The clinical impact of human cytomegalovirus (CMV) and progression to CMV disease in immunocompromised patients has been reduced by therapeutic strategies using ganciclovir, valganciclovir, foscarnet and cidofovir. However, extensive antiviral therapy increases the risk of antiviral resistance due to mutations in the UL97 protein kinase and UL54 DNA polymerase. Co-infection with HHV-6 or HHV-7 is also associated with increased CMV reactivation and disease. Genotypic CMV antiviral resistance was identified in 38% of Australian immunocompromised patients. While UL97 mutations only were identified in 23% of patients, additional UL54 mutations, with the potential to confer multidrug resistance, were detected in 15% of patients. Antiviral resistant CMV strains were found to emerge rapidly in highly immunocompromised patients, and some strains were able to persist in the absence of selective pressure. Three new mutations were identified (UL97 - N597D, UL54 - F412S, D485N). N597D was characterised by recombinant phenotyping and conferred minimal ganciclovir resistance. Neither baculovirus nor coupled transcription/translation yielded full-length UL54 protein (pUL54; ~140 kDa) for activity assays. However, truncated pUL54 (~66 kDa) was purified after prokaryotic expression. HHV-6 and HHV-7 co-infection was a common clinical occurrence; with 36% of liver transplant recipients infected with HHV-6 (11% persistent) and 80% with HHV-7 (52% persistent). ValGCV therapy did not significantly alter the incidence of HHV-6, HHV-7 or co-infection. The most prevalent co-infection pattern was CMV, HHV-6 and HHV-7 (46%) and both CMV and HHV-7 (38%). CMV reactivation was predominantly independent of HHV-6/HHV-7, although 27% of patients had initial HHV-7 reactivation. Despite frequent co-infection, HHV-6 and HHV-7 were not associated with clinical disease, with possible exception of HHV-7 and acute cellular rejection. CMV antiviral resistance remains a significant issue in transplantation, emphasising the importance of antiviral resistance testing in an era of widespread prophylaxis. New mutations in UL97 and UL54 continue to be identified. Further characterisation of UL54 mutations using polymerase activity assays would increase our knowledge of enzymological basis of antiviral resistance. Co-infection with HHV-6 and HHV-7 is common in transplant recipients, but does not play a significant role in disease. Similar co-infection rates between valGCV-treated and untreated patients indicate that valGCV is not highly effective against HHV-6 and HHV-7.

Bibliography: leaves 103-116. / vi, 116 leaves, [6] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Addresses the practical aspects of using nuclear transfer for the production of identical animals. Results from experiments provide improved understanding of the technical constraints of nuclear transfer. Also the flexibility of the methodology was increased with the use of in vitro culture and/or in vitro matured oocytes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1992

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Xenotransplantation is the final frontier of using vascularised organs or cellular grafts to treat end-organ disease and offers a potential solution to the worldwide shortage of human tissue available for transplantation. The main immunological barrier to xenografting from pig-to-primate is the antigen, Galactose-α1,3-Galactose (Gal) which is found in all species except humans and other higher primates. Even with the major advancement of deleting Gal from the potential pig donor species with the aid of cloning technology, complete elimination may be elusive as alternative genes yet to be fully characterised, may still produce Gal at low levels. Thus, the human immune response against Gal may continue to be a barrier to successful xenotransplantation. The aim of this project was to develop small animal models of the important components of xenograft rejection that largely relate to the anti-Gal immune response. These include models of hyperacute, acute vascular and chronic xenograft-like rejection that in turn, provide new insights in the immune mechanisms of the rejection processes. The role of antibody and both innate and cognate cellular immunity are explored. Both vascularised heart grafts and non-vascularised skin graft models are examined as rejection of solid organs may differ from cellular transplantation. The project also provides a platform for future studies in testing genetic and pharmacotherapeutic strategies to overcome the rejection processes uncovered.

Doctor of Philosophy (PhD) / The BCNU + O6benzylguanine (O6BG) driven selective enrichment strategy was first established for enhanced transplantation of hematopoietic stem cells. This study describes a novel application of this BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation. Furthermore, this study addresses the three main limitations observed in previously reported skeletal muscle stem cell transplantation strategies. Limitation of ineffective donor cells which lack the ability for successful engraftment was overcome by using a heterogeneous population of donor cells which are present during a normal skeletal muscle regeneration response. The limitation of donor cell death upon transplantation as a result of competition from the endogenous stem cells of the host muscles was overcome by elimination of host muscle stem cells with BCNU + O6BG treatment. Efficiency of elimination of host muscle stem cells was further demonstrated by the complete inhibition of a regeneration response up to 3 months in injured, BCNU + O6BG treated muscles. The limitation of localised engraftment as a result of intramuscular injection of donor cells was also addressed. The transplanted donor cells demonstrated the ability to migrate via systemic circulation. This characteristic of the donor cells would allow the transplantation of cells via intraarterial or intravenous delivery which would overcome the limitation of localised engraftment. Finally, application of the BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation demonstrated enhanced engraftment. This is the first reported attempt of enhanced stem cell transplantation in a solid tissue achieved upon application of the BCNU + O6BG driven selective enrichment strategy. This study provides the basis for application of the BCNU + O6BG driven selective enrichment strategy in other tissues where stem cell transplantation is considered.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.