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Biomedical subjects

C Platzer

Publications and source records attributed to C Platzer.

At least 19 recordsLinked to original sources
PubMed2002

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.

Breast Neoplasms↗
PubMed2001

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.

Adsorption↗
PubMed2000

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.

Catecholamines↗
PubMed2000

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.

Acute Disease↗
PubMed1999

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.

Cyclic AMP↗
PubMed1998

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.

Adrenergic beta-Antagonists↗
PubMed1996

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.

Antioxidants↗
PubMed1995

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.

Cells, Cultured↗
PubMed1995

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.

Base Sequence↗
PubMed1995

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)

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

5' noncoding sequence of human IL-10 gene obtained by oligo-cassette PCR walking.

A fragment of the human IL-10 gene starting from a Hind III site and including 744 previously unknown bases of the 5' regulatory region was isolated by oligo-cassette PCR walking and the nucleotide sequence was determined. It shows remarkable homology to the mouse IL-10 promoter in regions that have been associated with transcriptional regulation.

Animals↗
PubMed1992

Analysis of cytokine mRNA levels in interleukin-4-transgenic mice by quantitative polymerase chain reaction.

Interleukin (IL)-4-transgenic mice were used as a model system to study the consequences of low levels of IL-4 expression for the expression of other cytokines examined by quantitative polymerase chain reaction (PCR). For this purpose, a plasmid was constructed which contains, in tandem array, 5' and 3' primer sequences specific for the cytokine genes IL-1 to IL-6, tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN)-gamma and beta-actin. During co-amplification, target and control DNA compete for the primers and the amount of PCR product is proportional to the amount of input DNA. Competitive PCR was performed first to adjust the cDNA to be compared to identical concentrations of beta-actin cDNA and subsequently to determine cytokine mRNA levels from spleen cells of normal and IL-4-transgenic animals. The sensitivity of this approach was demonstrated by the capability to detect a twofold difference in IL-4 mRNA levels between IL-4-transgenic heterozygous and homozygous animals. Upon lipopolysaccharide activation, the IL-4 transgene which is expressed essentially in B lymphocytes was induced approximately 50-fold. Several cytokine mRNA such as those coding for IL-5, IL-6, IFN-gamma and also the IL-4 receptor were found to be up-regulated in IL-4-transgenic mice, whereas IL-1, IL-2, IL-3, TNF and LT mRNA levels did not seemed to be influenced by IL-4. A possible functional significance of the elevated IFN-gamma mRNA was demonstrated by showing that (a) CD23 expression was not increased, and (b) Mac-1+ cells were markedly increased in the spleen of transgenic mice.

Animals↗
PubMed1992

Interleukin-4-mediated tumor suppression in nude mice involves interferon-gamma.

The molecular events during the anti-tumor response induced by interleukin (IL)-4 were investigated by quantitative polymerase chain reaction. The growth of Chinese hamster ovary cells transfected to produce IL-4 (CHO.T1) was strongly suppressed when cells were injected intraperitoneally into nude mice and this suppression was accompanied by the rapid accumulation of activated macrophages. Peritoneal cells from such mice were analyzed for mRNA induced by IL-4. Correlating with a high local IL-4 concentration, several transcripts were found to be up-regulated during the early phase of the anti-tumor response [IL-4 receptor, IL-5, tumor necrosis factor (TNF) and interferon (IFN)-gamma]. The functional relevance of the elevated mRNA levels was analyzed by injection of CHO.T1 cells together with anti-cytokine monoclonal antibodies (mAb). In contrast to anti-IL-5 and anti-TNF mAb, an anti-IFN-gamma mAb interfered with the anti-tumor response demonstrating the involvement of IFN-gamma during the IL-4-induced tumor suppression. Tumor growth in anti-IFN-gamma mAb-treated animals was significantly delayed in comparison to anti-IL-4 mAb-treated mice, suggesting that IFN-gamma-independent effector cells may also be involved.

Animals↗
PubMed1991

Combined use of spin-echo and gradient-echo MR-imaging in cervical disk disease. Comparison with myelography and intraoperative findings.

In a prospective study we compared the diagnostic value of combined gradient-echo (GE) and spin-echo (SE) MR imaging with cervical myelography in 30 patients with clinical signs and symptoms of cervical radiculopathy and/or myelopathy due to disk disease. Only patients who subsequently underwent surgery (anterior interbody approach) were included. By means of MRI the clinically relevant segment was identified in all cases, by means of myelography in all but two patients. Using both spin-echo (SE) and gradient-echo (GE) techniques it was possible to differentiate between bone tissue and disk material by MR in all but one. It is concluded that MRI-especially gradient-echo imaging is a viable alternative to cervical myelography and can be considered as an initial diagnostic procedure for suspected cervical disk disease.

Adult↗