Molekularzytogenetische und physiologische Charakterisierung von Weichteilsarkomen und zellbiologische Rolle des Tyrosinkinase-Rezeptors EPHB4 in der Onkogenese der Weichteilsarkome

Weichteilsarkome sind eine seltene, jedoch heterogene Gruppe maligner Tumoren. Das Wissen über die Pathogenese und folglich über potenzielle Zielstrukturen für die Therapie ist nicht ausreichend bekannt. In der vorliegenden Arbeit wurde die Physiologie, Genetik und Therapie von Weichteilsarkomen in drei Kapiteln näher untersucht. Im ersten Kapitel wurden Sarkomzelllinien über 47 Wochen kultiviert, um anschließend ein mögliches, spontanes Auftreten von chromosomalen und genetischen Veränderungen zu überprüfen. Im nächsten Kapitel wurde die Zytogenetik des undifferenzierten pleomorphen Sarkoms (UPS) untersucht. Auffällige Veränderungen wurden bei acht Chromosomen festgestellt. Im letzten Kapitel wurde die Rolle des Rezeptors EPHB4 bei der Pathogenese von Weichteilsarkomen erforscht. Die EPHB4-Inhibierung resultierte in einer signifikanten Verminderung der Proliferation, Migration und Metastasierung einiger Sarkomentitäten und präsentierte EPHB4 als ein potenzielles Therapieziel

Maggot extract interrupts bacterial biofilm formation and maturation in combination with antibiotics by reducing the expression of virulence genes

Biofilms are aggregates of bacteria encased in an extracellular polymer matrix that acts as a diffusion barrier protecting the microbial community. Bacterial communication occurs by small signaling molecules called quorum sensing (QS) factors, which are involved in the activation of virulence genes and formation of biofilms. Larvae of the green bottle blowfly $\textit {Lucilia sericata}$ remove necrotic tissue by mechanical action (debridement) and proteolytic digestion. We produced a freeze-dried storable powder from larval extract and investigated its therapeutic effect on biofilms. Larval extract in concentrations of 6 and 12 mg/mL in combination with 0.5% antibiotics (≙50 U/mL penicillin and 50 $\mu$g/mL streptomycin) diminished free-floating (planktonic) $\textit {Pseudomonas aeruginosa}$ maintenance, while it showed no effect on $\textit {Staphylococcus aureus}$ and was not toxic to dermal cells. We established an ex vivo human dermal wound model. Larval extract in concentrations of 24 and 75 mg/mL in the presence of antibiotics (0.5%) significantly destroyed the biofilm stability of both $\textit {P. aeruginosa}$ and $\textit {S. aureus}$ biofilms. Furthermore, SEM analyses revealed crack and gap formations on $\textit {P. aeruginosa}$. biofilm surface and decreased expression of $\textit {P. aeruginosa}$ biofilm maturation and virulence genes ($\it lasR$, $\it rhlR$ and $\it rhlA$) was observed after treatment by larval extract in combination with antibiotics

Inhibition of GDF8 (Myostatin) accelerates bone regeneration in diabetes mellitus type 2

Metabolic diseases like diabetes mellitus cause bone healing deficiencies. We found significant impairment of bone regeneration, osteogenic differentiation and proliferation in diabetic bone. Moreover recent studies suggest a highly underestimated importance of GDF8 (Myostatin) in bone metabolism. Our goal was to analyze the role of GDF8 as a regulator of osteogenic differentiation, proliferation and bone regeneration. We used a murine tibial defect model in diabetic ($Lepr^{db-/-}$) mice. Myostatin-Inhibitor Follistatin was administered in tibial bony defects of diabetic mice. By means of histology, immunohistochemistry and QRT-PC osteogenesis, differentiation and proliferation were analyzed. Application of Myostatin-inhibitor showed a significant improvement in diabetic bone regeneration compared to the control group (6.5 fold, p < 0.001). Immunohistochemistry revealed a significantly higher proliferation (7.7 fold, p = 0.009), osteogenic differentiation (Runx-2: 3.7 fold, p = 0.011, ALP: 9.3 fold, p < 0.001) and calcification (4.9 fold, p = 0.024) in Follistatin treated diabetic animals. Therapeutical application of Follistatin, known for the importance in muscle diseases, plays an important role in bone metabolism. Diabetic bone revealed an overexpression of the catabolic protein Myostatin. Antagonization of Myostatin in diabetic animals leads to a restoration of the impaired bone regeneration and represents a promising therapeutic option

Pharmacological elevation of sphingosine- 1- phosphate by S1P lyase inhibition accelerates bone regeneration after post-traumatic osteomyelitis

Posttraumatic osteomyelitis and the ensuing bone defects are a debilitating complication after open fractures with little therapeutic options. We have recently identified potent osteoanabolic effects of sphingosine-1-phosphate (S1P) signalling and have now tested whether it may beneficially affect bone regeneration after infection. We employed pharmacological S1P lyase inhibition by 4-deoxypyrodoxin (DOP) to raise S1P levels in vivo in an unicortical long bone defect model of posttraumatic osteomyelitis in mice. In a translational approach, human bone specimens of clinical osteomyelitis patients were treated in organ culture in vitro with DOP. Bone regeneration was assessed by $\mu$CT, histomorphometry, immunohistology and gene expression analysis. The role of S1P receptors was addressed using S1PR3 deficient mice. Here, we present data that DOP treatment markedly enhanced osteogenesis in posttraumatic osteomyelitis. This was accompanied by greatly improved osteoblastogenesis and enhanced angiogenesis in the callus accompanied by osteoclast-mediated bone remodelling. We also identified the target of increased S1P to be the S1PR3 as $S1PR3^{−/−}$ mice showed no improvement of bone regeneration by DOP. In the human bone explants, bone mass significantly increased along with enhanced osteoblastogenesis and angiogenesis. Our data suggest that enhancement of S1P/S1PR3 signalling may be a promising therapeutic target for bone regeneration in posttraumatic osteomyelitis

Alterations in pectoralis muscle cell characteristics after radiation of the human breast insitu\textit in situ

The life-time risk of being diagnosed with breast cancer is ~12%, hence breast cancer is by far the most common cancer among women. The multimodal treatment concept of breast cancer often intends radiation. The utilized ionizing radiation leads changes in the tissue resulting in tissue damage due to an alteration of molecular factors. The goal of this study was to identify the role of muscle-catabolic proteins after radiation of human pectoralis major muscles in situ. Tissue of the pectoralis major muscle was collected in 12 breast cancer patients after radiation (maximum 3 years after radiation) undergoing a deep inferior epigastric perforator free-flap breast reconstruction. At the same time, an intraindividual comparison to rectus abdominis muscle was carried out upon free-flap elevation. Immunological properties, cell proliferation, differentiation as well as the expression profile of the muscle tissue were investigated through immunohistological reactions, a DNA-microarray and histology. We found significantly increased neutrophil immigration in the radiated muscle tissue. At the same time, proteins responsible for muscular atrophy and apoptosis were significantly elevated in immunohistochemistry. A DNA microarray detected immunological upregulation and myo-differentiative disorders in radiated muscle tissue. This novel study investigating catabolism in radiated muscle in situ can serve as a basis for the treatment of radiation-accompanied muscle disorders

Inhibition of pathological increased matrix metalloproteinase (MMP) activity for improvement of bone regeneration in diabetes

Patients with diabetes suffer from poor fracture healing. Molecular reasons are not fully understood and our previous gene expression microarray analyses of regenerating bones from mice with type 2 diabetes (db$^{−}$/db$^{−}$) revealed accelerated activation of pathways concerning matrix metalloproteases (MMPs). Thus, we picked out the pathological MMP acceleration as a target for profound gene expression analyses and additional therapeutic intervention in the present study. In the first part, gene expression of ECM degrading proteinases and inhibitors was investigated three and seven days postoperatively. $\it Mmp3$, $\it Mmp9$, $\it Mmp13$ and gene expression of MMP inhibitor $\it Timp2$ was significantly higher in regenerating bone fractures of db$^{−}$/db$^{−}$ compared to wild type animals. $\it Timp1$ and metalloproteinase $\it AdamTS4$ showed no differences. In the second part, we locally applied a single dose (1 $\mu$L of 5 $\mu$M solution) of the broad-spectrum molecular MMP inhibitor Marimastat on tibial defects in db$^{−}$/db$^{−}$. We performed immunohistochemical and histological stainings seven days post operation. Impaired bone healing, collagen content, angiogenesis, and osteoclast invasion in db$^{−}$/db$^{−}$ were restored significantly by application of Marimastat compared to PBS controls ($\it n$ = 7/group). Hence, local intervention of bone defects by the molecular MMP inhibitor Marimastat might be an alternative therapeutic intervention for bone healing in diabetes

Myostatin deficiency protects C2C12 cells from oxidative stress by inhibiting intrinsic activation of apoptosis

Ischemia reperfusion (IR) injury remains an important topic in clinical medicine. While a multitude of prophylactic and therapeutic strategies have been proposed, recent studies have illuminated protective effects of myostatin inhibition. This study aims to elaborate on the intracellular pathways involved in myostatin signaling and to explore key proteins that convey protective effects in IR injury. We used CRISPR/Cas9 gene editing to introduce a myostatin $\textit {(Mstn)}$ deletion into a C2C12 cell line. In subsequent experiments, we evaluated overall cell death, activation of apoptotic pathways, ROS generation, lipid peroxidation, intracellular signaling via mitogen-activated protein kinases (MAPKs), cell migration, and cell proliferation under hypoxic conditions followed by reoxygenation to simulate an IR situation in vitro (hypoxia reoxygenation). It was found that mitogen-activated protein kinase kinase 3/6, also known as MAPK/ERK Kinase 3/6 (MEK3/6), and subsequent p38 MAPK activation were blunted in C2C12-$it Mstn^{−/−}$ cells in response to hypoxia reoxygenation (HR). Similarly, c-Jun N-terminal kinase (JNK) activation was negated. We also found the intrinsic activation of apoptosis to be more important in comparison with the extrinsic activation. Additionally, intercepting myostatin signaling mitigated apoptosis activation. Ultimately, this research validated protective effects of myostatin inhibition in HR and identified potential mediators worth further investigation. Intercepting myostatin signaling did not inhibit ROS generation overall but mitigated cellular injury. In particular, intrinsic activation of apoptosis origination from mitochondria was alleviated. This was presumably mediated by decreased activation of p38 caused by the diminished kinase activity increase of MEK3/6. Overall, this work provides important insights into HR signaling in C2C12-$it Mstn^{−/−}$ cells and could serve as basis for further research

Deficiency of myostatin protects skeletal muscle cells from ischemia reperfusion injury

Ischemia reperfusion (IR) injury plays a pivotal role in many diseases and leads to collateral damage during surgical interventions. While most studies focus on alleviating its severity in the context of brain, liver, kidney, and cardiac tissue, research as regards to skeletal muscle has not been conducted to the same extent. In the past, myostatin (MSTN), primarily known for supressing muscle growth, has been implicated in inflammatory circuits, and research provided promising results for cardiac IR injury mitigation by inhibiting MSTN cell surface receptor ACVR2B. This generated the question if interrupting MSTN signaling could temper IR injury in skeletal muscle. Examining human specimens from free myocutaneous flap transfer demonstrated increased MSTN signaling and tissue damage in terms of apoptotic activity, cell death, tissue edema, and lipid peroxidation. In subsequent in vivo Mstn$_{Ln/Ln}$ IR injury models, we identified potential mechanisms linking MSTN deficiency to protective effects, among others, inhibition of p38 MAPK signaling and SERCA2a modulation. Furthermore, transcriptional profiling revealed a putative involvement of NK cells. Collectively, this work establishes a protective role of MSTN deficiency in skeletal muscle IR injury

A highly reliable convolutional neural network based soft tissue sarcoma metastasis detection from chest x-ray images

Introduction: soft tissue sarcomas are a subset of malignant tumors that are relatively rare and make up 1% of all malignant tumors in adulthood. Due to the rarity of these tumors, there are significant differences in quality in the diagnosis and treatment of these tumors. One paramount aspect is the diagnosis of hematogenous metastases in the lungs. Guidelines recommend routine lung imaging by means of X-rays. With the ever advancing AI-based diagnostic support, there has so far been no implementation for sarcomas. The aim of the study was to utilize AI to obtain analyzes regarding metastasis on lung X-rays in the most possible sensitive and specific manner in sarcoma patients. Methods: a Python script was created and trained using a set of lung X-rays with sarcoma metastases from a high-volume German-speaking sarcoma center. 26 patients with lung metastasis were included. For all patients chest X-ray with corresponding lung CT scans, and histological biopsies were available. The number of trainable images were expanded to 600. In order to evaluate the biological sensitivity and specificity, the script was tested on lung X-rays with a lung CT as control. Results: in this study we present a new type of convolutional neural network-based system with a precision of 71.2%, specificity of 90.5%, sensitivity of 94%, recall of 94% and accuracy of 91.2%. A good detection of even small findings was determined. Discussion: the created script establishes the option to check lung X-rays for metastases at a safe level, especially given this rare tumor entity

P2000 - a high-nitrogen austenitic steel for application in bone surgery

Optimal treatment of bone fractures with minimal complications requires implant alloys that combine high strength with high ductility. Today, TiAl6V4 titanium and 316L steel are the most applied alloys in bone surgery, whereas both share advantages and disadvantages. The nickel-free, high-nitrogen austenitic steel X13CrMnMoN18-14-3 (1.4452, brand name: P2000) exhibits high strength in combination with superior ductility. In order to compare suitable alloys for bone implants, we investigated titanium, 316L steel, CoCrMo and P2000 for their biocompatibility and hemocompatibility (according to DIN ISO 10993–5 and 10993–4), cell metabolism, mineralization of osteoblasts, electrochemical and mechanical properties. P2000 exhibited good biocompatibility of fibroblasts and osteoblasts without impairment in vitality or changing of cell morphology. Furthermore, investigation of the osteoblasts function by ALP activity and protein levels of the key transcription factor RUNX2 revealed 2x increased ALP activity and more than 4x increased RUNX2 protein levels for P2000 compared to titanium or 316 steel, respectively. Additionally, analyses of osteoblast biomineralization by Alizarin Red S staining exhibited more than 6x increased significant mineralization of osteoblasts grown on P2000 as compared to titanium. Further, P2000 showed no hemolytic effect and no significant influence on emocompatibility. Nanoindentation hardness tests of Titanium and 316L specimens exposed an indentation hardness (HIT) of about 4 GPa, whereas CoCrMo and P2000 revealed HIT of 7.5 and 5.6 GPa, respectively. Moreover, an improved corrosion resistance of P2000 compared to 316L steel was observed. In summary, we could demonstrate that the nickel-free high-nitrogen steel P2000 appears to be a promising alternative candidate for applications in bone surgery. As to nearly all aspects like biocompatibility and hemocompatibility, cell metabolism, mineralization of osteoblasts and mechanical properties, P2000 was similar to or revealed advantages against titanium, 316L or CoCrMo