22455 research outputs found
Sort by
Sampling-based approaches for the computation of reaction paths, stacking interactions, and IR-sepctra applied to the fields of origins of life and enzymatic catalysis
Get PDFComparative response of nine hepatoma cell lines to the four tyrosine kinase inhibitors, sorafenib, lenvatinib, regorafenib and cabozantinib
Get PDFRelevanz des ultragroßen und thrombozytären von Willebrand Faktors für die primäre Hämostase und in der Diagnostik des von Willebrand Syndroms
Get PDFDie primäre Hämostase kann als Abfolge von Thrombozyten und Protein Interaktionen beschrieben werden. Das Zusammenspiel mit dem von Willebrand Faktor (VWF) ist dabei unerlässlich. Um der Komplexität gerecht zu werden, die hinter diesem stark regulierten System steckt, muss die Mikroumgebung um eine Gefäßläsion betrachtet werden. Es spielt die initiale Phase, in der ein zeitlich und räumlich eng begrenztes, hoch thrombogenes Milieu geschaffen wird, eine wichtige Rolle. Es entsteht um Mikroläsionen des Endothels, ist aber kaum durch gängige Messmethoden erfassbar. Durch die Messung einzelner Parameter können dennoch klinisch relevante Rückschlüsse gezogen werden. Die Konzentration und Aktivität des VWF im Plasma sind Routineparameter bei der Beurteilung von Hämostasestörungen. Neben dem plasmatischen VWF ist bekannt, dass der VWF der Thrombozyten – synthetisiert in Megakaryozyten und gespeichert in den α-Granula der zirkulierenden Blutplättchen – zur primären Hämostase und in der Mikroumgebung der Thrombusbildung beiträgt. Der 2018 auch in der EU zugelassene rekombinante VWF (rVWF) Veyvondi® weist die multimere Verteilung von frisch sezerniertem VWF mit ultragroßen (ULVWF) und hochmolekularen Multimeren aus Endothelzellen und Megakaryozyten auf. Er war weder ADAMTS13 noch einem anderen proteolytischen Enzym ausgesetzt. Die Messung der Verschlusszeit (CT) mit dem Platelet Function Analyzer 200 (PFA-200) erwies sich als hochempfindlich für das Vorhandensein von ULVWF aus zugesetzten VWF-Konzentraten. Als ähnlich naiv wie der rVWF gegenüber Proteolyse stellt sich nur der VWF in Thrombozyten dar. Die laborchemische Bestimmung des platelet-derived VWF ist allerdings umständlich und hat sich nicht als Routineparameter durchgesetzt. Es wird daher im Rahmen dieser Arbeit eine Methode zur Laborbestimmung und Berichterstattung über den VWF in Thrombozyten vorgeschlagen, die für die Laborroutine und Forschung nützlich sein kann.
Aus der Datenbank der Gerinnungsambulanz des LMU Klinikums wurden 12 Patienten mit diagnostizierter schwerer von-Willebrand-Erkrankung (VWD) ausgewählt. Den Vollblutproben der VWD-Patienten wurden in vitro eine therapeutische Dosis des rVWF-Produkts (1 IE/ml) zugesetzt, und die PFA-CTs gemessen. Die PFA-CTs normalisierten sich daraufhin. Darüber hinaus führten steigende Dosierungen von rVWF (0,1; 0,2; 0,5 und 1,0 IE/ml) zu einer dosisabhängigen Verkürzung der PFA-CT. Die in vitro Daten zeigen, dass der PFA-200 ein nützliches Instrument zum Nachweis von rVWF ist. Da die PFA-CT-Korrektur dosisabhängig ist, könnte der rVWF Spiegel mit dieser Point-of-Care Analysemethode während der Ersatztherapie zuverlässig überwacht werden.
Zur Etablierung einer Methode zur Analyse des platelet-derived VWF wurden Thrombozyten aus dem Blut 20 freiwilliger Probanden isolierten. Daraus wurde der platelet-derived VWF extrahierter und ergab die Basis für einen Datensatz, mit deren Hilfe drei mögliche Modelle berechnet wurden. Diese Modelle erlaubten es, den platelet-derived VWF zu beschreiben als
1. die Konzentration des platelet-derived VWF im Vollblut,
2. die Menge des platelet-derived VWF in einer Probe mit einer definierten Konzentration von 1000 Thrombozyten pro Nanoliter und
3. die Konzentration des platelet-derived VWF in einem einzelnen Thrombozyten.
Die Ergebnisse legen nahe, dass bei gesunden Personen ein Anteil von 10 % der Aktivität des gesamt VWFs im menschlichen Plasma auf den platelet-derived VWF entfallen. Die Konzentration des platelet-derived VWF wird auf 0,4 IU/ml in einer Probe mit einer definierten Konzentration von 1000 Thrombozyten/nl und auf etwa 42 IU/ml in einem Thrombozyten berechnet (jeweils VWF:Ag).
Die Analyse des platelet-derived VWF bleibt methodisch komplex und der Handhabung in spezialisierten Laboren vorbehalten. Dennoch stellt diese und die Detektierbarkeit von ULVWF in VWF-defizientem Blutplasma von Patienten mit schwerer VWD mithilfe eines funktionellen Testgeräts einen Schritt Richtung Beurteilbarkeit der Mikroumgebung dar. Der PFA-200 als eine vollautomatische und zuverlässige Point-of-Care-Methode zur Bewertung der primären Hämostase, kann erheblichen klinischen Nutzen bringen. Eingesetzt zur Überwachung der rVWF-Ersatztherapie bei Patienten mit VWD, bietet er die Möglichkeit, Dosierungen des rVWF zu optimieren und trägt damit zur Patientensicherheit bei. Experimentell konnte gezeigt werden, dass Vollblutproben von VWD-Patienten, die mit rVWF versetzt wurden, eine Normalisierung im PFA-CT aufwiesen und dass eine Dosis-Wirkungs-Beziehung bestand.
Die Ergebnisse dieser Arbeit könnten zur Optimierung der diagnostischen Verfahren hinsichtlich des von Willebrand Faktors beitragen. Somit würden sie einen potentiellen Beitrag zum besseren Verständnis dieses Schlüsselproteins der primären Hämostase leisten.Primary hemostasis can be described as a sequence of platelet and protein interactions, in which the interplay with von Willebrand factor (VWF) is essential. To account for the complexity behind this highly regulated system, the microenvironment surrounding a vascular lesion must be considered. In this context, the initial phase, in which a temporally and spatially confined, highly thrombogenic milieu is created, plays an important role. It develops around microlesions of the endothelium, but is hardly detectable by common laboratory methods. Nevertheless, clinically relevant conclusions can be drawn by measuring individual parameters. The concentration and activity of VWF in plasma are routine parameters in the assessment of hemostasis disorders. In addition to VWF in plasma, VWF in platelets, synthesized by megakaryocytes and stored in the α-granules of circulating platelets, is known to contribute to primary hemostasis and in the microenvironment of thrombus formation. Veyvondi®, a recombinant VWF (rVWF), approved in 2018 also in the EU, exhibits the multimeric distribution of freshly secreted VWF with ultralarge (ULVWF) and high molecular weight multimers from endothelial cells and megakaryocytes, as it was not exposed to ADAMTS13 or any other proteolytic enzyme. Closure time (CT) measurement with the Platelet Function Analyzer 200 (PFA-200) was found to be highly sensitive to the presence of ULVWF from added VWF concentrates. Only VWF in platelets was found to be similarly to rVWF naive to proteolysis. However, laboratory determination of platelet-derived VWF is cumbersome and has not gained acceptance as a routine parameter. Therefore, a method for laboratory determination and reporting of VWF in platelets that might be useful for routine laboratory work and research is proposed in this work.
Twelve patients diagnosed with severe von Willebrand disease (VWD) were selected from the database of the Hemophilia Treatment Center of LMU Klinikum. A therapeutic dose of rVWF product (1 IU/ml) was added to the VWD patients' whole blood samples in vitro, and PFA-CTs were measured. PFA-CTs subsequently normalized. In addition, increasing doses of rVWF (0.1; 0.2; 0.5 and 1.0 IU/ml) resulted in a dose-dependent shortening of PFA-CTs. The in vitro data indicate that the PFA-200 is a useful tool for detecting rVWF. Because PFA-CT reduction is dose-dependent, rVWF levels could be reliably monitored by this point-of-care analysis method during replacement therapy.
To establish a method for analysis of platelet-derived VWF, platelets were isolated from the blood of 20 volunteers. From these, platelet-derived VWF was extracted and provided the basis for a data set that was used to calculate three alternative models. These models allowed platelet-derived VWF to be described as
1. the concentration of platelet-derived VWF in whole blood,
2. the amount of platelet-derived VWF in a sample with a defined concentration of 1000 platelets per nanolitre, and
3. the concentration of platelet-derived VWF in a single platelet.
The results suggest that in healthy individuals, platelet-derived VWF accounts for 10% of the activity of total VWF in human plasma. The concentration of platelet-derived VWF is calculated to be 0.4 IU/ml in a sample with a defined concentration of 1000 platelets/nl and approximately 42 IU/ml in a platelet (VWF:Ag, respectively).
Analysis of platelet-derived VWF remains methodologically complex and confined to specialized laboratories for handling. Nevertheless, this and the detectability of ULVWF in VWF-deficient plasma from patients with severe VWD using a functional analyzer is a step toward assessability of the microenvironment. The PFA-200, as a fully automated and reliable point-of-care method for assessing primary hemostasis, may provide significant clinical benefit. Used to monitor rVWF replacement therapy in patients with VWD, it provides the ability to monitor and optimize regimens of rVWF, contributing to patient safety. Experimentally, it was shown that whole blood samples from VWD patients spiked with rVWF exhibited normalization in PFA-CT and that a dose-response relationship existed.
The results of this work may contribute to the optimization of diagnostic procedures with regard to von Willebrand factor. Thus, they would potentially provide a contribution to a better understanding of this key protein of primary hemostasis
Rolle komplizierter Karotisplaques beim akuten ischämischen Schlaganfall
Get PDFKomplizierte Karotisplaques können die Ursache für einen akuten ischämischen Schlaganfall sein, auch wenn sie noch zu keiner >50%igen Stenose geführt haben. Dies ist insbesondere für Patienten mit einem kryptogenen Schlaganfall von Bedeutung, bei denen die Schlaganfall-Ursache nicht bekannt ist.
In der CAPIAS-Studie konnte gezeigt werden, dass komplizierte Karotisplaques bei Patienten mit einem kryptogenen Schlaganfall signifikant häufiger ipsilateral (31%) als kontralateral zum Schlaganfall (12%) vorhanden waren. Im Vergleich zu anderen Ätiologien waren komplizierte Plaques ipsilateral zum Schlaganfall signifikant häufiger bei Patienten mit einem kryptogenen Schlaganfall (31%) als bei Patienten aus der Referenzgruppe mit einem kardioembolischen oder mikroangiopathischen Schlaganfall (15%), aber seltener als bei Patienten aus der zusätzlichen Vergleichsgruppe mit einem makroangiopathischen Schlaganfall und >50-69%iger Stenose nach NASCET (68%).
Darüber hinaus wiesen ipsilaterale Plaques von Patienten mit einem kryptogenen Schlaganfall einen größeren Fettkern und eine größere Einblutungsfläche als ipsilaterale Plaques von Patienten mit einem kardioembolischen oder mikroangiopathischen Schlaganfall auf. Insgesamt ähnelte die Zusammensetzung ipsilateraler Plaques bei Patienten mit einem kryptogenen Schlaganfall eher der Zusammensetzung ipsilateraler Plaques bei Patienten mit einem makroangiopathischen Schlaganfall, ohne dass bereits eine Lumen-Einengung zu verzeichnen war. Pathophysiologisch ist von einem beginnenden Remodelling-Prozess auszugehen, d.h. diese nicht-stenosierenden Plaques befinden sich in einem Entwicklungsprozess hin zu stenosierenden Plaques mit noch größerer Plaque-Vulnerabilität.
Das Vorhandensein komplizierter Plaques ipsilateral zum Schlaganfall ist nicht nur eine potenzielle Schlaganfall-Ursache, sondern auch ein Risikofaktor für zukünftige zerebrale Ischämien. Für Patienten mit einem kryptogenen Schlaganfall und einer komplizierten Plaque ipsilateral zum Schlaganfall war das Rezidivrisiko 5,6fach erhöht, erneut einen ischämischen Schlaganfall oder eine TIA in einem Zeitraum von 3 Jahren zu erleiden. In der Gesamtkohorte war das Vorhandensein einer ipsilateralen komplizierten Plaque mit einem um den Faktor 2,5 erhöhten Rezidivrisiko im gleichen Zeitraum assoziiert.
Bisher ist die Untersuchung auf eine komplizierte Plaque nicht Bestandteil der Diagnostik nach einem akuten Schlaganfall. Die Plaque-Bildgebung, wie sie in der CAPIAS-Studie mit mehreren MRT-Sequenzen, Oberflächenspulen und Kontrastmittelgabe durchgeführt wurde, ist aufwendig und nicht in die klinische Routine übertragbar. Daher ist eine Vereinfachung der Plaque-Bildgebung notwendig.
Die Plaque-Einblutung ist das häufigste Merkmal ipsilateraler komplizierter Plaques. Als Untersuchungsmethode bietet eine T1-gewichtete, fettgesättigte Sequenz die Möglichkeit, eine Plaque-Einblutung zu identifizieren. Dazu sind weder Oberflächenspulen, noch die Gabe von Kontrastmittel notwendig. Damit ist es unkompliziert möglich, diese Sequenz in das MRT-Protokoll in der klinischen Routine zu implementieren.
Ob Patienten mit einer Plaque-Einblutung von einer spezifischen Sekundärprophylaxe wie einer Intensivierung der medikamentösen Therapie oder einer Intervention profitieren, ist derzeit noch unklar und wird in klinischen, randomisierten Studien untersucht werden müssen
Die Rolle von spannungsgesteuerten, neuronalen Natriumkanälen in der Epileptogenese von Pumilio2 defizienten Mäusen
Get PDFVergleich der FlexiCuff - und ShellCuff-Systeme für nichtinvasives hämodynamisches Monitoring
Get PDF
DKK3 driven Wnt pathway in radiation-induced inflammatory and fibrosing skin injury
Get PDFRadiation dermatitis is a common side effect of cancer radiotherapy. To some extent radiation dermatitis occurs in many patients, in particular if the lesion is close to the skin. The skin affections can range from mild and transient redness to severe inflammation, fibrosis, ulceration or necrosis, and may severely reduce the patients’ quality of life. The treatment of chronic radiation dermatitis and fibrosis is difficult, underscoring then unmet need for novel targets and treatment strategies. Previous studies have shown that the canonical Wnt signaling pathway plays an important role in driving fibrosis and represents a potential target for the treatment of radiationinduced dermatitis and fibrosis. The soluble factor Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3) modulates canonical Wnt activity in a cell-specific manner. Our group’s previous study (non-radiation) in inflammatory renal fibrosis suggested that epithelial DKK3 may activate canonical Wnt signaling, and through this, play a profibrotic and immune-polarizing role in renal fibrosis. Moreover, our group’s pilot studies of irradiated skin suggested that a global knockout of DKK3 may partially protect mice from radiation-induced hyperplasia and skin fibrosis. It was thus hypothesized for this thesis that following radiation damage, DKK3 expression in concert with canonical Wnt activation are linked to a compensatory hyper reactive epidermal hyperplasia, inflammatory infiltration and fibrosis.
To test this hypothesis, a model of radiation-induced skin hyperplasia and inflammatory fibrosis was developed based on previous experiments of the group investigating radiation-induced lung fibrosis. A single dose of 20Gy radiation delivered to C57BL/N wildtype mice was found to induce reproducible chronic radiation dermatitis. Four to eight weeks after radiation, significant hyperplasia, myeloid infiltration and fibrosis was seen in irradiated skin as compared to non-irradiated controls. To identify the relevant cellular source of DKK3, and to monitor the cellular activity of canonical Wnt signaling in irradiated mouse skin, a DKK3 promoter based, and canonical Wnt (TCF/LEF) dual reporter mouse (DKK3-LCh x TCF/LEF) was used. The activation of DKK3 expression and Wnt pathway activity was evaluated following a single dose of 20Gy. At 6 days and 14 days after radiation, increased DKK3 expression and canonical Wnt pathway activity was detected in basal keratinocytes of irradiated skin and in many instances was found to be coincident. To investigate the effect of targeted DKK3 depletion on radiation-induced dermatitis and inflammatory fibrosis in vivo, a series of DKK3 tissue-specific knockouts were evaluated following radiation injury. Both DKK3 global and DKK3 keratinocyte specific knockout mice were shown to be significantly protected from epithelial hyperplasia and fibrosis. Interestingly, both the DKK3 global knockout and DKK3 keratinocyte specific knockout mice showed even more myeloid infiltration at the radiation site than the already increased infiltration seen in WT mice. The results suggested that keratinocyte expression of DKK3 and accompanying activation of canonical Wnt pathway signaling may be linked to a tissue milieu conducive toward the establishment of a pro-fibrotic environment. By contrast, the absence of DKK3 expression by keratinocyte was linked to a non-fibrotic tissue environment. To evaluate the myeloid phenotypes seen in the presence or absence of DKK3, cells were isolated from the skin of both DKK3 global knockouts and DKK3 keratinocytespecific knockouts and compared to the cells present in WT mice skin at 4 weeks after radiation. FACS analysis of leukocyte and myeloid sub-group markers was employed to characterize the phenotype of the extracted cells. Both DKK3 global knockout and the keratinocyte-specific knockout mice exhibited a decreased CD206+ and CD163+ surface marker expression on macrophages that are known to represent M2-like profibrotic phenotypes in both human and mouse settings. To better characterize the potential effect of DKK3 on the keratinocyte secretome with accompanying effects on myeloid differentiation and polarization, a chemokine/growth factor array and RT-qPCR analysis was performed on human keratinocytes (N/TERT-1) after DKK3 modulation. The N/TERT-1 cell line was engineered using an in house developed vector platform using a doxycycline-inducible DKK3 over expression plasmid. DKK3 knockdown performed via siRNA transfection was found to increase expression of the M1-like phenotype activation factor of GM-CSF, and also increased expression of the proinflammatory factors CXCL8, CXCL10, and TNF-α, while over expression of DKK3 led to increased keratinocyte expression of the M2-like and profibrotic factor TGF-β1. The conditioned media from N/TERT-1 cells with DKK3 modulation was then used to mature human peripheral blood mononuclear cells (PBMC) from healthy donors. After seven days the resultant keratinocyte conditioned macrophages (KcMф) were collected for FACS analysis. DKK3 over-expression in the keratinocytes increased expression of the M2-like phenotype surface markers CD206 and CD163, while DKK3 knockdown decreased the expression of these markers in KcMф. By contrast, expression of MHCII, CD80 and CD86 in KcMф was increased in all settings with maturation of the monocytes, and was not significantly altered by modulation of the DKK3 expression status of the keratinocyte line.
The N/TERT-1 cell line was further modified to include a synthetic TCF-based reporter gene to monitor activation of canonical Wnt pathway signaling. Using this system it was demonstrated that radiation increased both endogenous DKK3 expression and canonical Wnt activity. Radiation also induced ROS and chemical ROS stimulation was able to increase DKK3 expression and canonical Wnt signaling activity suggesting a second messenger path for the phenomena. Fibroblasts are important drivers of inflammatory fibrosis. Interestingly, DKK3 fibroblast-specific knockout mice showed increased radiation-induced hyperplasia and fibrosis over that seen in WT mice. In line with this in vivo data, it could be demonstrated in vitro, that DKK3 over-expression in a dermal fibroblast cell line effectively inhibited canonical Wnt activity. These results further indicated the pleotropic nature of the DKK3 protein and its tissue specific mode of action. The present study thus shows that radiation-induced ROS can lead to enhanced DKK3 expression with activation of canonical Wnt activity. This activation in basal keratinocytes caused radiation-induced epithelial hyperplasia and subsequent tissue fibrosis, while loss of DKK3 in keratinocytes protected mice partially against chronic radiation dermatitis. Interestingly, while radiation increased myeloid infiltration in all mice under study, the level of infiltrate in the DKK3 global and keratinocyte specific knockout animals was even enhanced over that seen in the WT mice. Mechanistically, the results suggest that the presence of DKK3 in keratinocytes with accompanying epithelial hyperplasia drives macrophage polarization more towards a M2-like profibrotic phenotype, while the absence of DKK3 leads to macrophages showing a more M1-like pro-inflammatory phenotype. Overall, the results suggest that DKK3 has an important role in radiation-induced dermatitis and fibrosis, that blocking DKK3 may attenuate the disease, and that DKK3 may serve as a potential therapeutic target for radiation-induced dermatitis and fibrosis
Investigating the causes and consequences of altered subcellular spatial composition in the immune system and beyond
Get PDFNLRP3 priming by translocation
Working on the role of NEK7 in NLRP3 activation, we had discovered that, in con- trast to the role of NEK7 in mouse cells, human cells activate NLRP3 independently of NEK7. “Transplanting” mouse NLRP3 into a model of human monocytes rescued the activity of mouse NLRP3 in the absence of NEK7. From this result we concluded that rather than a difference between the two NLRP3 orthologues, a difference be- tween cellular signalling must be responsible for the differential requirement of NEK7 for NLRP3 activation. Coupled with the finding that TLR4 stimulation via LPS can bypass the requirement for NEK7 in mouse cells, we concluded that a pathway activated downstream of TLR4 can bypass NEK7 by priming NLRP3. Tracing the signalling cascade of TLR4 by genetically knocking out its components, we arrived at the kinase IKKβ. Indeed, experiments with knockouts of IKKβ in mouse and human cells explained both phenotypes: LPS could no longer bypass NEK7 in mouse cells and NLRP3 signalling in human cells was blunted. Why human cells are incapable of using NEK7 to prime NLRP3 in the absence of IKKβ remains unclear. Using human induced pluripotent stem cell-derived macrophages that we could genetically engineer to lack NEK7 as a model system, we confirmed that human cells in contrast to mouse cells do not require NEK7, but instead fully rely on IKKβ to prime NLRP3. Elucidating the mechanism by which IKKβ primes NLRP3 for NEK7-independent inflammasome activation, we found that IKKβ activity recruits a fluorescently tagged NLRP3 variant to the trans-Golgi network, a finding we corroborated by mass spec- trometry analysis of subcellular fractions. Our results define recruitment of NLRP3 to a specific organelle as a new priming modality of the NLRP3 inflammasome.
CRISPR screening for subcellular spatial phenotypes at genome scale
The development of charge coupled device (CCD) chips has enabled the acquisition of digital images at high resolution (Boyle and Smith 1970). In combination with modern microscopes the latest development of such chips has enabled the collection of large digital datasets representing the spatial composition of cells. A technology that can profile this composition and connect it to the genetic identity of individual cells at scale could generate insights into complex cellular biology. Here we developed a new genetic screening technology for image-based phenotypes. We first generated a library of 40 million human U2OS cells with one genetic knockout in each cell using CRISPR/Cas9. The cells in this library had been genetically engineered to express the fluorescently labelled autophagosome marker LC3 (mCherry-LC3). We stimulated these cells with the mTOR inhibitor Torin-1 to induce autophagy, during which LC3 gets redistributed onto autophagosomes. We then acquired microscopy images of this library and segmented these images into single cells using a nuclear stain to identify individual cells and a membrane stain to associate a the cytosol of a cell with its nucleus. This resulted in a dataset of single cell images across three channels: Nucleus and membrane that were used for segmentation and an image corresponding to the distribution of LC3 in each cell. Given that each cell in this library harboured a different genetic knockout, we expected some cells to have been unable to redistribute LC3 onto autophagosomes following Torin-1 stimulation owing to the lack of a gene that is essential for this process. We then sought to identify these cells based on their LC3 images. Since these data are inherently large and complex, we made use of the recent breakthrough in image analysis by machine learning models (LeCun et al. 2015). Using a knockout of ATG5, an essential autophagy gene, as a positive control, we trained a binary classifier based on a convolutional neural network to differentiate between images of cell undergoing autophagy and images of cells that had a blunted response to Torin-1 or were left unstimulated, and therefore not undergoing autophagy. With this classifier we were able to identify individual cells in our library that were incapable of forming autophagosomes in response to Torin-1. We then used fully automated laser microdissection to isolate the nuclei of these cells and subsequently sequenced their genetic perturbations. Here we found almost all genes known to be essential for autophagy to be defective in this pool of selected cells. This experiment demonstrates that our technology is capable of associating image-based phenotypes with the genotype of individual cells at genome scale