Die Rolle von Peroxisome Proliferator-Activated Receptor gamma Coactivator-1alpha (PGC-1α) in der Pathogenese der Sklerodermie

1) Background: Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology, characterized by vascular damage, autoimmunity and progressive fibrosis of the skin and internal organs. The mechanisms responsible for fibroblasts being falsely activated have only been depicted inadequately so far, effective therapeutic concepts for SSc are lacking. Recently, PGC-1α has been identified as an important transcriptional coactivator for the PDGF pathway. Since profibrotic cytokines such as PDGF and TGF-β are key players in activation and stimulation of fibroblasts, and since it has furthermore been shown that inhibiting PDGF results in strong antifibrotic effects, the present thesis aimed to investigate whether PGC-1α mediates profibrotic effects of these cytokines, and whether inhibition of PGC-1α would evoke an antifibrotic response, possibly posing a future therapeutic approach. 2) Methods: Different stages and phenotypes of SSc were represented by mouse models. Dermal thickness and inflammation cells were visualized histologically, PGC-1α, myofibroblasts and macrophages immunohistochemically. Hydroxyproline assay was utilized for quantification of collagen protein. In cell culture experiments human dermal fibroblasts were analyzed after stimulation with PDGF and TGF-β. Real-time PCR was used for the quantification of PGC-1α mRNA levels. 3) Results: Immunohistochemistry and real-time PCR analyses in SSc patients and various mouse models could demonstrate that PGC-1α expression was upregulated in fibrotic skin areas. This overexpression was seen both in early, inflammation-dependent and in late, largely inflammation-independent stages of SSc. Loss-of-function studies revealed that PGC-1α takes a central part in mediating profibrotic effects in vivo. In this case, PGC-1α-knockout mice were protected from the development of an experimentally induced fibrotic phenotype. Profibrotic effects – such as increased dermal thickness, upregulated collagen production as well as elevated numbers of myofibroblasts, inflammation cells and macrophages – were attenuated considerably. Cell culture experiments with human fibroblasts from normal and SSc-lesional skin suggested the conclusion that PGC-1α expression can be characterized as an exponential function of the duration of stimulation with profibrotic cytokines. 4) Conclusion: With regard to the potent antifibrotic effects, evoked by an inhibition of PGC-1α in an animal model, further characterization of the role of PGC-1α in the pathogenesis of fibrosis will be of great interest. Especially a more in-depth evaluation of the regulatory mechanisms between PGC-1α and profibrotic cytokines will be valuable. Based on the present results, PGC-1α may represent a possible target for future pharmacological approaches to the therapy of SSc.1) Wissenschaftlicher Hintergrund: Die Sklerodermie (SSc) ist eine Kollagenose unklarer Ätiologie, die durch Gefäßschäden, Autoimmunität und die progressive Fibrosierung der Haut und inneren Organe gekennzeichnet ist. Die der Fehlaktivierung von Fibroblasten zugrunde liegenden Mechanismen sind bisher nur unzureichend aufgeklärt, antifibrotische Therapiekonzepte für SSc fehlen. PGC-1α wurde kürzlich als ein wichtiger Transkriptions-Koaktivator im Signalweg von PDGF identifiziert. Da profibrotische Zytokine wie PDGF und TGF-β in der Aktivierung und Stimulation von Fibroblasten von entscheidender Bedeutung sind und darüber hinaus bereits gezeigt wurde, dass eine Hemmung von PDGF stark antifibrotisch wirkt, war das Ziel der vorliegenden Arbeit, zu untersuchen, ob PGC-1α die profibrotischen Effekte dieser Zytokine vermittelt und ob eine Hemmung von PGC-1α eine antifibrotische Antwort evoziert und somit einen möglichen zukünftigen Therapieansatz darstellen könnte. 2) Methoden: Unterschiedliche Stadien und Phänotypen von SSc wurden durch Mausmodelle abgebildet. Hautdicke und Entzündungszellen wurden histologisch, PGC-1α, Myofibroblasten und Makrophagen immunhistochemisch visualisiert. Die Quantifizierung der Proteinmenge von Kollagen erfolgte mithilfe des Hydroxyprolin-Assays. In zellkulturellen Experimenten wurden humane dermale Fibroblasten nach Stimulation mit PDGF und TGF-β untersucht. PGC-1α-mRNA-Level wurden durch Real-Time PCR quantifiziert. 3) Ergebnisse: Mittels Immunhistochemie und Real-Time PCR konnte für SSc-Patienten und verschiedene Mausmodelle gezeigt werden, dass PGC-1α in fibrotischen Hautarealen verstärkt exprimiert wird. Diese Überexpression wurde dabei sowohl in frühen, stark entzündungsabhängigen als auch in späten, von Entzündungsvorgängen weitgehend unabhängigen Stadien von SSc angetroffen. In Loss-of-function-Studien wurde nachgewiesen, dass PGC-1α bei der Vermittlung profibrotischer Effekte in vivo eine zentrale Stellung einnimmt. Dabei schützte ein Knockout von PGC-1α vor der Entwicklung eines experimentell hervorgerufenen fibrotischen Phänotyps. Profibrotische Effekte – etwa die Zunahme der Hautdicke, die gesteigerte Kollagen-Produktion sowie erhöhte Anzahlen von Myofibroblasten, Entzündungszellen und Makrophagen – wurden dabei deutlich abgeschwächt. Zellkultur-Experimente mit humanen Fibroblasten aus normaler und SSc-läsionaler Haut legten den Schluss nahe, dass die PGC-1α-Expression als eine exponentielle Funktion von der Stimulationsdauer mit profibrotischen Zytokinen charakterisiert werden kann. 4) Schlussfolgerung: Im Hinblick auf die potenten antifibrotischen Effekte, die durch Hemmung von PGC-1α im Tiermodell hervorgerufen wurden, ist die weitergehende Charakterisierung der Rolle von PGC-1α in der Pathogenese der Fibrose von großem Interesse. Insbesondere eine detailliertere Evaluation der Regulationsmechanismen zwischen PGC-1α und profibrotischen Zytokinen wird hierbei nützlich sein. Auf der Grundlage der vorliegenden Ergebnisse stellt PGC-1α ein mögliches Ziel für zukünftige pharmakologische Ansätze zur Therapie der SSc dar

Got milk? Breastfeeding and milk analysis of a mother on chronic hemodialysis

Purpose: Women on dialysis rarely become pregnant. However, the overall rate of successful pregnancies is increasing in this patient population and breastfeeding becomes an option for mothers on dialysis. In this study we performed a systematic breast milk composition analysis of a mother on chronic hemodialysis (HD). Methods: Specimens of breast milk and blood were collected in regular intervals before and after HD from a 39-year old woman starting on day 10 postpartum. Samples were analyzed for electrolytes, retention solutes, nutrients and other laboratory measurements. Breast milk samples from low-risk mothers matched for postpartum age were used as controls. Results: Significantly higher levels of creatinine and urea were found in pre-HD breast milk when compared to post-HD. A similar post-dialytic decrease was only found for uric acid but not for any other investigated parameter. Conversely, sodium and chloride were significantly increased in post-HD samples. Compared to controls creatinine and urea were significantly higher in pre-HD samples while the difference remained only significant for post-HD creatinine. Phosphate was significantly lower in pre- and post-HD breast milk when compared to controls, whereas calcium showed no significant differences. In terms of nutrient components glucose levels showed a strong trend for a decrease, whereas protein, triglycerides and cholesterol did not differ. Similarly, no significant differences were found in iron, potassium and magnesium content. Conclusion: To the best of our knowledge this is the first report on a breastfeeding mother on chronic dialysis. Although we found differences in creatinine, urea, sodium, chloride and phosphate, our general analysis showed high similarity of our patient’s breast milk to samples from low-risk control mothers. Significant variations in breast milk composition between pre- and post-HD samples suggest that breastfeeding might be preferably performed after dialysis treatment. In summary, our findings indicate that breastfeeding can be considered a viable option for newborns of mothers on dialysis

Got milk? Breastfeeding and milk analysis of a mother on chronic hemodialysis

Purpose: Women on dialysis rarely become pregnant. However, the overall rate of successful pregnancies is increasing in this patient population and breastfeeding becomes an option for mothers on dialysis. In this study we performed a systematic breast milk composition analysis of a mother on chronic hemodialysis (HD). Methods: Specimens of breast milk and blood were collected in regular intervals before and after HD from a 39-year old woman starting on day 10 postpartum. Samples were analyzed for electrolytes, retention solutes, nutrients and other laboratory measurements. Breast milk samples from low-risk mothers matched for postpartum age were used as controls. Results: Significantly higher levels of creatinine and urea were found in pre-HD breast milk when compared to post-HD. A similar post-dialytic decrease was only found for uric acid but not for any other investigated parameter. Conversely, sodium and chloride were significantly increased in post-HD samples. Compared to controls creatinine and urea were significantly higher in pre-HD samples while the difference remained only significant for post-HD creatinine. Phosphate was significantly lower in pre- and post-HD breast milk when compared to controls, whereas calcium showed no significant differences. In terms of nutrient components glucose levels showed a strong trend for a decrease, whereas protein, triglycerides and cholesterol did not differ. Similarly, no significant differences were found in iron, potassium and magnesium content. Conclusion: To the best of our knowledge this is the first report on a breastfeeding mother on chronic dialysis. Although we found differences in creatinine, urea, sodium, chloride and phosphate, our general analysis showed high similarity of our patient’s breast milk to samples from low-risk control mothers. Significant variations in breast milk composition between pre- and post-HD samples suggest that breastfeeding might be preferably performed after dialysis treatment. In summary, our findings indicate that breastfeeding can be considered a viable option for newborns of mothers on dialysis

A trapped single ion inside a Bose-Einstein condensate

Improved control of the motional and internal quantum states of ultracold neutral atoms and ions has opened intriguing possibilities for quantum simulation and quantum computation. Many-body effects have been explored with hundreds of thousands of quantum-degenerate neutral atoms and coherent light-matter interfaces have been built. Systems of single or a few trapped ions have been used to demonstrate universal quantum computing algorithms and to detect variations of fundamental constants in precision atomic clocks. Until now, atomic quantum gases and single trapped ions have been treated separately in experiments. Here we investigate whether they can be advantageously combined into one hybrid system, by exploring the immersion of a single trapped ion into a Bose-Einstein condensate of neutral atoms. We demonstrate independent control over the two components within the hybrid system, study the fundamental interaction processes and observe sympathetic cooling of the single ion by the condensate. Our experiment calls for further research into the possibility of using this technique for the continuous cooling of quantum computers. We also anticipate that it will lead to explorations of entanglement in hybrid quantum systems and to fundamental studies of the decoherence of a single, locally controlled impurity particle coupled to a quantum environment

Development of an Optimized LC-MS Method for the Detection of Specialized Pro-Resolving Mediators in Biological Samples

The cardioprotective and anti-inflammatory effects of long chain omega-3 polyunsaturated fatty acids (n3 PUFA) are believed to be partly mediated by their oxygenated metabolites (oxylipins). In the last two decades interest in a novel group of autacoids termed specialized pro-resolving mediators (SPMs) increased. These are actively involved in the resolution of inflammation. SPMs are multiple hydroxylated fatty acids including resolvins, maresins, and protectins derived from the n3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as well as lipoxins derived from arachidonic acid (ARA). In the present paper, we developed an LC-MS/MS method for a comprehensive set of 18 SPMs derived from ARA, EPA, and DHA and integrated it into our targeted metabolomics platform. Quantification was based on external calibration utilizing five deuterated internal standards in combination with a second internal standard for quality assessment of sample preparation in each sample. The tandem mass spectrometric parameters were carefully optimized for sensitive and specific detection. The influence of source parameters of the used AB Sciex 6500 QTRAP instrument as well as electronic parameters and the selection of transitions are discussed. The method was validated/characterized based on the criteria listed in the European Medicines Agency (EMA) guideline on bioanalytical method validation and method performance is demonstrated regarding recovery of internal standards (between 78 ± 4% and 87 ± 3% from 500 μL of human serum) as well as extraction efficacy of SPMs in spiked plasma (intra-day accuracy within ±20 and ±15% at 0.1 and 0.3 nM in plasma, respectively). Based on the lower limit of quantification of 0.02–0.2 nM, corresponding to 0.18–2.7 pg on column, SPMs were generally not detectable/quantifiable in plasma and serum supporting that circulating levels of SPMs are very low, i.e., <0.1 nM in healthy subjects. Following septic shock or peritonitis, SPMs could be quantified in the samples of several patients. However, in these studies with a small number of patients no clear correlation with severity of inflammation could be observed

Endogenous Renal Adiponectin Drives Gluconeogenesis Through Enhancing Pyruvate and Fatty Acid Utilization

Adiponectin is a secretory protein, primarily produced in adipocytes. However, low but detectable expression of adiponectin can be observed in cell types beyond adipocytes, particularly in kidney tubular cells, but its local renal role is unknown. We assessed the impact of renal adiponectin by utilizing male inducible kidney tubular cell-specific adiponectin overexpression or knockout mice. Kidney-specific adiponectin overexpression induces a doubling of phosphoenolpyruvate carboxylase expression and enhanced pyruvate-mediated glucose production, tricarboxylic acid cycle intermediates and an upregulation of fatty acid oxidation (FAO). Inhibition of FAO reduces the adiponectin-induced enhancement of glucose production, highlighting the role of FAO in the induction of renal gluconeogenesis. In contrast, mice lacking adiponectin in the kidney exhibit enhanced glucose tolerance, lower utilization and greater accumulation of lipid species. Hence, renal adiponectin is an inducer of gluconeogenesis by driving enhanced local FAO and further underlines the important systemic contribution of renal gluconeogenesis

Signal Transmission in the Auditory System

Contains table of contents for Section 3, an introduction and reports on six research projects.National Institutes of Health Grant RO1-DC-00194-11National Institutes of Health Grant PO1-DC00119 Sub-Project 1National Institutes of Health Grant F32-DC00073-3National Institutes of Health Contract P01-DC00119National Institutes of Health Grant R01 DC00238National Institutes of Health Grant P01-DC00119National Institutes of Health Grant T32-DC00038National Institutes of Health Contract P01-DC00361National Institutes of Health Grant R01-DC00235National Institutes of Health Contract NO1-DC2240

Conjugative Plasmids of Neisseria gonorrhoeae

Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1α, β, γ, δ and ε subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between N. gonorrhoeae strains, but did not enhance transfer of a genetic marker

Investigation of Association between PFO Complicated by Cryptogenic Stroke and a Common Variant of the Cardiac Transcription Factor GATA4

Patent foramen ovale (PFO) is associated with clinical conditions including cryptogenic stroke, migraine and varicose veins. Data from studies in humans and mouse suggest that PFO and the secundum form of atrial septal defect (ASDII) exist in an anatomical continuum of septal dysmorphogenesis with a common genetic basis. Mutations in multiple members of the evolutionarily conserved cardiac transcription factor network, including GATA4, cause or predispose to ASDII and PFO. Here, we assessed whether the most prevalent variant of the GATA4 gene, S377G, was significantly associated with PFO or ASD. Our analysis of world indigenous populations showed that GATA4 S377G was largely Caucasian-specific, and so subjects were restricted to those of Caucasian descent. To select for patients with larger PFO, we limited our analysis to those with cryptogenic stroke in which PFO was a subsequent finding. In an initial study of Australian subjects, we observed a weak association between GATA4 S377G and PFO/Stroke relative to Caucasian controls in whom ASD and PFO had been excluded (OR = 2.16; p = 0.02). However, in a follow up study of German Caucasians no association was found with either PFO or ASD. Analysis of combined Australian and German data confirmed the lack of a significant association. Thus, the common GATA4 variant S377G is likely to be relatively benign in terms of its participation in CHD and PFO/Stroke