Errata posted inside back end-paper (leaf 250). / Bibliography: leaves 204-249. / xxiv, 250 leaves, [8] 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. / Investigates the nature of the seminal vesicle-derived trigger(s) that stimulate the granulocyte-macrophage colony-stimulating factor release from the murine uterine epithelium. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1999

In the adult central nervous system (CNS) several factors are implicated in the failure of neurons to regenerate after spinal cord injury (SCI). However, this reduced ability of injured CNS neurons to regenerate can be improved by under certain conditions. For instance, in adult dorsal root ganglion (DRG) neurons, injury to its peripheral branch (unilateral conditioning lesion) prior to injury of its central DRG branch (dorsal column cut) enhances the intrinsic capability of some but not all CNS afferent neurons to regenerate. The exact mechanism mediating this type of response is not known. However, previous studies by other groups have proposed that the regeneration of these CNS afferent neurons might be associated with the inflammatory response following injury to the peripheral DRG branch. Our general aim, was to examine the involvement of the immune response in the regeneration of the CNS DRG branch, as part of the pre-conditioned lesion model. To test this, three questions/hypotheses were investigated. Firstly, we investigated the effects of vaccination in pre-conditioned lesion animals using a peripheral nerve homogenate (PNH, sciatic nerve) as the immunogen. Given the regenerative capabilities of peripheral nerves, we proposed that exposure to this homogenate could enhance the limited regeneration of CNS fibres, after pre-conditioning of DRG neurons. Our results showed that in adult and/or neonatal Sprague Dawley (SD) rats PNH-vaccinated, had greater number of regenerated fibres, as compared to injury matched saline-vaccinated controls. Conversely, passive exposure to PNH through parental vaccination resulted in the suppression of this regenerative trigger. This suppressed competence of CNS fibres to regenerate was indirectly correlated with a reduced number of macrophage cells throughout the SCI epicentre, as compared to greater macrophage numbers found in the adult and/or neonatal treated groups. Secondly, we explored the possibility that a systemic inflammatory effect originating from the peripheral conditioning lesion, might be able to contribute to the regeneration of other injured neurons within the matured CNS. Again, using adult SD rats, we pre-conditioned the peripheral DRG branch as previous and changed the location of the CNS injury from the spinal cord to the optic nerve. Where alike any other injured neuron within the CNS, fails to regenerate. Unfortunately, our results from anterograde or retrograde labelling did not find any regenerated optic nerve fibres, although, we did find macrophage numbers to be higher in pre-conditioned lesion animals as compared to sham-operated animals. Therefore, it is possible that the pre-conditioning peripheral lesion might be allowing for a greater macrophage infiltration into the CNS compartment. Finally, we determined whether an early macrophage infiltration into the CNS compartment could be correlated with the observed CNS regeneration, characteristic of the pre-conditioned lesion model. To test this, we temporarily depleted macrophages before, during and after peripheral nerve lesion, via liposomal clodronate delivery. Our results from anterograde and retrograde labelling of spinal cord fibres revealed no regenerated CNS fibres in macrophage depleted animals, only in injury matched controls. In conclusion, macrophage cells play a beneficial role in the regeneration of CNS afferent fibres of pre-conditioned lesion DRG neurons. This most likely occurs through activation of intrinsic somatic DRG responses, as well as, an increased macrophage activation. We believe this inflammatory response to be of favourable phenotypic characteristic to the regeneration of injured CNS neurons, especially those in proximity to the DRG cell body. In addition, we propose that the conditioning peripheral lesion permits an influx of macrophage cells into the CNS compartment before injury of the CNS DRG branch, which is also likely to be supporting regeneration of afferent fibres. Future studies should evaluate the possibility that activated inflammatory cells might be infiltrating into the CNS under minimal blood-brain barrier disruption. It is clear that a complex communication between the nervous and immune system is occurring after the initial peripheral injury.

Resident tissue macrophages are an integral component of many tissues and are important in development, homeostasis and repair. Macrophages are present at sites of both pathologic bone deposition and loss, and can produce osteo-active factors. These observations link macrophages to bone disease, however their contribution to bone dynamics is poorly understood. The molecular and cellular mechanisms driving osteoblast differentiation, matrix deposition and mineralization in vivo are incompletely understood and this deficiency is translated to limited ability to clinically manipulate bone formation. The emerging understanding of the bi-directional interactions between the osseus and immune systems (osteoimmunology) provides a novel avenue to identify mechanisms involved in the regulation of bone formation. In this study, the presence and distribution of macrophages on bone surfaces was systematically analysed and their functional contribution to the bone microenvironment was investigated. Using immunohistochemistry a discrete population of mature resident tissue macrophages was demonstrated throughout resting murine osteal tissues, termed OsteoMacs. Utilising MacGreen mice (csf1r promoter drives eGFP transgene expression in macrophages and other myeloid cells), it was demonstrated that OsteoMacs were intercalated amongst other bone lining cells in both the endosteum and periosteum. OsteoMacs were TRAPneg in situ and had limited osteoclastogenic ability in vitro therefore they are unlikely to serve as the immediate physiologic osteoclast precursors in vivo. Microarray gene expression profiling demonstrated that macrophage gene expression was regulated in response to a characteristic feature of the bone microenvironment, elevated extracellular calcium. Quantitative PCR validated upregulation of sphingosine kinase 1, interleukin 1 receptor antagonise, progressive ankylosis, vascular endothelial growth factor c and dipepetidase 2 mRNA in response to elevated extracellular calcium, suggesting the potential roles of these genes in this unique niche. GNF Symatlas microarray and quantitative PCR demonstrated the expression of macrophage-restricted genes throughout a 21-day primary osteoblast differentiation time course, suggesting co-isolation of OsteoMacs with primary osteoblasts. Flow cytometry analysis confirmed that over all 15.9% of the digested primary calvarial cell preparations were OsteoMacs. Immunocytochemistry demonstrated that OsteoMacs persisted and expanded in standard 21-day osteoblast differentiation assays. Contrary to previous studies, we demonstrated it was the OsteoMacs, and not osteoblasts, within calvarial preparations that selectively detected patho-physiological concentrations of the bacterial product lipopolysaccharide (LPS). A protocol was developed to deplete OsteoMacs from calvarial digests to determine if their presence within these cultures facilitates osteoblast differentiation or function. OsteoMac removal did not affect expression of the early osteoblast differentiation marker genes collagen type I or alkaline phosphatase. However, OsteoMac removal significantly decreased gene expression of the osteoblast mineralisation marker osteocalcin and mineralisation function, assessed by von Kossa staining. Microarray gene expression profiling demonstrated that osteoblast enrichment had a broad impact on transcription within the culture, identifying both candidate OsteoMac marker genes as well as osteoblast expressed genes that are regulated by OsteoMacs. Potential OsteoMac-enriched candidate genes insulin-like growth factor a, dipepetidase 2, glycoprotein NMB, and macrophage expressed gene 1 as well as osteoblast-specific genes bone sialoprotein and thrombospondin 1 were selected based on their potential involvement in osteoblast function. In a transwell co-culture system of enriched osteoblasts and macrophages, it was demonstrated that macrophages were required for osteoblast mineralisation in response to the physiologic remodelling stimulus, elevated extracellular calcium. A blocking soluble receptor strategy provided evidence that this is mediated in a BMP-2 and -4 independent manner. To investigate the relevance of OsteoMacs to bone formation in vivo, immunohistochemistry staining for the mature tissue macrophage marker F4/80 was performed in long bone sections from rapidly growing mice. OsteoMacs were closely associated with areas of bone formation in situ, forming a distinctive canopy structure over mature cuboidal osteoblasts (collagen type I+, osteocalcin+) on endosteal cortical surfaces. Using adapted histomorphometic analysis, we determined that 77 ± 2.1% (n = 7) of the endosteal mature osteoblast surface was covered by the F4/80+ OsteoMac canopy. This observation suggested that OsteoMacs are optimally located to regulate osteoblast function in vivo. In summary, we have demonstrated that OsteoMacs are an integral component of bone lining tissues and play a novel role in bone dynamics through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics. Further delineation of OsteoMac functions is likely to provide new avenues for treating bone disease and assisting bone repair.

Any lipoproteins and cells present in the inflammatory environment of atherosclerotic plaques are likely to be exposed to high levels of oxidative stress. As 7,8-dihydroneopterin (7,8-NP) is synthesized by interferon-γ (IFN-γ)-activated macrophages, this pteridine is also thought to exist at sites of inflammation. 7,8-NP s in vivo role remains controversial, but numerous in vitro studies have identified a radical scavenging activity. The possibility of 7,8-NP protecting against oxidative damage in inflammatory environments like plaque was investigated in this thesis. Both human monocyte-derived macrophages (HMDMs) and low density lipoprotein (LDL) were used as substrates. The extent of protein hydroperoxide formation in each model, and 7,8-NP s effect on this process, were specifically studied since most previous research has focussed on lipid rather than protein peroxidation. For the first time, neopterin (including oxidized 7,8-NP) was also directly detected by high performance liquid chromatography in the inflammatory environments of 19 pus and two atherosclerotic plaque samples. Peak concentrations even reached the low micromolar range. The positive correlation identified in the pus between neopterin and a well known antioxidant, vitamin E, further hinted at a potential antioxidant function. However, no significant association was noted between neopterin and markers of protein or lipid oxidation. Exposure of HMDMs to the AAPH peroxyl radical generator resulted in significant quantities of lipid hydroperoxides but not protein hydroperoxides, as detected by the FOX assays. This is likely due to the large accumulation of polyunsaturated fatty acidrich lipid in the primary HMDMs during differentiation in 10% human serum and is of relevance to atherosclerotic plaque, where macrophages also become lipid-loaded. The addition of up to 200μM 7,8-NP failed to prevent AAPH-induced lipid peroxidation and was also unable to inhibit a loss of cellular thiols or viability. This lack of effect suggests the damaging peroxyl radicals are not being scavenged by 7,8-NP. The high lipid content of HMDM cells appears to cause the AAPH and/or 7,8-NP to localize to a cellular site, where they are unable to interact. Macrophage-mediated oxidation of LDL in iron(II)-supplemented Hams F10 was associated with the formation of 30-40 moles of protein hydroperoxides per mole of LDL. The close parallel between protein and lipid peroxidation supports the theory that lipid-derived radicals are involved in protein hydroperoxide formation on LDL and indicates that protein hydroperoxides are an early product of LDL oxidation. Their detection during exposure of LDL to both the THP-1 macrophage cell line and primary HMDM cells confirms that protein hydroperoxides are also a normal consequence of macrophage-mediated LDL oxidation. Incubation of LDL with micromolar 7,8-NP prevented macrophage-mediated protein hydroperoxide formation in a concentration-dependent manner. Lipid oxidation and vitamin E loss were similarly inhibited by 7,8-NP during the cell-mediated attack of LDL. Kinetic analysis revealed protection due to extension of the lag phase, with 7,8-NP depletion and initiation of the propagation phase coinciding. This supports a radical scavenging activity for 7,8-NP, resulting in protection of the entire LDL particle. By contrast, the release of nanomolar quantities of 7,8-NP by IFN-γ-stimulated THP-1 macrophages failed to prevent LDL oxidation. HMDMs activated by IFN-γ did significantly inhibit LDL oxidation, including protein hydroperoxide formation, for up to 48 hours but this antioxidant effect was not due to the de novo synthesis of 7,8-NP. These results indicate that both the prevalence of protein hydroperoxides, and the ability of 7,8-NP to act as an antioxidant, depend on the system under investigation. Neopterin exists in inflammatory environments but, considering the lack of protection against AAPH-mediated HMDM oxidation and the 7,8-NP concentration required to inhibit macrophage-mediated LDL oxidation, strong evidence for an antioxidant activity of 7,8-NP in atherosclerotic plaque is currently lacking.

No description available.

Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1999? / Amendments to thesis in pocket on back cover. Copy of author's previously published article in pocket on back cover. Bibliography: leaves 124-172.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [210]-223).

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 119-147) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 119-147) Also available online.

Proefschrift Universiteit van Amsterdam. / Met lit.opg. en samenvatting in het Nederlands.