Browsing by Subject "Maus"

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Entwicklung und Testung neuer DNA- und Protein-basierter Multikomponentenvakzinen sowie regulatorischer Adjuvanzien gegen eine Infektion mit B. anthracis in Auszucht-Mäusen und Ziegen
(2015) Köhler, Susanne Melanie; Beyer, Wolfgang
The discovery of the Sterne spore live vaccine (SSLV) and subsequently its application in a veterinary context contributed to the global reduction of Anthrax related outbreaks since 1930. Nonetheless the causative agent Bacillus anthracis is still prevalent in some mediterranean countries, South and Central America, Africa and Central Asia, as well as the USA and Canada. Reasons for this are the persistence of the pathogen in the soil, as well as still undefined factors for an ongoing cycle of outbreak and spread of the disease and the limited applicability of the SSLV. This includes the necessity to revaccinate annually, the residual virulence in certain sensitive species (e. g. goats and llamas) and the incompatibility to treat and vaccinate simultaneously. To participate in the ongoing search for alternative vaccines this work was dedicated to evaluate protein- and DNA-based components as potential ingredients for a multi-component non-living vaccine formulation (NLV). For the protein-based NLV these included rPA83 as part of the Anthrax toxin, rBclA and Formalin inactivated spores (FIS) as spore specific antigens, a Capsule-Lipopeptide conjugate as part of the vegetative form of the pathogen and a Lipopeptide-adjuvant. The DNA-vaccines consisted of vector-backbones comprising signal sequences able to direct the integrated antigens (rPA83, PAD4LFD1 and BclAD1D3) to the MHCI, MHCII and the secretory pathway. A sperate vector encoding for a positive MHCII-regulator (CIITA) and a vector internal sequence for the Interferon-ß promotor stimulator (mIPS1) served as adjuvants for the DNA-vaccines. The majority of the groups showed detectable antibody titres against their respective antigens, with protein vaccines generally eliciting higher titres against rPA83 than the DNA-vaccines. Regarding rBclA equivalent high titres were measured for protein- and DNA-vaccines alike, which also corresponded to the anti-FIS titres for groups immunized with rBclA, FIS or both. The Capsule-Lipopeptide conjugate did not elicit high titres against the capsule, possibly due to an immune suppressing epitope. Survival rates ranged between 10 and 100 %, with full protection only achieved in a combination of all antigens including FIS. All DNA-vectors induced 30 – 50 % protectiveness when given alone. Notably DNA-vectors including BclAD1D3 elicited 50 % survival and sterile immunity. A combination of the most promising vectors encoding for toxin and spore specific antigens achieved 90 % protectiveness in mice. According to the results from the mice trials, the auspicious protein- and DNA-vaccine combinations were tested in goats in comparison to the SSLV in cooperation with our project partners in South Africa and Turkey. The efficacy of the SSLV was assessed in 3 groups which were challenged shortly after the first immunisation, one year after the first immunisation or after the revaccination. Apart from the comparison of immunogenicity and protectiveness between SSLV and NLV in goats, assessment of data concerning the titre development of SSLV-immunized goats during the course of a year as well as detailed diagnostic data during the infection (behavior, temperature, bacterial loads, correlations and minimal infective dose) were integral part of this study. Compared to one another the SSLV-immunized animals showed equal or higher antibody titres against the measured antigens, with FIS and rPA83 being the most immunogen antigens. Utilizing a higher dose (75 µg) the protein-based NLV protected equivalently to the SSLV (60 – 100 %) yielding 50 % protectiveness without FIS and 80 % if FIS was included. The DNA-vaccines showed little to no immunogenicity in goats, thus no challenge was performed on these animals. The humoral reaction against BclA was generally poor in goats, which has not been noted before and could be a basis for further improvements concerning the SSLV and NLV alike. The different immunizations with the SSLV revealed a broad range for the efficacy of the first vaccination as well as a notable difference in the antibody spectrum between first vaccination and revaccination. Together with the recorded data of the antibody titre development throughout a year a more optimal protocol for immunisation with the SSLV, possibly in combination with an NLV was postulated.
Goosecoid und Calponin : zwei neue Regulatoren des PCP-Signalwegs
(2012) Ulmer, Bärbel Maria; Blum, Martin
Vertebrate embryogenesis relies on morphogenetic movements such as cell migration and convergent extension (CE). The planar cell polarity (PCP) branch of non-canonical Wnt signaling governs the orientation of cells along embryonic axes. PCP-signaling leads to intracellular polarization of proteins such as Dishevelled, Prickle and Vangl2, resulting in activation of small GTPases such as Rho and Rac, and consequently oriented alignment of the cytoskeleton. This polarity is required for CE, namely for the intercalation of bipolar cells, during gastrulation and neurulation. CE promotes elongation of the notochord and the neural plate, which is a prerequisite of neural tube closure. Previous work had shown that misexpression of the transcription factor Goosecoid (Gsc) in the primitive streak of the mouse and in the dorsal marginal zone of the frog led to neural tube closure defects. The present work demonstrates that misexpression of Gsc inhibits CE in vivo and ex vivo. Gsc gain-of-function (Gsc-GOF) prevented the membrane localization of Dishevelled in the frog animal cap assay, suggesting a disturbance of the PCP pathway. The Gsc-induced phenotypes could be rescued by co-injection of core components of the PCP pathway, Vangl2 and Prickle. Overexpression of RhoA and the non-canonical Wnt11, rescued the effect of Gsc-GOF. Brachyury, a transcriptional activator of Wnt11 and known target of Gsc, was also able to rescue the effect of Gsc-GOF. Gsc thus acted as a repressor of PCP-mediated CE. Furthermore, loss of function experiments in Xenopus were conducted to reveal the endogenous function of Gsc. Due to the conserved and distinct expression of Gsc in Spemann's organizer and the induction of double axes upon injection of Gsc into the ventral marginal zone in Xenopus, a function of Gsc in the specification of dorsal tissue was predicted. The lack of gastrulation defects in the Gsc knock-out mouse, however, questioned an early role of Gsc. The repression of the PCP pathway by Gsc-GOF suggested a novel role of Gsc in the regulation of cell movements. Interestingly, Gsc is expressed in a distinct population of cells in the early organizer, which migrate out of the organizer during early gastrulation to form the prechordal mesoderm. In contrast, the subsequent involuting cells of the notochord undergo CE. Gsc knock-down in the frog reduced the prechordal plate resulting in a narrowing of eye distance. Furthermore, activin-induced CE in animal cap explants was enhanced by Gsc loss-of-function. These findings are consistent with a novel function of the organizer gene Gsc in the regulation of cell movements during early gastrulation, namely the repression of PCP-mediated CE as a prerequisite of active migration of the prechordal mesoderm. The directed migration of neural crest cells represents another embryological process which depends on PCP-signaling. Previous work showed expression of Calponin2 in neural crest cells. Moreover, inhibition of Calponin1 by the Rho-Kinase has been described. In Xenopus, Calponin2 localized to cell protrusion of delaminating and migrating neural crest cells. Loss of function of Calponin2 prevented the polarized outgrowth of cell extensions in neural crest explants and thus migration of neural crest cells. Moreover, additional stress fibers were formed in the central area of neural crest cells at the expense of the peripheral, cortical actin cytoskeleton. The PCP pathway directs migration via the activation of RhoA and inhibition of Rac in the cell compartment opposed to the leading edge. This suggested an interaction of PCP-signaling and Calponin2 during the migration of neural crest cells, which was examined by rescue experiments in vivo and in neural crest explants. Calponin2 knock-down rescued Wnt11 and Rho-Kinase loss-of-function, strongly suggesting that the actin-binding protein Calponin2 acts as an effector of the PCP pathway and directs the polarization of the actin cytoskeleton in migrating neural crest cells. In summary the present work involved two novel regulators of PCP-mediated CE, Gsc at the transcriptional level and Calponin2 as an effector of the actin cytoskeleton.
Influence of selenium on pancreatic carcinogenesis and the role of the selenoproteins cytosolic and mitochondrial thioredoxin reductase in the pancreas
(2007) Aichler, Michaela; Graeve, Lutz
Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive cancers in humans. It is the fourth leading cause of cancer related deaths in Germany and in the United States. Most PDA occurs sporadically, but there are also approximately 5-10% of patients with a family history of pancreatic cancer. The high mortality of PDA is attributed to a lack of early detection methods and poor efficacy in therapies for advanced disease. As an alternative, preventive strategies in individuals with familial pancreatic carcinoma should be considered. Several epidemiological studies showed an inverse correlation between selenium-intake and mortality of certain types of cancer and particularly in gastrointestinal cancers. To this end, in the first part of this study, the influence of selenium as a preventive nutritional additive was investigated in a genetically defined pancreatic cancer mouse model, the EL-TGFatg/+;p53+/- mouse strain. As a major finding, the differentiation grade of the pancreatic carcinomas was heavily influenced by the selenium status. In the selenium-deficient group there were more non-differentiated pancreatic carcinomas than in the selenium-adequate group, which highlighted the implication of selenium or selenoproteins in tumour differentiation. Unexpectedly, however, there was no protective effect of selenium on total or pancreatic tumour latency. Within the selenoproteins, the thioredoxin reductases are strong candidates which may influence cell death and differentiation in pancreatic carcinogenesis. Their function is generally associated with tumour proliferation and also linked to the activation of the tumour suppressor p53. Consequently, the role of the thioredoxin reductases in the pancreas was studied in the second part of this thesis. The enzymatic activity of cytosolic (TXNRD1) and mitochondrial (TXNRD2) thioredoxin reductase in the pancreas and other organs was determined in relation to the selenium-status. TXNRD1 activity in the pancreas was moderate and decreased under selenium deficiency. TXNRD2, instead, showed very high pancreatic activity in relation to other organs and its activity was even increased under selenium-deficiency emphasising its special role in this organ. To further investigate the function of Txnrd1 and Txnrd2 in the pancreas, tissue-specific knockout mice were created and characterized. The Txnrd1 knockout mice did not show an overt phenotype. Interestingly although, pancreatic acinus cells in one year old mice showed a disturbed rough endoplasmic reticulum and alterations in serum amylase and lipase. These mice also had an impaired glucose tolerance. The pancreas of Txnrd2 knockout mice showed severe chronic pancreatitis and pancreatic atrophy at the end of an observation period of one year. The progressive pathogenic process started with mild pancreatitis, developing spontaneously at an age of four weeks. The chronic stage was characterized by the formation of different types of acinar-to-ductal metaplastic lesions, which could be classified in part as early precursor lesions of pancreatic carcinomas. The endocrine pancreas was not affected. The pancreas-specific Txnrd2 knockout mouse strain is the first genetically modified mouse model spontaneously developing acute and chronic pancreatitis. This strain constitutes a unique and powerful tool to model pancreatic pathogenesis, especially the yet unresolved process of transformation from inflammatory to malignant disease.
The effect of aging in the murine gut microbiome
(2020) Hernández Arriaga, Angélica; Camarinha-Silva, Amélia
Aging is characterized by several physiological changes. During the lifespan, the biological systems from the body of humans and other animals remain dynamic. Throughout the early stages of life, the microbiome develops into a complex ecosystem with thousands of species. Variations related to diet, environmental changes, medications affect the diversity and composition of the microbiota through the lifespan. Some old individuals with higher incidence of chronic diseases have a loss of the stability of the microbiome and an imbalance occurs between the different colonizers of the gut, also named dysbiosis. One of the most distinctive changes occurring with age is the prevalent low grade inflammation, which is named inflamm-aging. This not only changes the microbial composition of the GIT but also affects the permeability. Murine models are well established and help us to understand the complex dynamics between the host and the microbial communities inhabiting the gastrointestinal tract. These models allow us to analyze microbial communities from tissue and mucosa, from all sections of the gut, which is limited in humans. Methods standardization is an important topic in microbiome research. In chapter 2 it was compared the efficiency of two sample methods, cotton swab and tissue biopsy, in characterizing the mouth microbiota. In recent years, the mouth microbiome is being seen as a diagnostic tool for not only oral diseases but also systemic diseases. As physiological changes occur with aging, the microbiome from the mouth is affected and there is an increase of pathogens present in the oral surfaces. In murine models, cotton swab is a common tool used for sampling the microbiome of the oral cavity. In our study, we observed similar microbial community structure using both methodologies. However, the species Streptococcus danieliae, Moraxella osloensis, and some unclassified members of Streptococcus were affected by the different sampling procedures. In this trial, we included mice at two different ages, 2 months old being considered young and 15 months old considered middle aged mice. We observed changes in the genera Actinobacillus, Neisseria, Staphylococcus, and Streptococcus related to the age of the animal and the sampling type. These results showed the importance of sampling standardization in microbiome research and that age has a strong effect on the microbial ecology of the oral cavity. In chapter 3, it was studied the bacterial communities from duodenum and colon of mice at 2, 15, 24 and 30 months of age in combination with the results of the expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function. Besides, in this chapter were also assessed the indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissues. At 24 and 30 months of age there was an increase in inflammation, they developed fibrosis and the levels of endotoxin in plasma were higher. Regarding changes in the microbiome, the duodenum had more changes than the colon related to age. Allobacullum, Bifidobacterium, Olsenella, Corynebacterium were the genera that differed statistically in the duodenum through the murine lifespan. Fewer changes were observed in the colon, as Allobaculum was the only genus that showed differences between young and old mice. Additionaly, it was analyzed the impact of aging in the active microbial communities of mouth, duodenum and colon at 2, 9, 15, 24 and 30 months of age (chapter 4). Changes were observed at every age and different taxonomical levels, with a greater shift at 15 months of age. This is related to the age of the mice, as at middle age systemic changes related to the aging process start to occur. At old ages, there was an increment of the pathobiontic species Helicobacter hepaticus and Helicobacter ganmani in the duodenum and colon. The oral, duodenal and colonic microbial communities are important pieces of information that can be related to the health status of the host. Research that focuses on assessing the changes in the different niches and not only in the feces, gives a broader overview of the microbial community of the host.