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Background: Regenerative therapies based on autologous mesenchymal stem cells (MSC) as well as stem cells in general are still facing an unmet need for non-invasive sampling, availability, and scalability. The only known adult source of autologous MSCs permanently available with no pain, discomfort, or infection risk is the outer root sheath of the hair follicle (ORS). Methods: This study presents a non-invasively-based method for isolating and expanding MSCs from the ORS (MSCORS) by means of cell migration and expansion in air–liquid culture. Results: The method yielded 5 million cells of pure MSCORS cultured in 35 days, thereby superseding prior art methods of culturing MSCs from hair follicles. MSCORS features corresponded to the International Society for Cell Therapy characterization panel for MSCs: adherence to plastic, proliferation, colony forming, expression of MSC-markers, and adipo-, osteo-, and chondro-differentiation capacity. Additionally, MSCORS displayed facilitated random-oriented migration and high proliferation, pronounced marker expression, extended endothelial and smooth muscle differentiation capacity, as well as a paracrine immunomodulatory effect on monocytes. MSCORS matched or even exceeded control adipose-derived MSCs in most of the assessed qualities. Conclusions: MSCORS qualify for a variety of autologous regenerative treatments of chronic disorders and prophylactic cryopreservation for purposes of acute treatments in personalized medicine.

Although several investigations have shown a relationship between increased epicardial adipose tissue (EAT) and atrial fibrillation (AF), the association between EAT and ventricular tachycardia (VT) has not been evaluated. We investigated the association between EAT and post-ablation VT recurrence. In this study, sixty-one consecutive patients (mean age=62.0±13.9) undergoing VT ablation with pre-procedural cardiac magnetic resonance imaging (MRI) were recruited. EAT thickness was measured using cardiac MRI in the right and left atrioventricular grooves (AVGs), RV free wall, anterior, inferior, and superior interventricular grooves (IVGs). During a mean follow-up period of 392.9±180.2 days, post-ablation VT recurrence occurred in 15 (24.6%) patients. EAT thickness was significantly higher in the VT recurrence group than that in the non-recurrent VT at the right (18.7±5.7 vs. 14.1±4.4 mm; p=0.012) and left (13.3±3.9 vs. 10.4±4.1; p=0.020) AVGs. The best cut-off points for predicting VT recurrence were calculated as 15.5 mm for the right AVG (area under ROC curve=0.74) and 11.5 mm for the left AVG (area under ROC curve=0.72). Multivariate Cox regression analysis showed that pre procedural right AVG-EAT (HR: 1.2; 95% CI: [1.06-1.39], p=0.004) was the only independent predictor of VT recurrence after adjustment for covariates. Kaplan–Meier analysis showed a difference for post-ablation VT recurrence between the two groups with right AVG-EAT thickness cut-off value of <15.5 mm versus ≥15.5 mm (log-rank, p=0.003). Based on the finding of this study, we suggested a new possible imaging marker for risk stratification of post-ablation VT recurrence. A higher EAT may be associated with VT recurrence after catheter ablation of VTs.:Epicardial Adipose Tissue Anatomy Embryology Physiology and Pathophysiology Measurement of EAT EAT and heart disorders Future direction VT Catheter Ablation History of VT ablation Catheter ablation for VT in structural and non-structural heart Outcome of VT catheter ablation Predictors of VT recurrence after catheter ablation Objectives of the thesis Publication Summary References

Colitis ulcerosa (CU) und Morbus Crohn (MC) zählen zur Gruppe der chronischen Darmerkrankungen (CED). Im Gegensatz zu CU lässt sich bei MC eine transmurale Entzündung und eine Ummantelung des entzündeten Dünndarms mit mesenterialen Fettgewebe, dem sogenannten “creeping fat”, feststellen. In dieser Arbeit wurden zwei verschiedene Mechanismen der Immunregulation in der Pathogenese von CED untersucht: 1) Der SOCE-Signalweg (store-operated Ca2+ entry) stellt eine wichtige Signalkaskade dar, die die Aktivierung von T-Zellen steuert. Wir haben die Auswirkung einer pharmakologischen Blockade von SOCE auf die Funktion von Immunzellen untersucht, die aus der intestinalen Mukosa therapierefraktärer CED-Patienten isoliert wurden. Anschließend konnten wir die Sicherheit einer systemischen Verabreichung von SOCE-Inhibitoren in vivo im einem murinen IBD-Modell bestätigen und zeigen, dass die Blockade von SOCE eine therapeutische Option für die Behandlung von CED darstellen könnte. 2) Darüber hinaus untersuchten wir, ob das Fehlen von Fettgewebe eine entzündungsfördernde oder -hemmende Rolle bei der Entstehung von CED spielt. Daher wurde die Zusammensetzung des mukosalen Immunsystems sowie die Funktion der intestinalen Epithelbarriere in einem Mausmodell mit Adipozytenatrophie sowohl im steady-state als auch nach induzierter Kolitis verglichen. Wir konnten zeigen, dass eine Fettgewebsatrophie vor dem Ausbruch einer Kolitis schützt, und führte zu einer erhöhten Resistenz der intestinalen Barriere. Schließlich verglichen wir die Merkmale des lipoatrophischen Mausmodells mit denen eines seltenen Patienten mit erworbener Lipodystrophie und MC und kamen zu dem Schluss, dass die chirurgische Resektion von mesenterialen Fettgewebes für Patienten mit einem MC, bei denen eine intestinale Resektionen durchgeführt wird, sinnvoll sein könnte. / Inflammatory bowel disease (IBD) is a group of chronic intestinal autoimmune disorders, including ulcerative colitis (UC) and Crohn's disease (CD). In contrast to UC, CD is characterized by transmural inflammation and mesenteric adipose tissue wrapping the inflamed small intestine, known as "creeping fat." Despite all currently available drug therapies, treating IBD remains a major challenge, underlying the necessity of identifying new therapeutic targets. In this work, two different mechanisms of immune regulation in the pathogenesis of IBD were investigated: First, because the store-operated Ca2+ entry (SOCE) signaling pathway is a crucial Ca2+ signaling cascade for T cell activation, we investigated the effect of pharmacological SOCE-blockade on intestinal immune cells isolated from therapy refractory IBD patients. Subsequently, we confirmed the efficacy and safety of systemic administration of SOCE inhibitors in vivo in an IBD murine model, demonstrating that the blockade of SOCE may represent a therapeutic option for treating IBD. Second, we investigated whether adipose tissue plays a pro- or anti-inflammatory role in the development of IBD. Therefore, we characterized the mucosal immune system and intestinal epithelial barrier in a murine model affected by adipocyte atrophy both at steady-state and after induction of colitis. We demonstrated that lipodystrophy protected against the onset of colitis and increased intestinal barrier resistance. Finally, we compared the lipoatrophic mouse model with a rare patient with acquired generalized lipodystrophy and CD, concluding that adipokines might play a pro-inflammatory role in IBD. Therefore, we suggest that surgical resection of mesenteric adipose tissue in CD patients might be a beneficial intervention in patients undergoing bowel resection.

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<p>Obesity is a major global health concern and is associated with the development of numerous non-communicable diseases. A thorough understanding of the onset of obesity is critical to the development of effective therapeutic strategies against this disease state. Recently, obesity has been described as a complex disease characterized by chronic low grade inflammation. Abnormal adipose tissue expansion is accompanied by an increased presence of proinflammatory immune cells, dysregulated adipokine expression, oxidative stress, and is associated with significant changes in the bacterial composition of the gut. While interleukin-15 (IL-15) has been studied extensively for its immunological effects, this cytokine has recently been shown to influence body weight and fat mass. The focus of this thesis was to elucidate the role of and mechanism by which IL-15 modulates adipose tissue. Our first study demonstrated that low levels of IL-15 expression are associated with adiposity and promotes an obese state in IL-15-/- mice and human subjects, while IL-15 overexpression was associated with a lean phenotype in IL-15tg mice when compared to appropriate controls. To uncover the underlining mechanisms by which IL- 15 mediates differences in body weight, we subsequently determined that IL-15 mediated weight loss occurred independently of lymphocytes. In another study, we showed that IL-15tg mice had increased mitochondrial activity and mass specific to adipose tissue compared to IL-15-/- and B6 mice, while acute IL-15 administration induced the expression of FAO markers in adipose tissue. Lastly, IL-15 treatment increased mitochondrial membrane potential and decreased lipid deposition in cultured adipocytes, suggesting that IL-15 may mediate its effects directly on adipose tissue. The experimental results presented in this thesis demonstrate that IL-15 is an important regulator of adipose tissue and body weight. Future studies examining the effects of IL- 15 on adipose tissue will further our knowledge on IL-15 biology, and may contribute to novel therapeutic strategies for the treatment and prevention of obesity.</p> / Doctor of Philosophy (PhD)

Background: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor beta(TGF-beta) and hypoxia-inducible factor 1-alpha (HIF1-alpha) signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.

Nicotinic acid (NA) has been clinically used for over 50 years to regulate lipid plasma levels. It is the only drug in current clinical use that significantly raises HDL cholesterol and reduces inflammatory markers. However, mechanistic understanding into its wide range of actions remains unclear. The recent identification of the Gi-coupled protein receptor HCAR2, for which NA is a potent agonist, provides intriguing insight due to its anti-lipolytic action and restricted, yet specific, expression in adipose tissue and immune cells. The HCAR2 gene is 96% homologous to HCAR3, but the HCAR3 receptor shares neither the specificity for NA, nor the range of functional effects. Moreover, the close homology makes it difficult to separate the genetic variability and regulation of the two genes. To this end, I resequenced HCAR2 and HCAR3 in a selected population to characterize the variability of the two genes and to inform the subsequent design of specific genotyping assays. The Oxford Biobank, which is a random population-based collection of 30-50 year old men and women in Oxfordshire with a wide range of collected phenotypes, was used to explore genetic associations. A preliminary trend with HDL and rs7314976 in HCAR2 motivated the further search associations. However after increasing the sample size, the HDL association did not reach significance. When looking at inflammatory phenotypes, a 20% lower level of systemic hsCRP was found in males with a promoter region variant in HCAR3 (N=1808, p=0.007 for rs55718746). Replication of this finding in two relevant cohorts (NPHS-II, N=2185 and Whitehall, N=4228) resulted in conflicting findings. After optimising the specific detection of both HCAR2 and HCAR3 transcripts, I characterized gene expression in human AT biopsies. This revealed an 18% increase in HCAR2 expression in the female abdominal depot (N=106, p<0.0001) and a reduction in abdominal HCAR2 in both males (β=-0.37, p<0.001, N=107) and females (β=-0.251, p=0.005, N=106) with increasing adiposity. The rs55718746 variant in HCAR3 was also seen to influence expression of both HCAR2 (N=182, p=0.018 in the abdominal depot) and HCAR3 (N=198, p=0.005) but surprisingly in opposite directions, establishing it as the first cis-eQTL for this genomic region. Finally, I used human adipocyte in vitro culture systems to setup a pilot to study the anti-inflammatory effects of NA. The gene expression of HCAR2 and HCAR3 increased significantly with adipocyte differentiation in vitro. NA led to a drop in IL-6 transcript abundance in two out of three of the in vitro differentiated human adipocytes. In conclusion, genetic variability in HCAR2 and HCAR3 shows weak associations with cardiovascular disease risk phenotypes relating to their respective pathways. The relevance of HCAR2 and HCAR3 gene expression and the role of the receptor in the control of inflammation will require further studies.