mTOR-Inhibierung und Gentherapie in chronischen Nierenerkrankungsmodellen

In the chronic allograft nephropathy and chronic kidney diseases fibrosis progresses, that means it leads to deposition of extracellular matrix and therefore to changes in the structure of tissue. The most common used calcineurin inhibitors are nephrotoxic, pro-fibrotic and kidney function can lead to aggravation. Thus, in chronic kidney disease their applicability is limited. A new category of immunsuppressives are the mTOR inhibitors. The mTOR inihibitors are anti-fibrotic, anti-proliferative, anti-angiogenetic and not nephrotoxic. Therefore, using the examples of the chronic anti-Thy1 nephritis and the streptozotocin-induced diabetes model in rats we investigated, how mTOR-inhibitors (Everolimus, Sirolimus) are suitable for an anti-fibrotic therapy and how they influence the progress of disease. The model of chronic anti-Thy1 nephritis in rats shows some features of the human situation of an mesangial proliferative glomerulonephritis, whose principal form is the IgA nephropathy. The everolimus therapy was started after the acute phase of the anti-Thy1 model and the study showed, that everolimus enhanced the time course of the anti-Thy1 model. Sixteen weeks of everolimus treatment led to a reduced interstitial fibrosis, FSGS-lesions, proteinuria, glomerular hypertrophy and influx of macrophages/monocytes as well as deposition of fibronectin. In the second chronic model, the influence of sirolimus and cyclosporin A on the streptozotocin-induced diabetic nephropathy in rats was investigated. By now the diabetic nephropathy is the most common renal disease, which ends in a renal replacement therapy. A further actual problem is the new-onset diabetes after transplantation, because it is caused by administration of calcineurin inhibitors and glucocorticoids. mTOR inhibitors are just as well diabetic like calcineurin inhibitors, but by their positive features they could reduced the diabetic complications like diabetic nephropathy. Nineteen weeks after diabetes induction in the rats the sirolimus therapy led to a reduced glomerular hypertrophy, glomerular and interstitial fibrosis, podocyte lost, deposition of glomerular and cortical fibronectin and deposition of glomerular laminin compared to the diabetic control group. Furthermore, sirolimus led to a decreased expression of glomerular TGF-β1 and 2 and glomerular p-smad 2/3, influx of macrophages/monocytes and expression of VEGF. In contrast cyclosporine A was incapable to intervene the process of diabetic nephropathy. Cyclosporine A induced an increase in deposition of cortical fibronectin and enhanced glomerular fibrosis. The last part of my work deals with gene therapy. Many kidney diseases are conditional upon genetic reasons. For these diseases the gene therapy would be a good approach. In cells the absent or mutated gene would be contributed by a functional copy, some diseases could be ameliorated or cured. For our gene therapy we choosed factor h as example. It plays an important role in the immune system, especially the protection of the somatic cells during immune responses. Patients with a deficiency of factor h or a mutation in factor h gene can be develop a membranoproliferative glomerulonephritis type II or the hemolytic-uremic syndrome. Factor h is generated mainly in the liver, but also in endothelial cells, glomerular mesangial cells and podocytes of rodents. The concentration of factor h is easily determined in the blood. To perform a safe gene therapy it is recommended to use a controllable system to regulate the formed gene product. The mostly used controllable system is the tetracycline inducible system (tet-system). There are two types of the tet-system, tet-on and tet-off. For our study we decided to use tet-off- system, in the absence of tetracycline/ doxicycline the continued expression of factor h is guaranteed. If tetracycline/ doxicycline is administrated, than the expression of factor h falls down to its background activity, which is ideally slight. Furthermore, we determined for a non-viral system to avoid complications of viral gene therapy. However, all components should be combined in one vector. Over different intermediate steps we were successful to generate a tet-controlled vector with factor h gene. In in vitro experiments the vector was able to transfect HEK293 cells. The regulation of the vector with tetracycline and doxicycline was tested. After 48 h of transfection a dose-dependent regulation of factor h under tetracycline administration was observed. A regulation under doxicycline administration is more difficult, because it is not so adjustable to control like under tetracycline administration.In der chronischen Allograft-Nephropathie und den chronischen Nierenerkrankungen kommt es zur Fibrosierung, d.h. es führt zu Ablagerungen von extrazellulärer Matrix und somit zu Strukturveränderungen des Gewebes. Die bisher meist eingesetzten Calcineurin-Inhibitoren sind jedoch nephrotoxisch, pro-fibrotisch und können zu einer Verschlechterung der Nierenfunktion führen. Daher ist ihre Anwendbarkeit bei chronischen Nierenerkrankungen begrenzt. Eine neue Klasse an Immunsuppressiva stellen die mTOR-Inhibitoren dar. Die mTOR-Inhibitoren sind anti-fibrotisch, anti-proliferativ, anti-angiogenetisch und nicht nephrotoxisch. Daher untersuchten wir am Beispiel der chronischen Anti-Thy1 Nephritis und der Streptozotozin-induzierten diabetischen Nephropathie der Ratte, inwiefern sich mTOR-Inhibitoren (Everolimus, Sirolimus) für eine anti-fibrotische Therapie eignen und wie sie den Krankheitsverlauf beeinflussen. Das Modell der chronischen Anti-Thy1 Nephritis der Ratte spiegelt die Situation einer humanen mesangialen proliferativen Glomerulonephritis wider, deren Hauptform die IgA-Nephopathie ist. Erst nach Abschluss der akuten proliferativen Phase des Anti-Thy1 Modells wurde mit der Everolimus-Therapie begonnen und es zeigte sich, dass Everolimus den Krankheitsverlauf im chronischen Anti-Thy1 Modell verbesserte. Everolimus führte nach 16 Wochen zu einer Verminderung von interstitieller Fibrose, FSGS-Läsionen, Proteinurie, glomerulärer Hypertrophie und Einwanderung von Makrophagen/Monozyten als auch Ablagerungen von Fibronektin. Im zweiten chronischen Modell wurde der Einfluss von Sirolimus und Ciclosporin A auf die STZ-induzierte diabetische Nephropathie im Rattenmodell untersucht. Die diabetische Nephropathie ist mittlerweile die häufigste Nierenerkrankung, welche in einer Nierenersatztherapie endet. Auch der neuentstehende (new-onset) Diabetes nach erfolgreicher Transplantation stellt ein großes Problem dar, denn er wird meist durch den Einsatz von Calcineurin-Inhibitoren und Glucocorticoiden hervorgerufen. Zwar sind die mTOR-Inhibitoren ebenso diabetogen, aber durch ihre anderen positiven Eigenschaften könnten sie die diabetischen Komplikationen mindern. Neunzehn Wochen nach Diabetesinduktion zeigte sich unter Sirolimus-Behandlung eine verbesserte glomeruläre Hypertrophie, verminderte glomeruläre und interstitielle Fibrose, geringerer Podozytenverlust, reduzierte Ablagerung von Fibronektin im Glomerulus und Kortex und verminderte Ablagerung an glomerulärem Laminin im Vergleich zur diabetischen Kontrollgruppe. Zudem führte Sirolimus zu einer geringeren Expression an glomerulärem TGF-β1 und 2 und weniger p-Smad 2/3 positiven Zellen im Glomerulus, vermindertem Makrophagen- und Monozyteneinstrom und geringerer Expression an VEGF. Im Gegensatz dazu war Ciclosporin A meist nicht in der Lage in den Prozess der diabetischen Nephropathie einzugreifen. Ciclosporin A führte sogar zu erhöhter Fibronektin-Ablagerung im Kortex und vermehrter glomerulärer Fibrose. Der letzte Teil meiner Doktorarbeit befasst sich mit der Gentherapie. Viele Nierenerkrankungen sind auch genetisch bedingt. Für diese Art von Erkrankungen wäre die Gentherapie ein guter Ansatzpunkt. Denn würde in die Zelle eine intakte Kopie des fehlenden oder mutierten Gens eingebracht, könnte sich manche Erkrankung verbessern oder auch heilen lassen. Als Beispiel für eine Gentherapie wählten wir Faktor H. Es spielt eine wichtige Rolle im Immunsystem, vor allem beim Schutz der Körperzellen vor der eigenen Immunantwort. Patienten mit einem Mangel an Faktor H oder einer Mutation des Faktor H-Gens können eine membranoproliferative Glomerulonephritis Typ II oder das hämolytisch-urämische Syndrom entwickeln. Die Konzentration an Faktor H lässt sich einfach im Blut bestimmen. Um die Gentherapie sicherer zu machen, benutzt man ein regulierbares System. Die Menge an gebildetem Genprodukt lässt sich so steuern. Das wohl am meisten genutzte regulierbare Expressionsystem ist das Tetrazyklin-induzierbare (Tet)-System. Es gibt zwei Arten des Tet-Systems, Tet-On und Tet-Off. Für unser System entschieden wir uns für das Tet-Off, in dem in Abwesenheit von Tetrazyklin (Tc)/Doxizyklin (Dox) ein kontinuierliche Expression des Zielgens Faktor H gewährleistet ist. Wird Tc oder Dox hinzugegeben, fällt die Expression des Zielgens auf die Basalaktivität zurück. Zudem entschieden wir uns für ein nicht virales System, um Komplikationen der viralen Genexpression zu vermeiden und alle notwendigen Komponenten sollten auf einem Plasmid vereint sein. Über verschiedene Zwischenschritte ist es uns gelungen dieses Tet-regulierbare Plasmid mit dem Faktor H-Gen herzustellen. Die Regulation des Plasmids wurde mit Tc und Dox getestet. 48 h nach Transfektion ist eine deutliche dosisabhängige Regulation von Faktor H unter Tetrazyklin zu erkennen. Eine Regulation unter Doxizyklin ist schwieriger, denn sie ist nicht so fein steuerbar wie unter Tetrazyklin

Beyond Janus Geometry: Characterization of Flow Fields around Nonspherical Photocatalytic Microswimmers

Catalytic microswimmers that move by a phoretic mechanism in response to a self-induced chemical gradient are often obtained by the design of spherical janus microparticles, which suffer from multi-step fabrication and low yields. Approaches that circumvent laborious multi-step fabrication include the exploitation of the possibility of nonuniform catalytic activity along the surface of irregular particle shapes, local excitation or intrinsic asymmetry. Unfortunately, the effects on the generation of motion remain poorly understood. In this work, single crystalline BiVO4 microswimmers are presented that rely on a strict inherent asymmetry of charge-carrier distribution under illumination. The origin of the asymmetrical flow pattern is elucidated because of the high spatial resolution of measured flow fields around pinned BiVO4 colloids. As a result the flow from oxidative to reductive particle sides is confirmed. Distribution of oxidation and reduction reactions suggests a dominant self-electrophoretic motion mechanism with a source quadrupole as the origin of the induced flows. It is shown that the symmetry of the flow fields is broken by self-shadowing of the particles and synthetic surface defects that impact the photocatalytic activity of the microswimmers. The results demonstrate the complexity of symmetry breaking in nonspherical microswimmers and emphasize the role of self-shadowing for photocatalytic microswimmers. The findings are leading the way toward understanding of propulsion mechanisms of phoretic colloids of various shapes

Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins

Intestinal mucins trigger immune responses upon recognition by dendritic cells via protein–carbohydrate interactions. We used a combination of structural, biochemical, biophysical, and cell-based approaches to decipher the specificity of the interaction between mucin glycans and mammalian lectins expressed in the gut, including galectin (Gal)-3 and C-type lectin receptors. Gal-3 differentially recognized intestinal mucins with different O-glycosylation profiles, as determined by mass spectrometry (MS). Modification of mucin glycosylation, via chemical treatment leading to a loss of terminal glycans, promoted the interaction of Gal-3 to poly-N-acetyllactosamine. Specific interactions were observed between mucins and mouse dendritic cell-associated lectin (mDectin)-2 or specific intercellular adhesion molecule–grabbing nonintegrin-related-1 (SIGN-R1), but not mDectin-1, using a cell-reporter assay, as also confirmed by atomic force spectroscopy. We characterized the N-glycosylation profile of mouse colonic mucin (Muc)-2 by MS and showed that the interaction with mDectin-2 was mediated by high-mannose N-glycans. Furthermore, we observed Gal-3 binding to the 3 C-type lectins by force spectroscopy. We showed that mDectin-1, mDectin-2, and SIGN-R1 are decorated by N-glycan structures that can be recognized by the carbohydrate recognition domain of Gal-3. These findings provide a structural basis for the role of mucins in mediating immune responses and new insights into the structure and function of major mammalian lectins.—Leclaire, C., Lecointe, K., Gunning, P. A., Tribolo, S., Kavanaugh, D. W., Wittmann, A., Latousakis, D., MacKenzie, D. A., Kawasaki, N., Juge, N. Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins

Beyond janus geometry : characterization of flow fields around nonspherical photocatalytic microswimmers

Catalytic microswimmers that move by a phoretic mechanism in response to a self-induced chemical gradient are often obtained by the design of spherical janus microparticles, which suffer from multi-step fabrication and low yields. Approaches that circumvent laborious multi-step fabrication include the exploitation of the possibility of nonuniform catalytic activity along the surface of irregular particle shapes, local excitation or intrinsic asymmetry. Unfortunately, the effects on the generation of motion remain poorly understood. In this work, single crystalline BiVO4 microswimmers are presented that rely on a strict inherent asymmetry of charge-carrier distribution under illumination. The origin of the asymmetrical flow pattern is elucidated because of the high spatial resolution of measured flow fields around pinned BiVO4 colloids. As a result the flow from oxidative to reductive particle sides is confirmed. Distribution of oxidation and reduction reactions suggests a dominant self-electrophoretic motion mechanism with a source quadrupole as the origin of the induced flows. It is shown that the symmetry of the flow fields is broken by self-shadowing of the particles and synthetic surface defects that impact the photocatalytic activity of the microswimmers. The results demonstrate the complexity of symmetry breaking in nonspherical microswimmers and emphasize the role of self-shadowing for photocatalytic microswimmers. The findings are leading the way toward understanding of propulsion mechanisms of phoretic colloids of various shapes

Zafirlukast Is a Dual Modulator of Human Soluble Epoxide Hydrolase and Peroxisome Proliferator-Activated Receptor γ

Cysteinyl leukotriene receptor 1 antagonists (CysLT1RA) are frequently used as add-on medication for the treatment of asthma. Recently, these compounds have shown protective effects in cardiovascular diseases. This prompted us to investigate their influence on soluble epoxide hydrolase (sEH) and peroxisome proliferator activated receptor (PPAR) activities, two targets known to play an important role in CVD and the metabolic syndrome. Montelukast, pranlukast and zafirlukast inhibited human sEH with IC50 values of 1.9, 14.1, and 0.8 μM, respectively. In contrast, only montelukast and zafirlukast activated PPARγ in the reporter gene assay with EC50 values of 1.17 μM (21.9% max. activation) and 2.49 μM (148% max. activation), respectively. PPARα and δ were not affected by any of the compounds. The activation of PPARγ was further investigated in 3T3-L1 adipocytes. Analysis of lipid accumulation, mRNA and protein expression of target genes as well as PPARγ phosphorylation revealed that montelukast was not able to induce adipocyte differentiation. In contrast, zafirlukast triggered moderate lipid accumulation compared to rosiglitazone and upregulated PPARγ target genes. In addition, we found that montelukast and zafirlukast display antagonistic activities concerning recruitment of the PPARγ cofactor CBP upon ligand binding suggesting that both compounds act as PPARγ modulators. In addition, zafirlukast impaired the TNFα triggered phosphorylation of PPARγ2 on serine 273. Thus, zafirlukast is a novel dual sEH/PPARγ modulator representing an excellent starting point for the further development of this compound class

Rationale and design of the PeriOperative ISchemic Evaluation-3 (POISE-3) : a randomized controlled trial evaluating tranexamic acid and a strategy to minimize hypotension in noncardiac surgery

Altres ajuts: Canadian Institutes of Health Research (CIHR, FDN-143302); General Research Fund (14104419), Research Grant Council, Hong Kong SAR, China; National Health and Medical Research Council, Funding Schemes (NHMRC Project Grant 1162362), Australia; McMaster University Department of Medicine Career Research Award and a Physicians' Services Incorporated (PSI) Foundation Mid-Career Clinical Research Award.Background: For patients undergoing noncardiac surgery, bleeding and hypotension are frequent and associated with increased mortality and cardiovascular complications. Tranexamic acid (TXA) is an antifibrinolytic agent with the potential to reduce surgical bleeding; however, there is uncertainty about its efficacy and safety in noncardiac surgery. Although usual perioperative care is commonly consistent with a hypertension-avoidance strategy (i.e., most patients continue their antihypertensive medications throughout the perioperative period and intraoperative mean arterial pressures of 60 mmHg are commonly accepted), a hypotension-avoidance strategy may improve perioperative outcomes. Methods: The PeriOperative Ischemic Evaluation (POISE)-3 Trial is a large international randomized controlled trial designed to determine if TXA is superior to placebo for the composite outcome of life-threatening, major, and critical organ bleeding, and non-inferior to placebo for the occurrence of major arterial and venous thrombotic events, at 30 days after randomization. Using a partial factorial design, POISE-3 will additionally determine the effect of a hypotension-avoidance strategy versus a hypertension-avoidance strategy on the risk of major cardiovascular events, at 30 days after randomization. The target sample size is 10,000 participants. Patients ≥45 years of age undergoing noncardiac surgery, with or at risk of cardiovascular and bleeding complications, are randomized to receive a TXA 1 g intravenous bolus or matching placebo at the start and at the end of surgery. Patients, health care providers, data collectors, outcome adjudicators, and investigators are blinded to the treatment allocation. Patients on ≥ 1 chronic antihypertensive medication are also randomized to either of the two blood pressure management strategies, which differ in the management of patient antihypertensive medications on the morning of surgery and on the first 2 days after surgery, and in the target mean arterial pressure during surgery. Outcome adjudicators are blinded to the blood pressure treatment allocation. Patients are followed up at 30 days and 1 year after randomization. Discussion: Bleeding and hypotension in noncardiac surgery are common and have a substantial impact on patient prognosis. The POISE-3 trial will evaluate two interventions to determine their impact on bleeding, cardiovascular complications, and mortality. Trial registration: ClinicalTrials.gov NCT03505723. Registered on 23 April 2018

A phylogenetic classification of the world’s tropical forests

Knowledge about the biogeographic affinities of the world’s tropical forests helps to better understand regional differences in forest structure, diversity, composition and dynamics. Such understanding will enable anticipation of region specific responses to global environmental change. Modern phylogenies, in combination with broad coverage of species inventory data, now allow for global biogeographic analyses that take species evolutionary distance into account. Here we present the first classification of the world’s tropical forests based on their phylogenetic similarity. We identify five principal floristic regions and their floristic relationships: (1) Indo-Pacific, (2) Subtropical, (3) African, (4) American, and (5) Dry forests. Our results do not support the traditional Neo- versus Palaeo-tropical forest division, but instead separate the combined American and African forests from their Indo-Pacific counterparts. We also find indications for the existence of a global dry forest region, with representatives in America, Africa, Madagascar and India. Additionally, a northern hemisphere Subtropical forest region was identified with representatives in Asia and America, providing support for a link between Asian and American northern hemisphere forests