, or in conjunction with SV-40 enhancer sequences in human cells. The recombinant gene used in oviduct cells was a fusion between the lysozyme promoter, including 1.4 kb of upstream sequences, and the coding region of the gene for SV 40 T-antigen (plys-T). The expression in oviduct cells was stimulated by dexamethasone or progesterone, whereas SV 40 enhanced expression of lysozyme sequences in human cells could not be regulated by steroids. Using several deletion mutants, a region between -220 bp and -140 bp upstream of the cap site was found to be essential for both regulation by glucocorticoids as well as by progesterone.

Cloning and transcriptional regulation of a novel adipocyte-specific gene, FSP27. CAAT-enhancer-binding protein (C/EBP) and C/EBP-like proteins interact with sequences required for differentiation-dependent expression.

We have reported previously the cloning of several cDNAs whose mRNAs are induced during differentiation of the adipogenic cell line TA1. Here we characterize an adipocyte-specific gene, which we refer to as FSP27 (formerly clone 47), that encodes a protein of 27 kDa, the sequence of which is unrelated to any in the current data banks. The FSP27 promoter confers adipocyte-specific expression to a heterologous reporter gene in transfected adipogenic cell lines, e.g. TA1 and 3T3-L1. Analysis of regulatory elements in the FSP27 promoter region indicates the presence of (a) a proximal palindromic sequence that is necessary for adipocyte-specific expression; and (b) a distal differentiation-independent enhancer-like element. The palindromic sequence TTCGAAA is protected from digestion by DNase I using nuclear extracts from TA1 preadipocytes and adipocytes. Heated rat liver nuclear extract, a very abundant source of the transcription factor CAAT-enhancer-binding protein (C/EBP) and related proteins, generates an equivalent footprint over the palindrome. However, C/EBP can account for only a portion of the protein-DNA complexes in TA1 cells because preadipocytes as well as adipocytes contain proteins distinct from C/EBP which interact with the same sequence. We suggest that C/EBP and other C/EBP-like proteins play a critical role in regulating the transcription of the fat-specific gene FSP27.

Differential effects of n-6 and n-3 polyunsaturated fatty acids on cell growth and early gene expression in Swiss 3T3 fibroblasts.

Dietary n-3 polyunsaturated fatty acids (PUFAs) have been found to reduce accelerated cell growth. To study the underlying molecular mechanisms, we evaluated the effects of the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) compared with the n-6 PUFA arachidonic acid (AA) on cell growth and early gene mRNA accumulation in Swiss 3T3 fibroblasts. AA significantly increased cell numbers and incorporation of [3H]-thymidine compared with cells treated with EPA and DHA, which did not stimulate cell growth. In contrast to AA and parallel to its effect on cell growth, EPA and DHA did not lead to a pronounced increase in Egr-1 and c-fosmRNA levels. When they were incubated together with AA, both DHA and EPA reduced AA-induced Egr-1 and c-fosmRNA accumulation and incorporation of [3H]-thymidine. We have recently shown that AA strongly increases Egr-1 and c-fosmRNA accumulation 3T3 fibroblasts through its metabolism to prostaglandin E2 (PGE2) and its subsequent activation of protein kinase C (Danesch et al., 1994, J. Biol. Chem., 269:27258-27263). Consistent with the notion that increased PGE2 formation is required for the AA-induced early gene mRNA accumulation, EPA and DHA reduced PGE2 formation from exogenous [14C]-AA by more than 60%, but they did not decrease mRNA levels following stimulation with PGE2. We suggest that, in 3T3 fibroblasts, EPA and DHA antagonize AA-induced early gene mRNA accumulation and cell growth by reducing PGE2 formation.

The first placebo-controlled trial of a special butterbur root extract for the prevention of migraine: reanalysis of efficacy criteria.

This is an independent reanalysis of a randomised, placebo-controlled parallel-group study on the efficacy and tolerability of a special butterbur root extract (Petadolex) for the prophylaxis of migraine. The original protocol and analysis had a number of major shortcomings. In order to follow regulatory requirements, an independent reanalysis of the original data was performed. Following a 4-week baseline phase, 33 patients were randomised to treatment with two capsules 25 mg butterbur twice a day and 27 to placebo. The mean attack frequency per month decreased from 3.4 at baseline to 1.8 after 3 months (p = 0.0024) in the verum group and from 2.9 to 2.6 in the placebo group (n.s.). The responder rate (improvement of migraine frequency > or =50%) was 45% in the verum group and 15% in the placebo group. Butterbur was well tolerated. This small trial indicates that butterbur may be effective in the prophylaxis of migraine.

Arachidonic acid increases c-fos and Egr-1 mRNA in 3T3 fibroblasts by formation of prostaglandin E2 and activation of protein kinase C.

Studying Swiss 3T3 fibroblasts, we report that arachidonic acid strongly stimulates mRNA levels of the growth-associated immediate early genes c-fos and Egr-1. Structurally related compounds like arachidonic acid methyl ester, arachidonyl alcohol, or eicosatetraynoic acid are ineffective, indicating a specific role of free unesterified arachidonic acid or an arachidonic acid metabolite in c-fos and Egr-1 mRNA accumulation. Blocking the conversion of arachidonic acid to prostaglandins by inhibiting cyclooxygenase abolishes arachidonic acid-induced accumulation of c-fos and Egr-1 mRNA. Inhibition of the lipoxygenase or cytochrome P-450 epoxygenase pathways has no significant effect on arachidonic acid-induced c-fos and Egr-1 mRNA levels, indicating that prostaglandin synthesis is necessary for the increase in c-fos and Egr-1 mRNA. Reversed phase high performance liquid chromatography revealed prostaglandin E2 (PGE2) as the major arachidonic acid metabolite in Swiss 3T3 fibroblasts. When added to the cells, PGE2 stimulates c-fos and Egr-1 mRNA levels to the same degree as arachidonic acid. Also, the inhibition of arachidonic acid-stimulated c-fos and Egr-1 mRNA accumulation by indomethacin is reversed by PGE2. Contrary to reports that PGE2 caused an increase in cAMP levels in Swiss 3T3 fibroblasts, we found that arachidonic acid and PGE2 only minimally increase cAMP levels as compared with untreated cells. In contrast, inhibition of protein kinase C by calphostin C and chelerythrine or down-regulation with phorbol 12-myristate 13-acetate drastically reduces PGE2 and arachidonic acid-induced c-fos and Egr-1 mRNA levels. These data indicate that arachidonic acid exerts its stimulatory effect on c-fos and Egr-1 mRNA via synthesis of PGE2 and subsequent activation of protein kinase C, probably through a PGE2 receptor coupled to phospholipase C.

Transcriptional regulation of the tryptophan oxygenase gene in rat liver by glucocorticoids.

The enzyme tryptophan oxygenase (EC 1.13.11.11), which is synthesized in rat liver, is induced by glucocorticoids. We have used cloned tryptophan oxygenase genomic and cDNA sequences to study the mechanism of induction. Rat liver poly(A+) RNA was separated on a formaldehyde gel, blotted to nitrocellulose, and hybridized to a nick-translated tryptophan oxygenase cDNA clone to analyze the kinetics of tryptophan oxygenase mRNA accumulation. Transcription in isolated rat liver nuclei was investigated to determine the relative rate of transcription of the tryptophan oxygenase gene. Analysis of the accumulation of albumin mRNA, which is unaffected by glucocorticoids, served as an internal control. We show here that the synthetic glucocorticoid dexamethasone causes a 10-fold increase in the concentration of tryptophan oxygenase mRNA sequences in rat liver, and that this is a consequence of transcriptional activation of the tryptophan oxygenase gene.

Glucocorticoid responsiveness of the transcriptional enhancer of Moloney murine sarcoma virus.

The long terminal repeat of Moloney Murine Sarcoma Virus (MoMSV) contains an imperfect direct repeat that serves as a strong transcriptional enhancer. The strength of the MoMSV enhancer is strongly dependent on the presence of glucocorticoid hormone. Mapping studies in combination with DNAase I footprinting experiments define the presence of glucocorticoid regulatory elements at the promoter-proximal ends of each enhancer repeat. These elements behave like inducible enhancers: their regulatory activity is independent of position and orientation when they are linked in cis to a heterologous promoter. These data demonstrate the modular nature of the MoMSV enhancer and identify the glucocorticoid receptor as one of the trans-acting factors that interact with the MoMSV enhancer and mediate its transcriptional activity.

Glucocorticoid induction of the rat tryptophan oxygenase gene is mediated by two widely separated glucocorticoid-responsive elements.

Transcription of the gene coding for tryptophan oxygenase (TO) in rat liver is induced 10-fold by glucocorticoids. To identify DNA elements mediating the glucocorticoid-regulated expression of the TO gene we transfected mouse L cells with a fusion gene consisting of 1.95 kb TO 5'-flanking sequences linked to the coding sequence of the gene for chloramphenicol acetyltransferase (CAT). CAT assay and RNA mapping experiments demonstrate that both transient and stable expression of the TO-CAT fusion gene are inducible by dexamethasone. Analysis of transcripts from 5'-deletion mutants identifies two glucocorticoid-responsive elements (GRE), located 450 bp and 1.2 kb upstream of the cap site. The purified rat glucocorticoid receptor binds to the sequence of each GRE as evidenced from footprinting experiments. Interestingly the protected sequence of the proximal footprint is by itself not sufficient for sequence induction, but requires sequences located immediately upstream.

Docosahexaenoic acid selectively attenuates induction of vascular cell adhesion molecule-1 and subsequent monocytic cell adhesion to human endothelial cells stimulated by tumor necrosis factor-alpha.

Incorporation of the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) but not eicosapentaenoic acid or n-6 arachidonic acid into human umbilical vein endothelial cell (HUVEC) phospholipids dose-dependently reduced tumor necrosis factor-alpha (TNF-alpha)-induced surface expression of vascular cell adhesion molecule-1 (VCAM-1). In parallel, DHA inhibited TNF-alpha-stimulated monocytic U937 cell adhesion to HUVECs but did not affect TNF-alpha- or interferon gamma-induced expression of intercellular adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 or VCAM-1 induction by interleukin-1 beta. DHA appeared to attenuate VCAM-1 transcription, as it reduced induction of VCAM-1 mRNA by TNF-alpha. VCAM-1 induction is regulated by activation of nuclear factor-kappa B, which can be mediated by a TNF-alpha-responsive phosphatidylcholine-specific phospholipase C (PC-PLC). Gel-shift analysis showed inhibition of TNF-alpha-induced nuclear factor-kappa B mobilization by DHA. While the PC-PLC inhibitor D609 dose-dependently prevented VCAM-1 induction by TNF-alpha, 1,2-diacyl-glycerol (DAG) stimulated VCAM-1 expression, suggesting that VCAM-1 induction by TNF-alpha may be mediated by activation of PC-PLC. Treatment with DHA resulted in a fourfold enrichment in PC. In addition, DHA or D609 but not eicosapentaenoic acid or arachidonic acid suppressed activation of PC-PLC by TNF-alpha, estimated as [14C]DAG synthesis in prelabeled HUVECs. Incorporation of DHA into phospholipids selectively attenuates VCAM-1 induction by TNF-alpha and subsequent monocytic cell adhesion by inhibition of TNF-alpha-stimulated PC-PLC activation in HUVECs.

Isolation and characterization of the rat tryptophan oxygenase gene.

Tryptophan oxygenase (TO, EC 1.13.1.12) from rat liver is subject to glucocorticoid and developmental control. To study the mechanism of regulation, TO mRNA sequences and the chromosomal TO gene were cloned. From a cDNA library prepared from rat liver poly(A)+ RNA enriched for TO mRNA, a recombinant plasmid containing TO cDNA sequences was identified by translation of hybrid-selected RNA and immunoprecipitation with antibodies directed against TO. This cDNA clone hybridizes to a mRNA 2000 bases long that is inducible by dexamethasone. With this clone as probe we isolated from a bacteriophage lambda rat DNA library genomic clones which together span a region of 32 kilobase pairs (kb). Heteroduplex analysis revealed that the gene extends over 19 kb and is interrupted by at least 11 introns. To characterize the presumptive control region the DNA sequence around the 5' end of the TO gene was determined. S1 nuclease protection experiments revealed two separate start sites for TO mRNA transcription within this region.