OPUS 4 | Universitätspublikationen

"A system for the intracellular generation of triple helix-forming oligonucleotides (TFOs) and the sequence-specific inhibition of human MCP-1 gene expression" (2005)

Kordula Kautz

Chemokines play a key role in the cellular infiltration of inflamed tissue. They are released by a wide variety of cell types during the initial phase of host response to injury, allergens, antigens, or invading microorganisms, and selectively attract leukocytes to inflammatory foci, inducing both migration and activation. Monocyte chemoattractant protein-1 (MCP-1), a member of the CC chemokine superfamily, functions in attracting monocytes, T lymphocytes, and basophils to sites of inflammation. MCP-1 is produced by monocytes, fibroblasts, vascular endothelial cells and smooth muscle cells in response to various stimuli such as tumour necrosis factor-a (TNF-a), interferon-g (IFN-g), and interleukin-1b (IL-1b). It also plays an important role in the pathogenesis of chronic inflammation, and overexpression of MCP-1 has been implicated in diseases including glomerulonephritis and rheumatoid arthritis. Oligonucleotide-directed triple helix formation offers a means to target specific sequences in DNA and interfere with gene expression at the transcriptional level. Triple helix-forming oligonucleotides (TFOs) bind to homopurine/homopyrimidine sequences, forming a stable, sequence-specific complex with the duplex DNA. Purine-rich sequences are frequent in gene regulatory regions and TFOs directed to promoter sequences have been shown to prevent binding of transcription factors and inhibit transcription initiation and elongation. Exogenous TFOs that bind homopurine/ homopyrimidine DNA sequences and form triple-helices can be rationally designed, while the intracellular delivery of single-stranded RNA TFOs has not been studied in detail before. In this study, expression vectors were constructed which directed transcription of either a 19 nt triplex-forming pyrimidine CU-TFO sequence targeting the human MCP-1 or two different 19 nt GU- or CA-control sequences, respectively, together with the vector encoded hygromycin resistance mRNA as one fusion transcript. HEK 293 cells were stable transfected with these vectors and several TFO and control cell lines were generated. Functional relevant triplex formation of a TFO with a corresponding 19 bp GC-rich AP-1/SP-1 site of the human MCP-1 promoter was shown. Binding of synthetic 19 nt CUTFO to the MCP-1 promoter duplex was verified by triplex blotting at pH 6.7. Underlining binding specificity, control sequences, including the GU- and CA-sequence, a TFO containing one single mismatch and a MCP-1 promoter duplex containing two mismatches, did not participate in triplex formation. Establishing a magnetic capture technique with streptavidin microbeads it was verified that at pH 7.0 the 19 nt TFO embedded in a 1.1 kb fusion transcript binds to a plasmid encoded MCP-1 promoter target duplex three times stronger than the controls. Finally, cell culture experiments revealed 76 ± 10.2% inhibition of MCP-1 protein secretion in TNF-a stimulated CU-TFO harboring cell lines and up to 88% after TNF-a and IFN-g costimulation in comparison to controls. Expression of interleukin-8 (IL-8) as one TNF-a inducible control gene was not affected by CU-TFO, demonstrating both highly specific and effective chemokine gene repression. Furthermore, another chemokine target, regulated upon activation normal T cell expressed and secreted (RANTES), which plays an essential role in inflammation by recruiting T lymphocytes, macrophages and eosinophils to inflammatory sites, was analysed using the triplex approach. A 28 nt TFO was designed targeting the murine RANTES gene promoter, and gel mobility shift assays demonstrated that the phosphodiester TFO formed a sequencespecific triplex with the double-stranded target DNA with a Kd of 2.5 x 10-7 M. It was analysed whether RANTES expression could be inhibited at the transcriptional level testing the TFO in two different cell lines, T helper-1 lymphocytes and brain microvascular endothelial cells (bend3 cells). Although there was a sequence-specific binding of the TFO detectable in the gel shift assays, there was no inhibitory effect of the exogenously added and phosphorothioate stabilised TFO on endogenous RANTES gene expression visible. Additionally, the small interfering RNA (siRNA) approach was tested as another strategy to inhibit expression of the pro-inflammatory chemokines MCP-1 and RANTES. Two different methods were pursuit, describing transient transfection with vector derived and synthetic siRNA. The vector pSUPER containing the siRNA coding sequence was used to suppress endogenous MCP-1 in HEK 293 cells. An empty vector without RNA sequence served as a control. Inhibition due to the siRNA was measured in stimulated and unstimulated cells. In TNF-a stimulated cells MCP-1 protein synthesis was decreased by 35 ± 11% after siRNA transfection. Using a synthetic double-stranded siRNA, the TNF-a induced MCP-1 protein secretion could be successfully inhibited about 62.3 ± 10.3% in HEK 293 cells, indicating that the siRNA is functional in these cells to suppress chemokine expression. The siRNA approach targeting murine RANTES in Th1 cells and b-end3 cells revealed no inhibition of endogenous gene expression. Gene therapy approaches rely on efficient transfer of genes to the desired target cells. A wide variety of viral and nonviral vectors have been developed and evaluated for their efficiency of transduction, sustained expression of the transgene, and safety. Among them, lentiviruses have been widely used for gene therapy applications. In order to improve the delivery of TFOs or siRNAs into the target cells, cloning of the lentiviral transfer vector SEW, the production of lentiviral particles by transient transfection were performed with the aim to generate lentiviral vector-derived TFOs in further experiments. Here, Th1 cells were transduced with infectious lentiviral particles and transduction efficacy was measured. Transduction efficacy higher than 82% could be achieved using the lentiviral vector SEW, opening optimal possibilities for the TFO or siRNA approach.

"And God Laughed ...": Indeterminacy, Self-Reference, and Paradox in Law (1989)

Gunther Teubner

"Antiflow" of antiprotons in heavy ion collisions (1994)

André Jahns Christian Spieles Heinz Sorge Horst Stöcker Walter Greiner

In the framework of the relativistic quantum dynamics approach we investigate antiproton observables in Au-Au collisions at 10.7A GeV. The rapidity dependence of the in-plane directed transverse momentum p(y) of p's shows the opposite sigh of the nucleon flow, which has indeed recently been discovered at 10.7A GeV by the E877 group. The "antiflow" of p's is also predicted at 2A GeV and at 160 A GeV and appears at all energies also for pi's and K's. These predicted p anticorrelations are a direct proof of strong p annihilation in massive heavy ion reactions.

"Differential regulation of IL-12p70 and IL-23 in murine dendritic cells" (2009)

Cornelia Richter

Dendritic cells are the sentinels between the innate and the adaptive immunity. They are professionals that capture invading pathogens, recognize specific microbial structures and induce naïve T lymphocytes to polarize into a specific T cell subset. To initiate the T cell polarization DCs secrete cytokines which are induced upon Toll-like receptor activation by microbial structures. The recognition of these structures and the discrimination between non-self and self structures by TLRs is fine tuned, but under defined circumstances deregulation of immune responses appears. Consequently, this can result in immune disorders such as autoimmunity, chronic inflammatory diseases or cancer. In this thesis the investigations are focused on the regulation of the IL-12 family members IL-12p70 and IL-23 in DCs. The objective was to investigate three different endogenous and exogenous factors that regulate IL-12p70 or IL-23. In the first part Selenium, an essential trace element and important factor in several metabolic pathways including the cellular redox status and reactive oxygen species (ROS) dependent signaling was applied as supplement in immature Langerhans cell culture. Because Selenium also plays a role in the immune system the TLR-induced IL-23 production of the DCs upon Selenium treatment was analyzed. In the immature Langerhans cell line XS-52 the strongest inducer of IL-23 was TLR4 ligand LPS. Furthermore increased levels of TLR4-induced IL-23 in cells treated with Selenium were detected in a concentration dependent manner. Whereas the IL-23 subunit p40 was upregulated upon Selenium treatment the second subunit p19 was completely unaffected. This effect was detected on mRNA and protein level. In addition, as expected, IFN-gamma inhibited the TLR4-induced IL-23 secretion of both, Selenium treated and untreated cells. In the second part of this thesis p47phox, an organizing protein of the NADPH oxidase was analyzed regarding its potential to regulate IL-12p70 and/or IL-23 secreted by different DC subtypes. Since it was demonstrated that p47phox deficiency is associated with enhanced autoimmunity and chronic inflammation we wanted to prove whether it has a function in addition to that within the NADPH oxidase. We found some hints that p47phox may be interact with proteins of the TLR signaling pathway and thus we hypothesized that p47phox may have a function for the regulation of TLR-mediated cytokine production in DCs. In several experiments with DCs from the spleen of different p47phox deficient mice we detected an increased production of TLR9-induced IL-12p70 compared to wild type cells. In contrast TLR4 stimulation with LPS displayed no significant differences between p47phox deficient and wild type cells. In spleen cells IL-23 was not detected. Confirming the results of this new negative feedback by p47phox on IL-12p70 rats, with a single nucleotide polymorphism in the p47phox gene, were investigated. Interestingly this polymorphism is located in the phosphorylation site of IRAK4, an important kinase in the TLR pathway. In rats with a methionine residue at this position in the p47phox protein enhanced IL-12p70 level were found, compared to the rats with threonine, which can be phosphorylated by IRAK4. All analyzed mice and rats have defects in the NADPH oxidase function due to a non functional p47phox protein which results in a defective ROS production. To determine whether the observed negative feedback mechanism is connected to the lack of ROS production experiments with gp91phox deficient mice, which also have a defective NADPH oxidase function, were performed. In several experiments the enhanced IL-12p70 production in cells from p47phox deficient mice could be confirmed, but no differences between gp91phox deficient and wild type mice have been observed. In further studies was found that the inhibition of the NADPH oxidase function did not alter the negative feedback on TLR9-induced IL-12p70 secretion by p47phox. Interestingly upon treatment with the inhibitor a feedback mechanism in wild type cells also after TLR4 stimulation was observed. Hence, blocking a ROS-dependent TLR4 pathway by the inhibitor uncovered the LPS induced ROS-independent pathway of the TLR4 signaling. These findings strongly approve a NADPH oxidase/ROS-independent function of p47phox in DCs. Because splenic DCs do not secrete IL-23, in vitro differentiated DCs from the bone marrow were investigated regarding the negative feedback mechanism. In DCs from p47phox deficient mice, differentiated with GM-CSF, the upregulation of IL-12p70 was confirmed, whereas Flt3-L cultured DCs did not display the negative feedback. In contrast to IL-12p70 no difference for the IL-23 production between wild type and p47phox deficient cells has been detected. Thus, we concluded that IL-23 production is not regulated by p47phox. IL-12p70 is the major cytokine in the Th1 polarization whereas IL-23 is important for the maintenance and survival of Th17 cells. To prove whether the regulation of IL-12p70 influences the T cell response immunization experiments closely resembling the classical DTH-like protocols were performed. Groups of p47phox deficient and wild type mice received either PBS, OVA alone or mixed with TLR9 ligand CpG2216 in IFA s.c. to activate and polarize naïve T cells towards Th1 or Th17 cells. After ten days isolated lymph node cells were incubated in an ELISA spot assay with or without OVA and the frequency of IFN-gamma and IL-17 producing T cells was quantified. In vitro recall of OVA immunization of wild type and p47phox deficient mice resulted in an increased IFN-gamma and IL-17 frequency in the p47phox deficient cells. The combination with CpG2216 as adjuvant and inducer of the 3rd signal enhanced the frequency of IFN-gamma and IL-17 producing T cells in wild type mice significantly. However, in p47phox deficient cells the IFN-gamma and IL-17 response, being already detectable without in vitro OVA re-stimulation, was strongly augmented upon OVA restimulation. These findings confirmed our in vitro data for IL-12p70. Hence, the data supports our hypothesis that the p47phox dependent regulation of IL-12p70 and the consequences for the T cell response is an important mechanism to prevent uncontrolled immune responses. In the last part of this thesis the immunomodulatory property of vitamin D3 on the IL-12p70 production of DCs was examined. Since it was shown that VD3 influences the differentiation and maturation of monocytes and DCs, splenic DCs from C57BL/6 and BALB/c mice were investigated regarding their IL-12p70 production after VD3 treatment. Spleen cells, stimulated with LPS or CpG2216, exhibited a decreased IL-12p70 production when treated with VD3 before stimulation phase. In contrast treatment with VD3 only during TLR stimulation had no influence on the IL-12p70 production. Since it was demonstrated that VD3 stimulates the expression of p47phox mRNA cells from p47phox deficient mice were also treated with VD3. In initial experiments only a slight inhibition of IL-12p70 has been detected in p47phox deficient cells compared to the wild type. In summary the thesis displays three different possibilities to influence the TLR-induced cytokine secretion of DCs, although with different intensities and specificities.

"Do credit rating agencies add to the dynamics of emerging market crises?" (2003)

Roman Kräussl

The experience in the period during and after the Asian crisis of 1997-98 has provoked an extensive debate about the credit rating agencies' evaluation of sovereign risk in emerging markets lending. This study analyzes the role of credit rating agencies in international finan-cial markets, particularly whether sovereign credit ratings have an impact on the financial stability in emerging market economies. The event study and panel regression results indicate that credit rating agencies have substantial influence on the size and volatility of emerging markets lending. The empirical results are significantly stronger in the case of government's downgrades and negative imminent sovereign credit rating actions such as credit watches and rating outlooks than positive adjustments by the credit rating agencies while by the market participants' anticipated sovereign credit rating changes have a smaller impact on financial markets in emerging economies. Klassifikation: E44, E47, G15. This version August 2003.

"Expectations, learning and monetary policy" : conference organized jointly by Athanasios Orphanides (Federal Reserve Board, Washington D.C.), John C. Williams (Federal Reserve Bank of San Francisco), Heinz Herrmann (Deutsche Bundesbank), and Volker Wieland (Center for Financial Studies and University of Frankfurt) on August 30 - 31, 2003 in Eltville (2004)

Conference Reader zur gemeinsam von Athansios Orphanides (Federal Reserve Board, Washington D.C.), John C. Williams (Federal Reserve Bank of San Francisco), Heinz Hermann (Deutsche Bundesbank), und Volker Wieland (Center for Financial Studies and Goethe University Frankfurt) organisierten Konferenz, die vom 30. - 31. August, 2003 in Eltville stattgefunden hat. Inhaltsverzeichnis: * Volker Wieland (Director Center for Financial Studies): Foreword * Hans Georg Fabritius (Member of the Executive Board of the Deutsche Bundesbank): Opening Remarks * Charles Goodhart (Norman Sosnow Professor of Banking and Finance at the London School of Economics and External Member of the Bank of England's Monetary Policy Commitee): After Dinner Speech * Paper Abstracts * List of Participants

"Foreign banks and the regulation of financial holding companies" (2002)

Michael Gruson

It is an established policy in the United States to separate commercial banking (the business of taking deposits and making commercial loans) from other commercial activities. The separation of banking and commercial activities is achieved by federal and state banking laws, which enumerate the powers that banks may exercise, the activities that banks may engage in, and the investments that banks may lawfully make, and expressly exclude banks from certain activities or relationships. Some of these provisions could be circumvented if a nonbank company could carry on banking activities through a banking subsidiary and nonbanking activities either itself or through a nonbanking subsidiary.

"Irresponsible lending" with a better informed lender (2009)

Roman Inderst

We present a simple model of personal finance in which an incumbent lender has an information advantage vis-a-vis both potential competitors and households. In order to extract more consumer surplus, a lender with sufficient market power may engage in "irresponsible"lending, approving credit even if this is knowingly against a household’s best interest. Unless rival lenders are equally well informed, competition may reduce welfare. This holds, in particular, if less informed rivals can free ride on the incumbent’s superior screening ability.

"It´s hard for me to cope with life. With the dead it´s easier" : Dina Wardi´s book about the children of the Holocaust (2000)

Frederik van Gelder

"Modus scolipetarum et reportisarum" : Pronuntiatio and Fifteenth-Century University Hands (1988)

Gerhardt Powitz

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