by Ying-tat Mak. / Thesis (M.Phil.)--Chinese University of Hong Kong. / Bibliography: leaves 59-62.

Monocytes and their subsets play an important role in immune and inflammatory diseases. In this study the focus was on their role of driving pathogenesis in asthmatic patients. The hypothesis was that although monocytes have not been identified as key players in atopic disease they are likely to be pro-inflammatory, be readily recruited to tissue and have a major role in the exacerbation of disease. This may relate to the prevailing pro-inflammatory microenvironment. Chemokines such as CCL2 and CX3CL1 are involved in monocyte recruitment and survival respectively in the inflamed tissues. Three subsets of monocytes namely CD14++CD16-, CD14++CD16+ and CD14+CD16++ monocytes are known to express chemokine receptors. Alterations if any in the numbers or in the molecular inflammatory markers of these three monocyte subsets with contrasting potential of modulating inflammatory responses in the peripheral blood may provide some insight in the disease process. It is also believed that circulating fibrocytes in allergic asthma are the cause of fibrosis in chronically inflamed pulmonary tissues resulting in refractory asthma. Fibrocytes, also derived from the bone marrow, produce connective tissue components such a collagen-1 and can adopt a mesenchymal phenotype and contribute to granulomas, scar tissue and tissue remodeling. They appear to play an important role in chronic and refractory asthma but have also been found in lungs of patients with mild asthma indicating that they may play an as yet undefined role in the development of inflammation. Fibrocytes also express chemokine receptors and their recruitment in tissues may also depend upon chemokines. Using six-color flow-cytometry phenotypic analysis of human blood monocyte and fibrocyte populations from asthma patients was performed along with the isolation of mRNA from CD14+ monocytes for assessment of inflammatory markers expression and the data was compared with the normal healthy individuals. Based on the findings of gene expression an attempt was made to demonstrate the effect of routine treatment used by our patients on gene expression of selectively isolated monocytes from healthy individuals. Finally serum levels of chemokines between patients and normal healthy controls were also determined. XXI From the flow-cytometry analysis we demonstrated a significant increase in both CD45-positive monocytes and circulating fibrocytes in severe asthmatic patients. While CCR2 percentage was significantly increased, the CX3CR1 percentage was significantly decreased in the intermediate and non-classical monocyte subsets in both mild and moderate patients, however these changes were only observed in the non-classical monocytes in severe patients. In the gene expression the proinflammatory receptors CCR2, CCR5, CX3CR1, IL-17RA, and cytokine inhibitor SOCS3 in monocytes were all significantly reduced in the severe asthma patients. While CCR1 and CD36 were significantly decreased in mild and moderate asthmatic patients. No significant change in serum pro-inflammatory cytokines were detected although IL-5 was significantly increased in moderate and severe asthmatic patients. Analysis of monocyte gene expression in asthma by other groups although not highlighting a specific gene did indicate that inflammatory and TNF pathways might be involved. In conclusion there was a tendency toward more inflammatory monocytes in our asthma patients, however this was not clearly reflected in the patients serum profile given that inflamatory monocytes make up only a small percentage of the leukocytes in human blood. Finally increases in total monocytes and fibrocytes correlated with asthma severity.

No description available.

Mechanical stress has been shown to play a role in bone remodelling during orthodontic tooth movement. Receptor activator of nuclear factor kβ - ligand (RANKL), osteoprotegerin (OPG), monocyte colony stimulating factor (M-CSF) and interleukin 1-β (IL-1β) play key roles in the regulation of bone remodelling, but the role of these cytokines in orthodontic tooth movement is poorly understood. Aim: The aim of this experiment was to examine the response of periodontal ligament (PDL) fibroblasts in monolayer culture to intermittent tensile stress as regards RANKL, OPG, M-CSF and IL-1β production. Methods: Human PDL fibroblasts were dissected from premolars extracted for orthodontic purposes. Explants were seeded out in 1cm wells and grown to confluence in Dulbecco�s modification of Eagle�s medium, containing 10% foetal calf serum and antibiotics, at 37�C in a humidified atmosphere of 5% CO₂/95% air. Upon reaching confluence, the cells were passaged into sequentially larger flasks. Fibroblasts were passaged 6 times. After reaching confluence in T175 flasks, the cells were detached and plated at a cell density of 10⁵/dish in 35mm Bioflex� Plates coated with type 1 collagen. The cells were placed under a continuous uni-axial strain of 12% for 6s of every 90s by a Flexercell FX 4000C[TM] for 0, 12, 24 and 48 hours. Cells were then detached and stored in RNAlater. Quantitative RT-PCR was used to determine the mRNA of the cytokines of interest. Results: Tensile force led to the down regulation of mRNA expression for OPG and IL-1β at 12 and 24 hours respectively, while M-CSF was up-regulated at 6 hours. RANKL was not detected at a significant level for quantification. Conclusion: This osteoclastic-type response indicates the complexity of mechanotransduction in an in vitro setting. Acknowledgments: This research was supported by the New Zealand Dental Research Foundation, the New Zealand Lottery Grants Board and the New Zealand Association of Orthodontists.

La sénescence prématurée est induite chez des fibroblastes diploïdes humains entre deux et trois jours après un traitement avec une concentration sublétale de peroxyde d’hydrogène. Ce phénotype est notamment caractérisé par une morphologie étalée, un arrêt irréversible du cycle cellulaire et une activité ß-galactosidase associée à la sénescence. Selon des études précédentes, il semble que la néosynthèse protéique soit importante pour l’établissement de la morphologie aplatie, élargie et irrégulière des cellules en sénescence induite par H2O2. De plus, il a été montré que la neutralisation du TGF-ß1 par des anticorps spécifiques altère cette morphologie induite par le traitement de fibroblastes avec H2O2. Étant donné que les mécanismes impliqués dans l’apparition de la morphologie sénescente sont peu compris, nous avons étudié la néosynthèse de protéines cytoplasmiques dépendante ou indépendante du TGF-ß1, entre le jour deux et trois après le traitement de fibroblastes avec H2O2 et avons identifié des protéines impliquées dans l’organisation du cytosquelette : l’ezrine, la caldesmone et HSP27. Rac1, Cdc42 et la cavéoline 1 ont été définies dans la littérature comme étant impliquées dans la morphogenèse typique lors de la sénescence réplicative et de la sénescence prématurée induite par H2O2 (dans le cas de la cavéoline 1). Nous avons étudié l’activation de Rac1 et Cdc42, la phosphorylation de la cavéoline 1 suite au traitement H2O2 et avons étudié l’effet de l’invalidation de ces protéines ainsi que celle de p38αMAPK sur la morphologie des fibroblastes. De plus, nous avons montré que Cdc42 pouvait activer p38αMAPK vice-versa, pendant le traitement avec H2O2. En parallèle de cette étude, nous avons tenté d’analyser l’abondance différentielle de protéines membranaires au jour 3 après le traitement des fibroblastes avec H2O2. Ceci nous a permis d’étudier la surexpression d’une protéine pouvant être associée aux membranes et au cytosquelette d’actine : l’annexine 2. Ce travail permet de mieux comprendre les mécanismes mis en place par les fibroblastes pour entrer en sénescence prématurée, et plus particulièrement pour présenter une morphologie étalée lors de ce processus.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Porphyromonas gingivalis is a keystone periodontopathogen and its lipopolysacharide (LPS) is strongly associated with periodontal disease. A long-standing controversy occurs on the role of P. gingivalis LPS in induction of innate host response in different cell types. It has recently been found that P. gingivalis LPS displays remarkable heterogeneity with both tetra- (LPS1435/1449) and penta-acylated (LPS1690) lipid A structures. However, the potential effects of heterogeneous structures of P. gingivalis LPS on modulating host innate responses in human gingival fibrobalsts (HGFs) - the most abundant cells in gingiva remain unclear. To fulfill this research gap, a comprehensive study on the P. gingivalis LPS-HGFs interations was undertaken. The effects of P. gingivalis LPS1435/1449 and LPS1690 on the expression profiles of pro-inflammatory cytokines were investigated (Chapter III). P. gingivalis LPS1690 (not LPS1435/1449) significantly upregulated the expression of IL-6, IL-8 and TNF-α, suggesting that P. gingivalis LPS may differentially modulate the expression of pro-inflammatory cytokines. The effects of P. gingivalis LPS on the expression of MMPs 1-3 and TIMP-1, and regulation of MMP-3 were then determined (Chapter IV). P. gingivalis LPS1690 markedly induced MMP-3 expression through p38 MAPK and ERK signal pathways, whereas TIMP-1 was greatly upregulated by P. gingivalis LPS1435/1449. These findings suggest that P. gingivalis LPS heterogeneity may differentially modulate the expression and regulation of MMP-3. Based on these findings, the involvements of TLR2/4 and the downstream signaling pathways were explored (Chapter V). P. gingivalis LPS1690 induced TLR4 expression, whereas TLR2 was upregulated by P. gingivalis LPS1435/1449. NF-κB pathway played a dominant role in P. gingivalis LPS1690-induced expression of IL-6 and IL-8. These findings suggest that the two isoforms of P. gingivalis LPS critically interact with TLR2 and TLR4, and may determine the subsequent activation of signal transduction cascades that differentially modulate immuno-inflammatory response. P. gingivalis could thereby evade innate host defense and contribute to periodontal pathogenesis. To obtain a holistic profile of heterogeneous P. gingivalis LPS-HGFs interactions, a systems biology-based study through proteomics, metabolomics and bioinformatics approaches was undertaken (Chapter VI). Pro-inflammatory proteins (e.g. Cyclophilin, Annexins, IL-6 and Cathepsins) were induced by P. gingivalis LPS1690. In contrast, anti-inflammatory proteins (e.g. ANXA1, ANXA2 and Gal-1) were upregulated by P. gingivalis LPS1435/1449. P. gingivalis LPS1690 also induced antioxidant defense molecules like MnSOD and PRDXs. Secretomic analysis showed that immuno-inflammatory mediators, extra-cellular proteases and matrix proteins were differentially modulated by the two isoforms of P. gingivalis LPS as well. These findings demonstrate that host responses such as immuno-inflammatory activity, oxidative stress and anti-oxidant defense may be differentially modulated and regulated by the heterogeneous P. gingivalis LPS. Further study shows that P. gingivalis LPS1435/1449 and LPS1690 differentially modulate oxidative stress response and antioxidant expression, and differential regulation of MnSOD could be a critical determinant of periodontal homeostasis (Chapter VII). The present findings may bring new insight into the molecular mechanisms of periodontal pathogenesis. Targeting the mechanisms of shift in lipid A structures of P. gingivalis LPS may be a potential strategy to develop novel approaches to control and prevent periodontal diseases. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

The ability of light to affect and modify human activity, both physiologically and psychologically has been known for many centuries. The treatment of wounds with light with varying properties has seen a dramatic rise over the last twenty years, from initial reports of the "magic laser" in the 1970's, to recent well controlled clinical trials. However, one of the major problems facing researchers and clinicians in elucidating the mechanisms by which light can modulate wound repair, is that of the different parameters of the light sources used, be they true laser devices, monochromatic light sources or. broadband wavelength units. This thesis describes a series of in vitro studies of the effects of varying the parameters of three light sources a) directly on fibroblast proliferation and b) indirectly, via supernatant from light-irradiated macrophage-like U-937 cells. The effect of U-937 cells was investigated because, as well as playing a pivotal role in wound debridement, macrophages playa central role in mediating the body's inflammatory and immune responses, mainly through the release of various polypeptide growth factors and cytokines and these can modulate wound healing. The experiments reported here show that there is a clear dose-response effect on the proliferation of fibroblasts when grown in macrophage-conditioned medium in which U-937 cells had been subjected to energy densities of 2.4, 4.B and 7.2J/cm2 using a monochromatic light source of 660nm at a frequency of 5kHz. Earlier work had shown that of a number of wavelengths examined, this wavelength evoked the greatest response. 2.4, 4.Band 7.2J/cm2 produced an increase in fibroblast proliferation above that of the sham-irradiated sample, 7.2J/cm2 producing the greatest effect. 9.6 J/cm2 was found to be inhibitory. The relationship between energy density and power density was investigated using the same model, but with a pure laser light source (820nm, frequency 5kHz). The U-937 cells were exposed to either 400mW/cm2 or 800mW/cm2 at energy densities of either 2.4 or 7.2J/cm2 (the doses giving the lowest and highest response in the previous experiment). There was a statistically significant difference in fibroblast proliferation between the 400mw/cm2 and Boomw/cm2 treatments, BoomW/cm2 producing the greatest cell proliferation at an energy density of 2.4J/cm2• There was no significant difference between the sham-irradiated sample and the 400mw/cm2 treated group. In contrast, using an energy density of 7.2J/cm2, the 400mW/cm2 treatment produced a greater increase in fibroblast proliferation than the sham-irradiated sample and there was no significant difference in cell number between the sham irradiated sample and 800mw/cm2 sample. The 400mW/cm2 sample produced a greater increase in cell proliferation than the 800mW/cm2 sample. Using the same model as above, the effects of two broadband (400-2000nm, continuous wave) light sources, of which one was 95% and the other 14% polarised, were investigated using energy densities of 1.44 and 2.88 Jjcm2• The proliferative response was greatest in the cultures exposed to supernatants from macrophages treated with the 95% polarised light source for both the energy densities used. The direct effect of 860 nm laser light (continuous wave) on the proliferation and succinic dehydrogenase levels of human forearm fibrobasts was investigated using energy densities of 2 and 16 Jjcm2• At an energy density of 2Jjcm2, succinic dehydrogenase and fibroblast proliferation levels increased to above that of the sham-irradiated sample, however, at an energy density of 16Jjcm2, succinic dehydrogenase levels and fibroblast proliferation were inhibited. The results presented in this thesis indicate either stimulation or inhibition of cellular activity which can be induced by specific levels and types of light irradiation and helps to elucidate the mechanism of action of light-producing devices at the cellular level.

No description available.

Mitochondrial myopathies are a group of diverse neuromuscular disorders. Defects in electron transport chain (ETC) subunits have been implicated in pediatric and adult onset cases. Skin fibroblasts from four patients were studied to elucidate the biochemical defects. / Cells from two patients with ETC complex I deficiency, showed reduced oxidation of alanine with normal oxidation of succinate. Analysis of complex I subunits indicated deficient synthesis of the 20 kDa subunit in the severely affected patient. In the milder patient, subunit abnormalities were not detected. / Fibroblasts from a patient with facioscapulohumeral disease (FSHD), showed reduced oxidation of alanine and succinate through the ETC. / A fourth patient, with decreased activity in several complexes in muscle and liver, was found to have a heteroplasmic mtDNA population in fibroblasts. / These studies exemplify the heterogeneity of mitochondrial myopathies and demonstrate the utility of fibroblasts in the investigation of these disorders.