Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 17/19 Archiv

Background: Stigma can be detrimental to the quality of life, as well as the treatment and rehabiltation process of people with mental illness. The purpose of this study was to measure the extent and determine correlates of public and self-stigma against people with mental illness (PWMI) and their families in Jimma Zone, Southwest Ethiopia. Methods: Community and institution based quantitative and qualitative cross-sectional studies were conducted among 845 randomly selected community members at GGFRC, consecutive 422 PWMI and 422 family members of PWMI at Jimma University Specialized Hospital. Univariate, bivariate and multivariate linear regression analyses were done. Results: The mean scores of public stigma against PWMI and their family members were 2.62 (+0.34) and 2.16 (+0.49), respectively, on a range of 1 to 5. The mean self-stigma score among PWMI, on a range of 1 to 4, was 2.32 (+0.30). Place of residence, belief in the supernatural, psychosocial and biological explanations of mental illness were associated with stigma towards PWMI and family members of PWMI. Level of education and income predicted PWMI public stigma. A higher number of perceived signs of mental illness was correlated with lower stigma against family members of PWMI. Females, individuals with history of traditional treatment, individuals experiencing higher number of drug side-effects, and individuals who subscribed to more signs and supernatural explanations had significantly higher levels of self-stigma. In contrast, patients with higher education level and higher self esteem showed significantly lower levels of self-stigma. Supporting supernatural explanations of mental illness was associated with greater care-givers’ self-stigmatization. Conclusion: High public stigma against PWMI and high levels of patients’ self-stigma were found. Care-givers demonstrated reluctance to be identified with PWMI. Systematic forms of discrimination against PWMI and their family members were identified. PWMI and their family members faced behavioral and structural challenges. Thus, reducing stigma against patients may help to reduce stigma against family members. Developing strategies to improve patients’ self esteem, and developing policies and guidelines about mental illness may be helpful in reducing stigma. Effective intervention strategies that target patients, their families, as well as the public need to be designed to reduce stigma.

Neurodegenerative disease patients suffer from cognitive decline and/or motor dysfunctions, depending on the different regions affected by the neuron loss. With aging being the major risk factor and a society with increased life expectancy, there is an urgent need to develop new effective treatments to alleviate the situation faced by patients, their families and society. Although neurodegenerative diseases including Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) lead to different clinical symptoms, they share common pathomechanisms, such as protein aggregation and altered RNA metabolism. A subset of ALS and FTD cases, for instance, is pathologically characterized by neuronal cytoplasmic inclusions containing aggregated Fused in Sarcoma (FUS) protein. There is also a genetic link, since FUS mutations cause ALS with FUS pathology. FUS is a DNA/RNA-binding protein known to regulate different steps of RNA metabolism, however, its exact function and target genes in neurons were unknown. In this study, I evaluated the neuronal role of FUS in alternative splicing using a candidate approach focusing on the microtubule-associated protein TAU. TAU is one of the most widely studied proteins in neurodegeneration research due to its aggregation in different tauopathies, most notably AD. Mutations in the TAU gene MAPT, that affect alternative splicing of exon 10, are known to cause another subtype of FTD. Here, I demonstrate that FUS depleted rat neurons, although having normal viability, show aberrant alternative splicing of TAU, with increased inclusion of exon 3 and exon 10, resulting in higher expression of the 2N and 4R TAU isoforms. Importantly, reintroduction of human FUS rescues aberrant splicing of TAU in FUS depleted neurons. Accordingly, overexpression of FUS decreases expression of 2N and 4R TAU isoforms. In mouse brain lysates, I detected direct FUS binding to TAU pre-mRNA, with strong binding around the regulated exon 10, often at AUU-rich RNA stretches. Since TAU splicing is regulated differently in humans and rodents, I also confirmed the role of human FUS in TAU exon 10 splicing using a TAU minigene and a human neuronal cell line. In addition, I analyzed the morphology and development of axons to evaluate the functional consequences of FUS depletion in neurons. Although FUS depleted neurons develop neurites normally, their axons are significantly shorter than in the control cells. Similar to observations in TAU/MAP1B knockout neurons, axons of FUS depleted neurons develop significantly larger growth cones with abnormal cytoskeletal organization. The development of growth cones in vivo is an essential step in axonal maintenance and repair. Altogether, this study identified TAU as the first physiological splice target of FUS in neurons. The newly discovered role of FUS in regulating the axonal cytoskeleton indicates that aberrant axonal function could contribute to the neuron loss seen in ALS/FTD cases with FUS aggregates.

Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor that is characterized by abundant tumor stroma and a potent immunosuppressive microenvironment. Further studies to clarify why T cells infiltrate the tumor but are not able to perform effector functions as well as to find new effective therapies to overcome immunosuppression are urgently needed. The aims of the present study were (1) to characterize different murine PDAC tumor models in regard to their utility for studying novel immunotherapeutic approaches, (2) to assess the therapeutic efficacy of a novel bifunctional ppp-siRNA that combines silencing of the immunosuppressive molecule galectin-1 and RIG-I-mediated immune activation in murine PDAC and (3) to characterize the immunosuppressive mechanisms leading to T cell inhibition in the tumor microenvironment. (1) This study revealed that the pancreatic cancer cell line T110299, which was developed from a primary tumor of the KPC mouse model, is a new valuable tool for studying novel treatment strategies for PDAC. The histological appearance of T110299 tumors reflects in many aspects the primary tumors in KPC mice, which harbor mutations in the Kras oncogene and p53, and the human disease with regard to tumor differentiation, extensive tumor stroma development, poor vascularization and expression of immunosuppressive molecules, like indoleamine 2,3-dioxygenase (IDO) and galectin-1 (Gal-1). In contrast, Panc02 tumors were found to have a sarcomatoid architecture with very little tumor stroma. In Panc02 cells, galectin-1 was strongly expressed by the tumor cells, which differs from the situation found in humans, KPC mice and T110299 tumors, where galectin-1 is preferentially expressed in tumor-associated pancreatic stellate cells (PSC). However, expression of the cytosolic helicase RIG-I was functional in Panc02 cells, but defective in T110299 cells. As RIG-I is expressed in all human PDAC cell lines tested, the Panc02 model appears to be better suited to study RIG-I-based immunotherapies (Ellermeier et al., 2013). Thus, the histological and functional characterization of the tumor models in this thesis will allow selecting the best-suited tumor model for addressing specific aspects of immunotherapy. (2) Treatment studies of PDAC were performed with the 5’ppp-modified siRNA molecule ppp-Gal-1 in the orthotopic Panc02 tumor model. The dual activities of this molecule were confirmed in vitro, leading to (i) reduced galectin-1 expression via RNAi; and (ii) production of CXCL10 and IFN-, MHC-I up-regulation and apoptosis of tumor cells via RIG-I activation. Treatment of mice with orthotopic pancreatic tumors with ppp-Gal-1 significantly prolonged survival, as compared to unmodified OH-Gal-1 or control RNA. In addition, 20% of the mice completely rejected their tumors leading to long-term tumor control. Thus, bifunctional 5’ppp-modified siRNA is a promising treatment strategy for PDAC deserving further pre-clinical evaluation. (3) Pancreatic tumor cells employ multiple mechanisms for suppression of T cell responses. This study identified TGF-β and IDO as two potent mechanisms leading to inhibition of T cell proliferation. Minute amounts of PDAC supernatants effectively blocked T cell proliferation induced by CD3 and CD28 triggering. This could be partially prevented by SD-208, a small molecule inhibitor of TGF-β receptor signaling, or by blocking IDO activity with D-1-MT. Interestingly, tumor supernatants induced up-regulation of IDO mRNA expression in T cells. Furthermore, blocking IDO activity in T cells appeared to be more effective than blocking IDO in tumor cells. This leads to a new hypothesis that factors secreted by the tumor cells induce IDO expression in T cells, which in turn leads to auto-intoxication of the T cells via kynurenine production and eventually T cell apoptosis. Further studies confirming this hypothesis are warranted.

Obesity is a major public health challenge. Modeling growth and identifying children at risk of being overweight in early life is essential for effective prevention and intervention. To date, longitudinal studies from birth to adolescent are rare, and crucial period in childhood for overweight in future life is unclear. In addition, apart from known risk factors of overweight or obesity, further research on other potential risk factors is necessary. Even though it is well accepted that obesity is mainly a consequence of an unbalanced energy status, the impact of specific food item or food group on growth has long been controversial, which calls for more efforts. In order to contribute to the aforementioned questions and research problems, we conducted a series of studies using data from two ongoing German birth cohorts. 1) Using longitudinal anthropometric data from birth to 5 years, we established a 10-year-overweight prediction model. Our results suggest that from 5 years onwards being overweight become predictive for 10-year overweight. 2) We investigated the association between mode of delivery and childhood obesity using cross-sectional data at age 2, 6 and 10 years. Children delivered by cesarean section were more likely to be obese at 2 years compared to those delivered vaginally, but not at age 6 and 10 years. These results do not support the hypothesis that increasing rates of cesarean section contribute to the childhood obesity epidemic. 3) We analyzed data from food frequency questionnaires which were completed during the 10-year follow-up, where we grouped 82 food items into 11 food groups. Our results suggest that intakes of meat, fish and beverages are important correlates of body weight status. In contrast, confectionery intake conversely associated with being overweight. Further study on the influence of possible reverse causation is needed. 4) We investigated the association between maternal BMI and child food intake at the age of 10 years. Our results suggest that maternal BMI and maternal overweight are important correlates of a child’s intake of energy, meat and eggs. Potential impact of mother’s weight status should be considered in diet counseling. Moreover, mother’s participation in dietary counseling might be helpful to improve offspring diet pattern and weight status. These studies contributed to the current knowledge on early identification of children at risk of overweight and the potential influence of diet. In addition, our results suggest more efforts on longitudinal studies and more attention on reverse causation and misreport in dietary assessments in future studies.

Multiple Sclerosis is most probably an autoimmune inflammatory disease of the central nervous system. Demyelination of neurons and axonal loss occur in temporal and spatial resolution in multiple areas of the brain and spinal cord. This impairment manifests in neurological symptoms. The course of disease varies between individuals and the causing mechanisms still remain elusive. Environmental factors as well as genetic predispositions are widely discussed. Recent studies stressed the prominent role of CD8+ T lymphocytes in the autoimmune pathomechanism. They notably exceed the amount of CD4+ T cells in acute lesions yet target cells and activating antigens remain elusive. A genetic linkage between the human leukocyte antigen gene locus and disease susceptibility was observed. Carrying the HLA A*0301 allele or the HLA A*0201 allele correlates with a risk factor or protective effect for Multiple Sclerosis susceptibility, respectively. Thus during this thesis two main questions were followed: 1. Which CD8+ T lymphocytes participate in the autoimmune attack on central nervous system tissue and what are their receptors for antigen recognition? 2. How does the expression of HLA-A2 molecules lead to a decreased disease susceptibility? In the first part, the T cell receptor molecules of potentially disease-related single CD8+ T cells from frozen patient tissue samples were characterized. T lymphocytes were considered disease-related when they either belonged to a clonally expanded T cell population or expressed an activation marker on their cell surface. In a clone-specific approach, the T cell receptor beta chains of pre-analyzed, clonally expanded T cell populations were investigated. Further an unbiased approach independent of pre-analyses was established. In the second part, antigen recognition of the probably disease-related T cell receptor 2D1 was investigated. This receptor was isolated from a Multiple Sclerosis patient and was known to be activated by a myelin-derived peptide presented on HLA-A3 molecules. In an animal model double-transgenic mice expressing HLA-A3 and the 2D1 T cell receptor developed a Multiple Sclerosis-like disease after immunization with the known peptide. Surprisingly not a single triple-transgenic mouse expressing HLA-A3, the T cell receptor 2D1 and HLA-A2 showed symptoms after immunization. In these mice 2D1 T lymphocytes were shown to be depleted in the thymus. To characterize HLA-A2-bound peptides which mediate this protective effect a novel technology for unbiased identification of antigenic peptides recognized by human leukocyte antigen class I-restricted T lymphocytes was employed. 28 peptides presented on HLA-A2 molecules were found to be recognized by the T cell receptor 2D1. Those peptides displayed very closely related sequences. Eight possible parent proteins existing in mouse, therefrom even four equally expressed in humans were identified. Finally those putative parent proteins were further characterized and first investigations of antigen processing in different antigen presenting cell lines were performed.