Modulators of the hyaluronan matrix during extrinsic skin aging

Klinisch manifestiert sich die extrinsisch gealterte Haut durch das Auftreten von Falten, Gewebeschlaffheit und einem ledrigen Erscheinungsbild. Kausal verknüpft mit diesen Symptomen sind histologische und ultrastrukturelle Veränderungen der extrazellulären Matrix (EZM), im Speziellen eine deutliche Abnahme des Hyaluronsäure(HA)-Gehalts in der Dermis. Welche regulatorischen Mechanismen diesen Veränderungen zugrunde liegen ist bislang weitgehend unbekannt. Ziel der vorliegenden Untersuchungen war es daher, potentielle Modulatoren der HA Matrix zu identifizieren. Ein Hauptmerkmal der Umstrukturierung der EZM durch Sonneneinstrahlung ist die Entstehung von Kollagenfragmenten (KF) durch Matrix Metalloproteinasen. Die Stimulation von dermalen Fibroblasten mit KF in vitro führte zu einer verminderten HA-Synthese und einer Abnahme der HA-Synthase 2-mRNA-Expression. Es konnte gezeigt werden, dass KF αvβ3-Integrine aktivieren und über eine Inhibition der RhoA-Kinase eine Degradation des Aktin-Zytoskeletts bedingen. Dies führt im Anschluss zu einer verminderten Translokation von pERK1/2 in den Zellkern und einer nachgeschalteten reduzierten HAS2-Transkription. KF wirken folglich als negative Modulatoren der HA-Matrix, die die extrazelluläre und perizelluläre HA-Matrix vermindern. Im Gegensatz zu KF wird Estradiol (E2) ein positiver Effekt auf die strukturellen und funktionellen Eigenschaften der Haut zugeschrieben. Zur Untersuchung des Einflusses von E2 auf die HA-Matrix wurde bei skh-1 Nacktmäusen die endogene Hormonproduktion durch Ovariektomie depletiert. Anschließend wurde den Tieren ein Placebo- oder E2-freisetzendes-Pellet implantiert. Der Einfluss der extrinsischen Alterung wurde durch UVB-Bestrahlung imitiert. Extrinsisch gealterte Tiere zeigten einen deutlichen Verlust an HA, der durch die Behandlung mit E2 aufgehoben werden konnte. Versican, ein HA assoziiertes Proteoglykan, lag in UVB-bestrahlten und E2–behandelten Tieren erhöht vor. Einhergehend mit der Erhöhung des HA-Gehalts der Dermis konnte eine Induktion der HA-Synthase 3 beobachtet werden. Es zeigte sich, dass diese Regulation auf einem indirekten Mechanismus beruhte. Hierbei führt E2 vermutlich zu einer erhöhten Sekretion von EGF in Keratinozyten, welches im Anschluss auf parakrinem Weg die Induktion der HAS3 und Versican hervorruft und somit eine positive Modulation der HA-/Versican-Matrix herbeiführt. Diese veränderte Matrix ist vermutlich an den proliferationssteigernden und inflamationshemmenden Effekten von E2 beteiligt. Zusammenfassend konnten somit zwei völlig neue Mechanismen, welche an der Regulation der HA-Matrix im Rahmen der extrinsischen Alterung beteiligt sind, charakterisiert werden. Zudem gelang es erstmalig einen direkten negativen Regulator der HA-Synthase 2 zu identifizieren.Extrinsically aged skin is clinically characterized by wrinkling, laxity and a leather like appearance. These symptoms of actinic aging are accompanied by histological and ultrastructural changes of the connective tissue, specifically by a loss of hyaluronan (HA) in the dermis. The regulatory mechanisms underlying these alterations are up to date largely unknown. Aim of the present study was therefore to identify modulators of the extracellular matrix (ECM) with respect to their effect on the papillary dermis during photoaging. One major characteristic of UVB-evoked structural changes of the ECM are the emergence of collagen fragments due to the upregulation of matrix metalloproteinases. Stimulation of dermal fibroblast with collagen fragments (CF) in vitro led to reduced HA-synthesis and downregulation of HA-synthase 2 mRNA expression. It was found, that CF activate αvβ3-Integrins and thereby let to resolution of the actin cytoskeleton by inhibition of the RhoA-kinase. Consequently translocation of pERK1/2 into the nucleus was inhibited which resulted in decreased HA-synthase 2 transcription. Thus, CF act as negative regulators of HA, by reducing the extracellular and pericellular HA-Matrix. In comparison to CF, a positive effect on the structural and functional characteristics of the skin is attributed to estradiol (E2). To investigate the influence of E2 on the HA-Matrix the endogenous hormone production of hairless skh-1 mice was depleted by ovariectomy. Subsequently placebo or E2 controlled release pellets were implanted. UVB-irradiation was employed to mimic extrinsic aging. Extrinsically aged mice showed a marked decline of HA in the papillary dermis which could be abolished by E2 treatment. Versican, a HA-binding proteoglycan, was augmented after both UVB and E2 treatment. Accompanied by the induction of HA an increase of HA-synthase 3 was detected. Furthermore, it was found that this effect was dependent on a parakrine mechanism: E2 led to an upregulation of EGF-secretion in keratinocytes, which in turn induced HA-synthase 3 and Versican expression in dermal fibroblast. E2 therefore is a positive modulator of the HA-/Versican-Matrix. The altered matrix might be part of the pro-proliferative and anti-inflammatory response of the dermal matrix to E2. In conclusion two novel regulatory mechanism are presented, which are involved in the response of the HA-Matrix to extrinsic aging. Furthermore CF are the first direct inhibitors of the HA-synthase 2 expression

Stochastic processes as surrogate models for dynamical systems in magnetic confinement fusion

When designing machine learning (ML) models for scientific applications, a key point is to incorporate a priori domain-specific information in the model. Especially, when constructing reduced complexity models as surrogates, we need to ensure that the mathematical and physical properties of the underlying system are reflected correctly by the ML model. The first part of this thesis focuses on physics-consistent Gaussian processes (GPs) that respect laws of physics by design. This stands in contrast to so-called physics-informed regressors that incorporate physical constraints weakly through the loss function. In scenarios where data originate from underlying linear partial differential equations (PDEs) with localized sources, the proposed model is a superposition of a Gaussian process with a specialized kernel that is constructed to exactly fulfill the homogeneous part of the PDE while a linear model is used for sources. The specialized kernel ensures an exact correspondence and physical interpretability of hyperparameters allowing insights into the underlying physical characteristics. Physics-consistent GPs are then extended to model mappings in the phase space of Hamiltonian systems. Here, we propose a surrogate model based on multi-output GPs deploying derivative information with a matrix-valued covariance function to fully preserve the symplecticity of the Hamiltonian flow and thus conserve integrals of motion. The proposed method is related to geometric integration methods, but models the flow map with larger time steps, accelerating long-term computations. In chaotic systems, the symplectic surrogate model can not only be used for faster computations but also for early classification of chaotic versus regular trajectories, based on the calculation of Lyapunov exponents directly available from the surrogate model. One particular challenge in applying ML models to problems in plasma physics is the lack of labeled data for training larger models. Usually, physical experiments are extremely expensive and with regard to future fusion reactors, sufficient data will not be available until operations start. The second part of this thesis treats data augmentation via robust surrogate models of multivariate time series data to mitigate this problem. We apply Student-$t$ process regression in a state space formulation to ensure reliable uncertainty estimates despite outliers. This reduces computational complexity and allows us to use the model for high-resolution time series. We are using different approaches in this regard. One approach assumes uncorrelated input signals and induces correlations and cross-correlations via coloring transformations in a post-processing step. Another technique immediately incorporates correlations by using a multivariate Matérn kernel. Both approaches are found to be well-suited for data imputation and augmentation for multichannel time series sensor data with outliers

Effect of Protein Binding on the Pharmacological Activity of Highly Bound Antibiotics▿

During antibiotic drug development, media are frequently spiked with either serum/plasma or protein supplements to evaluate the effect of protein binding. Usually, previously reported serum or plasma protein binding values are applied in the analysis. The aim of this study was to evaluate this approach by experimentally measuring free, unbound concentrations for antibiotics with reportedly high protein binding and their corresponding antimicrobial activities in media containing commonly used protein supplements. Free, unbound ceftriaxone and ertapenem concentrations were determined in bacterial growth medium with and without bovine/human serum albumin, as well as adult bovine serum and human plasma using in vitro microdialysis. The corresponding antimicrobial activity was determined in MIC and time-kill curve experiments using Escherichia coli ATCC 25922 and Streptococcus pneumoniae ATCC 6303 as test strains. A semimechanistic maximum effect model was simultaneously fitted to the data and respective EC50 (concentration at half-maximum effect) values compared. Protein binding differed significantly for ceftriaxone (P < 0.05) between human plasma (76.8 ± 11.0%) and commercially available bovine (20.2 ± 8.3%) or human serum albumin (56.9 ± 16.6%). Similar results were obtained for ertapenem (human plasma, 73.8 ± 11.6%; bovine serum albumin, 12.4 ± 4.8%; human serum albumin, 17.8 ± 11.5%). The MICs and EC50s of both strains were significantly increased (P < 0.05) for ceftriaxone when comparing human and bovine serum albumin, whereas the EC50s were not significantly different for ertapenem. Free, unbound antibiotic concentrations differed substantially between plasma and protein supplements and correlated well with antimicrobial efficacy. Therefore, free, active concentrations should be measured in the test system instead of correcting for literature protein binding values

Improved Detection of Circulating Tumor Cells in Metastatic Colorectal Cancer by the Combination of the CellSearch® System and the AdnaTest®.

Colorectal cancer (CRC) is one of the major causes of cancer-related death and reliable blood-based prognostic biomarkers are urgently needed. The enumeration and molecular characterization of circulating tumor cells (CTCs) has gained increasing interest in clinical practice. CTC detection by CellSearch® has already been correlated to an unfavorable outcome in metastatic CRC. However, the CTC detection rate in mCRC disease is low compared to other tumor entities. Thus, the use of alternative (or supplementary) assays might help to itemize the prognostic use of CTCs as blood-based biomarkers. In this study, blood samples from 47 mCRC patients were screened for CTCs using the FDA-cleared CellSearch® technology and / or the AdnaTest®. 38 samples could be processed in parallel. We demonstrate that a combined analysis of CellSearch® and the AdnaTest® leads to an improved detection of CTCs in our mCRC patient cohort (positivity rate CellSearch® 33%, AdnaTest® 30%, combined 50%). While CTCs detected with the CellSearch® system were significantly associated with progression-free survival (p = 0.046), a significant correlation regarding overall survival could be only seen when both assays were combined (p = 0.013). These findings could help to establish improved tools to detect CTCs as on-treatment biomarkers for clinical routine in future studies

Inhibitory role of the small leucine-rich proteoglycan biglycan in bladder cancer.

Urothelial bladder cancer is the ninth most common cancer. Despite surgical and chemotherapeutic treatment the prognosis is still poor once bladder cancer progresses to a muscle-invasive state. Discovery of new diagnostic markers and pathophysiologic effectors might help to contribute to novel diagnostic and therapeutic options. The extracellular matrix microenvironment shaped by the extracellular matrix critically affects tumor cell and stroma cell functions. Therefore, aim of the present study was to assess the possible implication of the small leucine-rich proteoglycan biglycan in progression of human urothelial bladder cancer.For this purpose tumor biopsies of 76 bladder cancer patients with different tumor stages (pTa, pT1-T4) were investigated with respect to biglycan expression and correlated with a long-term (10 years) clinical follow-up. Interestingly, higher biglycan mRNA expression was associated with higher tumor stages and muscle invasiveness. In vitro knock-down of endogenous biglycan in human urothelial carcinoma cells (J82 cells) increased proliferation, whereas addition of recombinant biglycan and overexpression of biglycan inhibited tumor cell proliferation. In line with this growth-inhibitory effect of biglycan, transplantation of J82 cells after knock-down of biglycan resulted in significantly increased growth of subcutaneous xenograft tumors in nude mice in vivo. Furthermore, treatment with two anti-proliferative, multi-receptor tyrosine kinase inhibitors-sunitinib and sorafenib-strongly upregulated biglycan expression. Collectively, the experimental data suggest that high biglycan expression is associated with reduced tumor cell proliferation. In accordance, Kaplan-Meier analysis revealed higher 10-year survival in patients with high biglycan mRNA expression in tumor biopsies.In conclusion, the present data suggest that biglycan is an endogenous inhibitor of bladder cancer cell proliferation that is upregulated in response to anti-proliferative tyrosine kinase inhibitors. In addition, high biglycan expression is associated with favorable prognosis

The impact of the receptor of hyaluronan-mediated motility (RHAMM) on human urothelial transitional cell cancer of the bladder.

Hyaluronan (HA) is a carbohydrate of the extracellular matrix with tumor promoting effects in a variety of cancers. The present study addressed the role of HA matrix for progression and prognosis of human bladder cancer by studying the expression and function of HA-related genes.Tissue samples of 120 patients with different stages of transitional cell bladder cancer, who underwent surgical treatment for bladder cancer at the University Hospital of Essen were analysed. mRNA-expression levels of HA synthases (HAS1-3) and HA-receptors (RHAMM and CD44) were evaluated by real time RT-PCR in comparison to healthy bladder tissue as control. In uni- and multivariate cox proportional hazard survival regression analysis, the impact of the gene expression levels on survival was assessed. In vitro knock-down of RHAMM, CD44 and HAS isoenzymes was achieved by siRNA and lentiviral shRNA in J82 bladder cancer cells. Transfected cells were analysed in vitro with regard to proliferation, cell cycle and apoptosis. J82 cells after knock-down of RHAMM were xenografted into male nu/nu athymic mice to monitor tumor progression in vivo.In invasive tumor stages RHAMM-, HAS1 and HAS2 mRNA-expression levels were elevated whereas HAS3v1 was reduced as compared to non-invasive tumors. Subsequently, Kaplan-Meier analysis revealed reduced bladder cancer specific survival in patients with high RHAMM mRNA and low HAS3v1 expression. Elevated RHAMM in invasive tumors was confirmed by RHAMM immunohistochemistry. Furthermore, multivariate analysis revealed that only RHAMM expression was associated with poor prognosis independent from other survival factors (HR=2.389, 95% CI 1.227-4.651, p=0.01). Lentiviral RHAMM knock-down revealed reduced J82 cell proliferation in vitro and reduced xenograft tumor growth in vivo.The data suggest that RHAMM plays a crucial role in mediating progression of muscle-invasive bladder cancer and recommends RHAMM for further evaluation as a prognostic marker or therapeutic target in bladder cancer therapy

Kaplan-Meier curves for overall survival according to the CTC status using the AdnaTest^® (A), CellSearch^® (B), or both assay in combination (C).

<p>Kaplan-Meier curves for overall survival according to the CTC status using the AdnaTest^® (A), CellSearch^® (B), or both assay in combination (C).</p

Patient characteristics.

<p>Patient characteristics.</p

Detailed patient characteristics and CTC results.

<p>Detailed patient characteristics and CTC results.</p

Kaplan-Meier curves for progression-free survival according to the CTC status using the AdnaTest^® (A), CellSearch^® (B), or both assay in combination (C).

<p>Kaplan-Meier curves for progression-free survival according to the CTC status using the AdnaTest^® (A), CellSearch^® (B), or both assay in combination (C).</p