Molecular characterization of breast cancer cell lines by expression profiling.

PURPOSE: Gene expression patterns provide detailed insights into cellular regulation that reflect minor differences of cellular capacity not accessible by standard descriptions of the cellular phenotype or origin. METHODS: To identify fundamental differences and similarities we analyzed the gene expression patterns of four breast cancer cell lines: MCF-7, SK-BR-3, T-47D, and BT-474. RESULTS: Although only a small subset of genes (597) is represented on the Atlas-cDNA-Array (Clontech) used, clear differences in the expression of a number of genes could be detected. For example, unique high levels of expressions were found for the HLH-protein ID-1 (MCF-7) and the receptor tyrosine kinase erbB2 (SK-BR-3 and T-47D). Most genes analyzed were expressed at comparable levels in all cell lines studied. CONCLUSIONS: For interpretation of the results sets of genes that show similar variation of expression among the cells were grouped together. Furthermore, our analysis allows the assignment of similarity values that lead to a relation profile of the cell lines. How these results correlate with known biological properties of the cell lines is discussed. Additionally, we demonstrate that results obtained by cDNA-Array hybridization for expression of the ErbB receptor family correlate well with competitive RT-PCR, thus confirming the reliability of the cDNA-Array analysis.

Nutrient removal in subsurface flow constructed wetlands for application in sensitive regions.

One of the most interesting sites for research on CWs in Germany has been established in Wiedersberg (Saxonia). The multi-stage concept with primary settling, vertical and horizontal flow reed bed followed by UV-disinfection and a special phosphorus filter bed, allows numerous ways of operation and investigations. Denitrification can be improved by recirculation through VF bed and sedimentation tank or by means of adding carbonaceous water from the primary stage to a second level within the VFB or directly to the following HF bed. In order to investigate the efficiency of P-elimination four kinds of natural sands containing different amounts of iron have been used. To maintain a long-term capacity for P-reduction an additional filter bed is filled with gravelly sand which had been used for the precipitation of iron from drinking water before. After saturating with P this filter medium can be exchanged easily. A result of more than one year of operation is the high performance rate for adsorption of phosphorus by enriched iron on drinking water filter sand. At a total loading rate of 350 g P/m3 filter medium 250 g P/m3 have been adsorbed. Design considerations can not be given yet. The median denitrification rate at VFB is 1.3 g N m(-2) d(-1) and at HFB is 0.25 g N m(-2) d(-1). The low denitrifcation rate of HFB might be due to a very high quota of wastewater dilution by storm- and ground-water of 100 to 200 percent. The investigations on this wastewater treatment plant will be continued until June 2001 and experiments with filter columns will be added.

Catecholamines induce IL-10 release in patients suffering from acute myocardial infarction by transactivating its promoter in monocytic but not in T-cells.

The anti-inflammatory cytokine IL-10 is up-regulated in response to TNF-alpha suggesting a control mechanism of inflammation. In addition, we recently found systemic IL-10 release in response to acute stress reactions in the absence of any systemic inflammation. In vitro and in vivo studies in experimental models suggest that catecholamines induce IL-10 release via a cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) dependent pathway. Here we studied patients for plasma IL-10 after acute myocardial infarction, a very stressful event without significant signs of systemic inflammation. In fact, the activation of the sympathetic system initiated by cardiac infarction was accompanied by a temporary systemic release of IL-10. Catecholamine induced IL-10 may be released by different cells. Recently, we demonstrated that catecholamines directly stimulate the IL-10 promoter/enhancer via a cAMP/PKA pathway in monocytic cells. A cAMP responsive element (CRE) was identified as major target. Here we show that there is no influence of catecholamines on the IL-10 promoter activity in T-cells. In contrast to monocytic cells, in T-cells cAMP-induced PKA-dependent phosphorylation of the CRE-binding protein 1 (CREB-1) seems to play a marginal role in IL-10 induction, which was reflected by a low cAMP-dependent IL-10-promoter/enhancer stimulation in reporter gene assays. Thus, catecholamines are directly involved in the regulation of IL-10 expression in monocytic but not in T-cells after acute stressful conditions.

Delayed-type skin reaction to the heparin-alternative danaparoid.

Eczematous, infiltrated plaques at the site of subcutaneously administered heparin appear to be common. Heparinoids cannot be recommended in general as a substitute for heparin or low molecular weight heparin because delayed-type skin reactions to these molecules can also occur, as demonstrated in this case report.

GB virus C/hepatitis G virus infections in traumatologic outpatients, chronic non-A-E hepatitis and extrahepatic malignancies.

GB virus C/hepatitis G virus (GBV-C/HGV) is a recently discovered flavivirus of still unknown pathogenic relevance. We examined traumatologic outpatients to determine GBV-C/HGV viremia for further epidemiological studies, as blood donors hitherto used as controls represent healthy individuals without risk factors. Anti-GBV-C/HGV antibodies were detectable in 13.2% (95% confidence interval [CI] 9.3-18.2) and GBV-C/HGV RNA was detectable in 4.5% (95% CI 2.4-8.2) of the outpatients. In chronic non-A-E hepatitis patients GBV-C/HGV viremia was detectable at a significantly higher level of 16.1% (95% CI 6.1-34.5), while the prevalence of anti-GBV-C/HGV antibodies was 12.9% (95% CI 4.2-30.8). The rate of GBV-C/HGV viremia in patients with malignant diseases (different types of tumors, blood recipients were excluded) was 12.5% (95% CI 8.4-18.1), a significant elevation compared to traumatologic outpatients. The seroprevalence in the tumor group was 22.1% (95% CI 16.7-28.6), also significantly elevated. Thus, there are two messages. Firstly, testing for GBV-C/HGV may be a useful extension of the diagnostic procedure of viral hepatitis. Secondly, common risk factors or etiologic relations of GBV-C/HGV and extrahepatic malignancies should be discussed.

Catecholamines trigger IL-10 release in acute systemic stress reaction by direct stimulation of its promoter/enhancer activity in monocytic cells.

Acute stress reactions (e.g. linked with trauma, major surgery, psychic stress and myocardial infarction) are accompanied with temporary systemic release of the anti-inflammatory cytokine IL-10 followed by immunodepression. Since an association between activation of the sympathetic system and IL-10 release has been described, we studied the influence of catecholamines on its promoter activity in vitro. Using reporter gene assays we demonstrated that catecholamines in monocytic cells directly stimulate the IL-10 promoter/enhancer via a cAMP/protein kinase A-dependent pathway. A cAMP responsive element was identified as major target. Thus, catecholamines are directly involved in the regulation of immunoresponsiveness under stressful conditions.

Face dermatitis from contaminants on a mask for anaesthesia.

Allergic and irritant contact reactions to face masks for anaesthesia have rarely been reported. We present a 55-year-old female patient who developed facial allergic contact dermatitis after an operation requiring general anaesthesia. Patch tests showed positive reactions to cocospropylenediamin-guanidinium-diacetate (trivial name Dodigen 3558), a preservative used in disinfectants for medical instruments. It could be proven that residues of the causative allergen in the disinfectant adhered to the mask. This is the first report of a clinically relevant sensitization to this increasingly widely used agent.

Cyclic adenosine monophosphate-responsive elements are involved in the transcriptional activation of the human IL-10 gene in monocytic cells.

IL-10 plays an important role in the regulation of immune responses. We and others have demonstrated recently that cyclic adenosine monophosphate (cAMP)-elevating substances up-regulate monocytic IL-10 expression in vitro and in vivo. Computer analysis of the IL-10 promoter/enhancer region localized four putative cAMP-responsive elements (CRE1- 4) with homology to the CRE consensus motif. In electrophoretic mobility shift assays CRE1 and CRE4 bound protein complexes consisting of transcription factors CREB-1 and ATF-1, while CRE3 bound only marginal amounts of CREB-1/ATF-1 in combination with unknown protein(s). CRE2 showed no protein binding activity. In vitro mutation of CRE1 and CRE4 reduced the level of cAMP-stimulated transactivation in reporter gene assays in comparison to the wild-type promoter by 20 % and 50 %, respectively, while mutation of CRE3 had no effect. The main action of CRE4 on cAMP-dependent stimulation is probably based on its adjacent localization to the TATA box and its sequence comprising a perfect half site. Experiments with double and triple mutants and with deleted promoter fragments indicated the participation of additional elements beside the CRE motifs in the cAMP-dependent stimulation. Our data suggest that intracellular cAMP may directly affect expression of the immunoregulatory cytokine IL-10 in monocytic cells via activation of the eukaryotic transcription factors CREB-1 and ATF-1 and their binding to CRE1 and CRE4 in the upstream enhancer of the IL-10 promoter.

Sympathetic activation triggers systemic interleukin-10 release in immunodepression induced by brain injury.

The mechanism of immunodepression after brain injury is not yet clear. Here we demonstrate rapid systemic release of the immunoinhibitory cytokine interleukin-10, monocytic deactivation and a high incidence of infection in patients with 'sympathetic storm' due to acute accidental or iatrogenic brain trauma. In vitro studies showed that within minutes catecholamines trigger the secretion of interleukin-10 from unstimulated monocytes through a beta-adrenoreceptor-mediated, cAMP/protein kinase A-dependent pathway. We found that in a rat model of acute brain injury, the beta-receptor antagonist propranolol prevented the increase of interleukin-10 plasma levels. Rapid monocytic interleukin-10 release after sympathetic activation may represent a common pathway for immunodepression induced by stress and injury.

Epidemiological significance of bufexamac as a frequent and relevant contact sensitizer.

Bufexamac-containing ointments and creams are widely used by many patients with eczematous disorders as an alternative to topical corticosteroids. Recent studies provide evidence of a notable prevalence of contact sensitization in patch test populations. The aim of this study was to assess the frequency of use of this topically-applied drug by eczema patients in general, and to evaluate its potential to cause allergic contact reactions. 500 routinely patch tested patients (f:m = 377:123) were tested with bufexamac 5% and Parfenac ointment (the only commercial product available in Austria) in addition to the standard and other series of the German Contact Dermatitis Group. The packaging of the commercial product was shown to the entire study population, to decide whether or not they had ever used this product. In addition, their general practitioner was contacted to verify the anamnestic data. A total of 30 patients agreed that they had definitely used bufexamac, 5 others having probably applied it. The indication for and the duration of treatment were noted. Positive and relevant patch test reactions to bufexamac, as well as the bufexamac-containing ointment, were seen in 20 out of these 35 patients (57%), and sensitization occurred even after short-term application. Our study demonstrates that bufexamac has to be assumed to be a topical drug with a very high sensitization rate in an unselected patch test population (4% of 500 patients), and should therefore be added to the standard series.

Differential regulation of monocytic tumor necrosis factor-alpha and interleukin-10 expression.

Activation of human monocytes by bacterial endotoxin (LPS) results in an initial burst of inflammatory cytokines like tumor necrosis factor (TNF)-alpha which is followed by the secretion of anti-inflammatory mediators like interleukin (IL)-10. The signaling pathways in IL-10 induction are unknown. Here, we show that the regulation of IL-10 expression is more complex than that of TNF-alpha. LPS-induced TNF-alpha and IL-10 expression requires early activation of protein tyrosine kinases (PTK). Moreover, delayed addition of PTK inhibitors blocked IL-10, but not TNF-alpha, suggesting the impact of a late PTK activity. Two inducers of PTK activity are the downstream mediators of LPS activation, TNF-alpha and cyclic adenosine monophosphate (cAMP). Both mediators synergistically up-regulate IL-10 expression. Downstream of PTK activation, they use distinct pathways. TNF-alpha, but not cAMP-induced IL-10 gene expression was inhibited by pyrrolidine dithiocarbamate, suggesting the involvement of reactive oxygen species. Inhibition of protein kinase C (PKC) suppressed LPS-induced TNF-alpha and IL-10 expression as well, but, unlike TNF-alpha, direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) did not induce IL-10 expression. Furthermore, PKC is not involved in late events of IL-10 activation, as delayed addition of PKC inhibitors did not suppress LPS-induced IL-10 expression and did not influence cAMP- or TNF-alpha-induced IL-10. The modulation of IL-10 expression by inflammatory mediators suggests a regulatory circuit of the inflammatory response.

Effect of topical laurocapram (Azone) on the in vitro percutaneous permeation of sodium lauryl sulfate using human skin.

The probability of simultaneous cutaneous exposure to surfactants and penetration enhancers could occur frequently during routine skin treatment. This study ascertains whether pre-exposure of skin to laurocapram would affect the penetration of a model surfactant, sodium lauryl sulfate (SLS). In vitro experiments with human skin were performed to compare the penetration of SLS after pretreatment with (1) different concentrations of laurocapram, (2) after repeated SLS treatments, (3) untreated controls, and (4) water-control. Pre-exposure to laurocapram enhanced penetration of SLS compared to all other treatments (p < 0.05). Since subsequent pre-exposure of skin to laurocapram increased SLS penetration, the chances of an elevated skin irritation reaction at the exposed site may therefore be possible. Pre-exposure of the skin with SLS did not increase the SLS flux values significantly, compared to the laurocapram pretreated skin. From these results it can be proposed that proper care and precautions may be necessary after exposure of skin to laurocapram and also to various other percutaneous enhancers. Further in vivo correlations are essential to define the clinical implications of this study, especially as related to irritant dermatitis.

Epidermal enzymes as penetration enhancers in transdermal drug delivery?

Epidermal enzymes play an important role in the process of differentiation of keratinocytes. The present preliminary in vitro study was undertaken to observe if topical enzyme treatment influenced permeation of compounds across the skin. Due to the noted function and importance of phosphatidylcholine metabolism during maturation of the barrier lipids, the effects of topical application of the phosphatidylcholine dependent phospholipase C enzyme (not present in epidermis) on skin penetration of three model drugs, viz. benzoic acid, mannitol and testosterone, were studied. Similar studies were also carried out using epidermal enzymes like triacylglycerol hydrolase, acid phosphatase, and phospholipase A2 (present in epidermis). Pretreatment of skin with phospholipase C significantly enhanced permeation of benzoic acid, mannitol, and testosterone relative to untreated skin. Triacylglycerol hydrolase (neutral) increased the penetration of mannitol 3-fold and had no effect on benzoic acid penetration. Topical application of acid phosphatase did not alter the permeation of any of these compounds. Phospholipase A2 significantly enhanced permeation of benzoic acid and mannitol while it did not have any effect on the penetration of testosterone. These results for the first time demonstrate that enzymes may remarkably affect and/or regulate the permeation of topically applied compounds.

Mechanism of endotoxin desensitization: involvement of interleukin 10 and transforming growth factor beta.

Tolerance of monocytes/macrophages to endotoxin (lipopolysaccharide [LPS]) can be induced both in vivo and in vitro by LPS itself. Exposure to LPS, even at a very low dose, induces a downregulation of cytokine response to a second high dose LPS challenge. To learn more about the unknown mechanisms of this phenomenon, we studied the role of antiinflammatory cytokines in this process. Preculture of human peripheral blood monocytes for 24 hours with low concentrations of LPS induced hyporesponsiveness to high-dose LPS rechallenge with respect to tumor necrosis factor (TNF) alpha and interleukin (IL) 10 but not IL-1RA production. These results suggest that LPS tolerance reflects a functional switch of monocytes rather than a general LPS hyporesponsiveness. IL-10 and transforming growth factor (TGF) beta 1 showed additive effects in replacing LPS for induction of LPS hyporesponsiveness in vitro. Additionally, neutralizing anti-IL-10 and anti-TGF-beta monoclonal antibodies prevented induction of LPS tolerance. In vitro induced LPS tolerance looks like the ex vivo LPS hyporesponsiveness of monocytes from septic patients with fatal outcome: downregulation of LPS-induced TNF-alpha and IL-10 production but not of IL-1RA secretion. LPS hyporesponsiveness in septic patients was preceded by expression of IL-10 at both the mRNA and protein level. In summary, our data suggests that IL-10 and TGF-beta mediate the phenomenon of LPS tolerance in vitro and perhaps in vivo (septic patients), too.

Up-regulation of monocytic IL-10 by tumor necrosis factor-alpha and cAMP elevating drugs.

It is well established that endotoxin [lipopolysacharide (LPS)] induces pro-inflammatory cytokine production in monocytes, which is followed by secretion of the anti-inflammatory cytokine, IL-10. IL-10 down-regulates inflammatory response [tumor necrosis factor (TNF)-alpha, IL-1, IL-6, IL-8] as well as IL-10 synthesis itself. We wondered whether pro-inflammatory cytokines such as TNF-alpha may be involved in the regulation of human IL-10 synthesis. TNF-alpha induced de novo IL-10 mRNA expression in a dose-dependent manner but no IL-10 protein in human peripheral blood mononuclear cells. Furthermore, LPS-induced IL-10 gene and protein expression was significantly inhibited by neutralizing anti-TNF-alpha mAb. On the basis of these results, we conclude that TNF-alpha is involved in the up-regulation of its antagonist IL-10. Paradoxically, drugs that effectively inhibit expression of TNF-alpha via the elevation of intracellular cAMP level (iloprost, pentoxifylline, prostaglandin E2 and N6,2-O-dibutyryl cAMP) augmented the endotoxin-induced IL-10 synthesis at both protein and mRNA levels. In order to provide a basis for the analysis of the transcriptional regulation of the human IL-10 gene, we isolated a fragment of the human IL-10 gene containing 1308 bp of the 5' non-coding sequence. It shows remarkable homology to the mouse IL-10 promoter in regions that have been associated with transcriptional regulation, including a cAMP responsive element which could explain the cAMP-mediated effects. The lack of a NF-kappa B-like binding site in the human sequence suggests a NF-kappa B-independent mechanism of TNF-alpha-induced IL-10 gene activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of the human interleukin 10 promoter. Characterization of the promoter activity in Burkitt's lymphoma cell lines.

Interleukin 10 (IL-10) is a pleiotropic growth and differentiation factor with potent suppressor functions on macrophages, T cells and NK cells and contributes to the regulation of proliferation and differentiation of B cells. The expression of IL-10 appears to be tightly regulated, as the levels of constitutive expression in normal cells is extremely low. In contrast to normal haematopoetic cells, Epstein-Barr virus (EBV)-immortalized B cells and EBV-positive Burkitt's lymphoma cells express high levels of IL-10 constitutively. In this report we have cloned and sequenced IL-10 promoter fragments and analysed their activity in EBV-positive Burkitt's lymphoma cells. A nested set of DNA fragments from the IL-10 gene 5'-flanking region was placed upstream of the luciferase gene and assayed for their ability to direct luciferase expression in Burkitt's lymphoma cells. We have identified elements within the 5'-flanking region of the human IL-10 gene which can activate or suppress the constitutive expression of IL-10. The essential promoter of the IL-10 gene, which induces low levels of luciferase expression, was found to require the major start site of transcription (+1), a TATA-box (-77) and up to 150 additional 5' nucleotides. Positive regulatory sequences are located between -1100/-900. Negative regulatory elements which abolish luciferase activity were identified between -800/-300.

A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats.

Competitive polymerase chain reaction (PCR) is a sensitive method for quantification of cytokine mRNA expression. Co-amplification of an internal standard serves as control for comparing the efficiency of PCR in different samples. We have developed a novel control fragment for multiple analyses of rat cytokine gene expression containing primers for IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, TNF-alpha, TGF-beta 1, IFN-gamma and MIP-2. Additional primers were incorporated to analyse the content of T cells (CD3), activated T cells (CD25) and housekeeping genes (beta-actin and HPRT). As an example we demonstrate analysis of IL-2 mRNA expression in small pieces of kidney tissue obtained from rats after kidney allotransplantation. The IL-2 expression decreased tenfold during treatment with an anti-rat CD4 monoclonal antibody as compared to untreated animals.