@phdthesis{Kohl2009,
  author    = {Kohl, Bianca Dorothea},
  title     = {PMP22-overexpressing mice as a model for Charcot-Marie-Tooth 1A neuropathy implicate a role of immune-related cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-43066},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2009},
  abstract  = {Charcot-Marie-Tooth disease (CMT) is a cohort of human hereditary disorders of the peripheral nervous system (PNS) which exhibit symptoms like sensory dysfunction, muscle weakness and gait disturbances. Different mutations are described as causation for this neuropathy, such as a duplication of chromosome 17 comprising the gene for the peripheral myelin protein-22 (PMP22). Based on different animal models former studies identified immune cells, i.e. macrophages and T-lymphocytes, as crucial mediators of pathology in these neuropathies. In this study, PMP22-overexpressing mice (PMP22tg, C61), serving as a model for a specific type of CMT - CMT1A - were crossbred with immune-deficient mutant mice to examine the impact of the immune system on nerve pathology. Crossbreeding of PMP22tg mice with recombination activating gene-1 (RAG-1) deficient mice, lacking mature T- and B-lymphocytes, caused no striking alterations of pathogenesis in peripheral nerves of mutant mice. In contrast, crossbreeding of PMP22tg myelin mutants with mice deficient in the chemokine monocyte chemoattractant protein-1 (MCP-1, CCL2) caused an amelioration of the demyelinating phenotype of peripheral nerves when MCP-1 was either reduced or completely absent. Furthermore, functional investigations, i.e. neurographic recordings and examinations of the grip strength of the extremities, revealed an amelioration in PMP22tg/MCP-1-/- mice in regard to a symptomatic improvement in the compound action muscle potential (CMAP) and stronger grip strength of the hindlimbs. Interestingly, peripheral nerves of PMP22tg mice showed an irregular distribution of potassium channels in presence of MCP-1, whereas the absence of MCP-1 in the myelin mutants rescued the ion channel distribution and resulted in a more wild type-like phenotype. Having shown the impact of MCP-1 as an important mediator of nerve pathology in PMP22/MCP-1 double mutants, the regulation of this chemokine became an important target for potential treatment strategies. We found that the signaling cascade MEK1/2/ERK1/2 was more strongly activated in peripheral nerves of PMP22tg mice compared to nerves of wild type mice. This activation corresponded to an increase in MCP-1 mRNA expression in peripheral nerves at the same age. Furthermore, a MEK1/2-inhibitor was used in vivo to confirm the regulation of MCP-1 by the MEK1/2/ERK1/2 pathway. After a treatment period of three weeks, a clear reduction of ERK1/2-phosphorylation as well as a reduction of MCP-1 mRNA expression was observed, accompanied by a decline in macrophage number in peripheral nerves of PMP22tg mice. These observations suggest that the expression of MCP-1 is crucial for the neuropathological progression in a mouse model for CMT1A. Therefore, this chemokine could provide a basis for a putative treatment strategy of inherited neuropathies.},
  subject      = {Myelin},
  language  = {en}
}
@phdthesis{Galmbacher2008,
  author    = {Galmbacher, Katharina Monika},
  title     = {Caspase-1 as a target of bacterial tumor therapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-33506},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2008},
  abstract  = {Tumorstroma und Tumor-assoziierte Makrophagen (TAMs) spielen in neoplastischen Erkrankungen eine wichtige Rolle im Bezug auf Tumorwachstum und Progression. In einigen Krebsarten besteht zwischen den Tumor-assoziierten Makrophagen und den Krebszellen eine intensive Interaktion, welche zu vermehrter Angiogenese und zur Unterdr{\"u}ckung lokaler Immunantworten f{\"u}hrt. Aus diesem Grund stellen TAMs einen vielversprechenden Angriffspunkt f{\"u}r eine Krebstherapie da. In dieser Arbeit wird gezeigt, dass intrazellul{\"a}re Bakterien wie Salmonella und Shigella haupts{\"a}chlich TAMs im Tumorgewebe infizierten. Um dieses Verhalten n{\"a}her zu untersuchen, konstruierten wir einen im Wachstum abgeschw{\"a}chten Shigella Stamm, welcher jedoch noch die F{\"a}higkeit hat, Apoptose in Makrophagen zu induzieren. Shigellen sind invasive Bakterien, die in das Darmgewebe einwandern und dort eine massive Inflammation induzieren. Intrazellul{\"a}re Shigellen aktivieren Caspase-1 und induzieren dadurch Apoptose in Makrophagen durch den sekretierten Virulenzfaktor IpaB. Durch eine Deletion des genomischen aroA-Gens, wurde ein Shigella Stamm konstruiert, der Defekte im intrazellul{\"a}ren Wachstum aufweist. Dennoch war dieser Stamm noch f{\"a}hig eukaryotische Zellen zu infizieren, sich interzellul{\"a}r fortzubewegen, Caspase-1 zu aktivieren und Apoptose in Makrophagen zu induzieren. Es wurde gezeigt, dass dieser Shigellen Stamm nach i.v. Injektion haupts{\"a}chlich die TAMs im 4T1-induzierten und transgenen MMTV-HER2/neu Brustkrebsmodel infizieren. Diese attenuierten Shigellen wurden im Tumorgewebe haupts{\"a}chlich intrazellul{\"a}r detektiert, im Gegensatz dazu wurden attenuierte Salmonellen zu sp{\"a}ten Zeitpunkten (7 d p.i.) auch extrazellul{\"a}r im Tumorgewebe aufgefunden. Der metabolisch aber nicht in der Virulenz attenuierte Shigella Stamm konnte in beiden Brustkrebsmodellen zu allen Zeitpunkten (4 h, 6h and 7 d p.i.) Caspase-1 in TAMs aktivieren und Apoptose induzieren. Diese Apoptose f{\"u}hrte in beiden Brustkrebsmodellen zu einer langandauernden und hoch signifikanten Reduktion der TAMs-Anzahl (bis zu 70 \%). Im Gegensatz dazu konnten Salmonellen nur zu fr{\"u}hen Zeitpunkten (6 h p.i.) Apoptose in TAMs induzieren und dies f{\"u}hrte in beiden Modellen zu keiner Reduzierung der TAMs-Anzahl. In dem 4T1-induzierten Tumormodel wurden die M{\"a}use mit dem attenuierten Shigella Stamm behandelt, was zu einer kompletten Blockierung des Tumorwachstums f{\"u}hrte, dies traf aber nicht f{\"u}r den avirulenten Stamm zu. Dar{\"u}berhinaus infizierte Shigella haupts{\"a}chlich die Makrophagen Fraktion eines Ovarkarzinoms ex vivo und induzierte in diesen Zellen Caspase- 1 Aktivierung und Apoptose. Zusammenfassend zeigen diese Daten, dass im Wachstum attenuierte intrazellul{\"a}re Bakterien dazu f{\"a}hig sind Apoptose in TAMs zu induzieren. Dadurch werden sie zu einem vielversprechenden Therapeutikum zur Behandlung von betimmten Krebserkrankungen, bei denen TAMs eine erwiesene Rolle im Tumorwachstum und der Tumorprogression spielen.},
  subject      = {Shigella flexneri},
  language  = {en}
}
@phdthesis{Fischer2008,
  author    = {Fischer, Stefan Martin},
  title     = {Regulation and functional consequences of MCP-1 expression in a model of Charcot-Marie-Tooth 1B disease},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-29189},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2008},
  abstract  = {Charcot-Marie-Tooth 1B (CMT1B) is a progressive inherited demyelinating disease of human peripheral nervous system leading to sensory and/or motor function disability and is caused by mutations in the P0 gene. Mice heterozygously deficient for P0 (P0+/-) are an adequate model of this human disorder showing myelin degeneration, formation of onion bulbs, remyelination and a reduced motor conduction velocity of around 30m/s similar to patients. Previously, it had been shown that T-lymphocytes and macrophages play a crucial role during pathogenesis in peripheral nerves of P0+/- mice. Both, T-lymphocytes and macrophages increase in number in the endoneurium and deletion of T-lymphocytes or deletion of a macrophage-directed cytokine ameliorates the disease. In this study the monocyte chemoattractant protein-1 (MCP-1) was identified as an early regulated cytokine before onset of disease is visible at the age of six months. MCP-1 mRNA and protein expression could be detected in femoral quadriceps and sciatic nerves of P0+/- mice already at the age of one month but not in cutaneous saphenous nerves which are never affected by the disease. MCP-1 was shown to be expressed by Schwann cells and to mediate the immigration of immune cells into peripheral nerves. Deletion of MCP-1 in P0+/- mice accomplished by crossbreeding P0 and MCP-1 deficient mice revealed a substantial reduction of immune cells in peripheral nerves of P0+/-/MCP-1+/- and P0+/-/MCP-1-/- mice at the age of six months. In twelve months old mice reduction of immune cells in peripheral nerves is accompanied by amelioration of demyelinating disease in P0+/-/MCP-1+/- and aggravation of demyelinating disease in lumbar ventral roots of P0+/ /MCP-1-/- mice in comparison to P0+/ /MCP 1+/+ mice. Furthermore, activation of the MEK1/2-ERK1/2 signalling cascade could be demonstrated to take place in Schwann cells of affected peripheral nerves of P0+/- mice overlapping temporarily and spatially with MCP-1 expression. An animal experiment using a MEK1/2-inhibitor in vivo, CI-1040, revealed that upon reduction of ERK1/2 phosphorylation MCP-1 mRNA expression is diminished suggesting that the activation of the MEK1/2-ERK1/2 signalling cascade is necessary for MCP-1 expression. Additionally, peripheral nerves of P0+/- mice showing reduced ERK1/2 phosphorylation and MCP-1 mRNA expression also show reduced numbers of macrophages in the endoneurium. This study shows a molecular link between a Schwann cell based mutation and immune cell function. Inhibition of the identified signalling cascade might be a putative target for therapeutic approaches.},
  subject      = {Schwann-Zelle},
  language  = {en}
}
@phdthesis{FernandezMora2005,
  author    = {Fern{\´a}ndez-Mora, Eugenia},
  title     = {Analysis of the maturation of Rhodococcus equi-containing vacuoles in macrophages},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-14049},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2005},
  abstract  = {Rhodococcus equi is a Gram-positive intracellular pathogen which can cause severe bronchopneumonia in foals. In recent years, the role of this bacterium as human pathogen has been noted, as R.equi infections in humans have increase in frequency. This increase is associated with the rise in immunosupressed individuals, specially AIDS patients, where infection leads to symptoms and pathology similar to those seen in foals with a high mortality rate. Due to its capability to survive and multiply in murine and equine macrophages, R.equi has been classified as a facultative intracellular bacterium. R.equi is found frequently in macrophages in alveolar infiltrate from infected animals. The pathogenicity of R.equi depends on its ability to exist and multiply inside macrophages and has been associated with the presence of virulence plasmids. It has been observed that, inside foal alveolar macrophages, R.equi-containing vacuoles (RCVs) do not mature into phagolysosomes. However, most of the intracellular events during R.equi infection have not been investigated in detail. The aim of this study was to elucidate the intracellular compartmentation of R.equi and the mechanism by which the bacteria avoid destruction in host macrophages. The importance of the virulence-associated plasmids of R.equi for the establishment of RCVs was also evaluated. Furthermore, the intracellular fate of viable and non-viable R.equi was compared in order to study whether viability of R.equi influeciantes the establishment of RCVs. In this study, the RCV was characterized by using a variety of endocytic markers to follow the path of the bacteria trhough murine macropages. Transmission electron microscopy-base analysis showed that R.equi was found equally frequently in phagosomes with loosely or thightly apposed membranes, and RCV often contains numerous membranous vesicles. Laser scanning microscopy of infected macrophages showed that the majority of phagosomes containing R.equi acquired transiently the early endosomal markers Rab5, Ptlns3P, and EEA-1, suggesting initially undisturbed phagosome maturation. Although the RCV acquired some late endosomal markers, such as Rab7, LAMP-1, and Lamp-2, they did not acquired vATPase, did not interact with pre-labeled lysosomes, and failed to acidify. These data clearly suggest that the RCV is a compartment which has left vacuoles that resemble multivesicular body compartments (MVB), which are transport intermediates between early and late endosomes and display internal vesicles very similar to the ones observed within RCVs. Analyisis of several R.equi strains containing either VapA- or VapB-expressing plasmids or neither demonstrated that the possession of the virulence-associated plasmids does not affect phagosome trafficking over a two hour period of infection. The finding that non-viable R.equi was still able to inhibit phagosome maturation (although not to the same extent as viable R.equi did) suggests that heat-insensitive factors, such as cell periphery lipids, may play a major role in inhibition of phagosome maturation, although heat-sensitive factors may also be involved.},
  subject      = {Rhodococcus equi},
  language  = {en}
}