Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 72-79).

The cytokines GM-CSF, IL-3 and IL-5 are central regulators of haemopoietic cell functions and are pivotal in the regulation of haemopoiesis and inflammatory responses of myeloid cells. In particular, these cytokines have been shown to perform essential functions in host defence against foreign pathogens through their ability to regulate innate immune responses in myeloid cells. As key regulators of such important processes, these cytokines play an important role in human inflammatory pathologies such as rheumatoid arthritis, asthma, multiple sclerosis and psoriasis as well as a number of leukemias such as JML and CMML. GM-CSF, IL-3 and IL-5 signal through receptors containing α subunits specific to each cytokine and a common β subunit (βc). Cytokine stimulation leads to tyrosine phosphorylation of the βc and promotes specific responses such as proliferation, survival and activation of haemopoietic cells. Mouse knockout studies identified a key function of these cytokines in the activation of effector functions of myeloid cells, including production of reactive oxygen species (ROS) and phagocytosis. These earlier studies provide a link between cytokine signalling and inflammation, but the molecular mechanisms by which βc activation regulates effector cell functions, and the receptor motifs involved, are unknown. The aim of this thesis was to address two broad questions with regard to βc signalling: (1) Does βc regulate specific cellular responses by phosphotyrosine-independent mechanisms? (2) What are the molecular mechanisms by which βc initiates signalling to promote specific biological responses such as activation of effector cell functions? To address the first question, we have focussed on Serine 585, a potential 14-3-3 binding site which lies in the cytoplasmic potion of huβc. Out results show that the mutation huβc S585G disrupted the interaction of 14-3-3ζ with βc, whilst not affecting receptor tyrosine phosphorylation. Both mouse and human βc were shown to interact with 14-3-3 proteins, indicating that this interaction is conserved between these species. Significantly, a huβc S585G mutant was unable to promote haemopoietic cell survival in response to IL-3. These results identify a new mechanism by which cytokine receptors are able to couple to downstream signalling pathways that regulate cell survival. An approach was developed and optimised to analyse specific GM-CSF-mediated responses in monocytes/macrophages expressing wildtype or mutant huβc, (including huβc S585G that was defective in regulating survival). Bone marrow-derived muβc -/-;muβIL-3 -/- monocytes/macrophages were retrovirally transduced with constructs expressing wildtype or mutant huβc, along with huGMRα, then purified by FACS. Two assays were established to measure effector functions in the transduced monocyte/macrophages; (1) a flow cytometry assay for ROS production, and (2) an assay for phagocytosis. The capacity for GM-CSF to prime (i.e. enhance effector functions) ROS production and phagocytosis was investigated in huGMRα-transduced monocytes/macrophages. Our results have identified two key residues in the cytoplasmic domain of βc subunit: Tyrosine 577 (required for huβc interaction with the adaptor protein Shc) and serine 585 (required for 14-3-3 association), that are essential for the ability of GM-CSF to regulate key effector functions in monocytes/macrophages. These novel findings are significant in that they establish a molecular link between the GM-CSF/IL-3/IL-5 receptor and the regulation of both haemopoietic cell survival and inflammatory responses, and therefore have important implications in our understanding of inflammatory diseases such as rheumatoid arthritis and asthma. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317007 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

O quelóide é um tumor fibroso benigno que ocorre durante a cicatrização da pele em indivíduos geneticamente predispostos. A cicatrização é um processo biológico complexo e depende da interação de diferentes estruturas teciduais e de um grande número de tipos celulares residentes e infiltrativos, que produzem citocinas. A interleucina 8 (IL-8), citocina pró-inflamatória, é super-expressa pelos fibroblastos durante o desenvolvimento do tecido de granulação, acelerando o processo de cicatrização. Como o quelóide resulta de uma reparação tecidual anormal após lesão da pele, o presente estudo teve por objetivo determinar a expressão dos receptores da IL-8, CXCR1 e CXCR2, e a capacidade proliferativa, pelo ciclo celular, dos fibroblastos queloideanos cultivados e extraídos ex vivo, por citometria de fluxo. Fibroblastos de cicatriz queloideana e de pele normal foram obtidos de 21 pacientes da raça negra, com idade variando entre 10 e 40 anos, de lesões com até 2 anos de evolução. Em nosso estudo constatamos expressão reduzida dos receptores para a IL-8, CXCR1(35,7%±11,2) e CXCR2 (27,8%±11,3), em fibroblastos de cicatriz queloideana cultivados, comparando com a pele normal (44,1±16,2 e 46,3±27,1 respectivamente). Entretanto, essa diferença só foi significante para o receptor CXCR2. A baixa expressão desses receptores poderia ser decorrente da atividade de metaloproteinases, que regulam a expressão de proteínas da superfície celular, através de clivagem enzimática, ou a capacidade reduzida de internalização e a reciclagem de receptores, mantida por filamentos de actina do citoesqueleto, que nos fibroblastos do quelóide estão diminuídos. Em relação ao ciclo celular de fibroblastos cultivados do quelóide e da pele normal, verificamos diferenças não significantes da capacidade de replicação (fase S do ciclo celular) e de apoptose. No quelóide observamos significante aumento de células na fase G2/M, indicando aumento da velocidade de divisão celular. Para confirmar esses achados estudamos o ciclo celular de fibroblastos extraídos ex vivo, da porção periférica e central do quelóide e da pele normal. Os fibroblastos da porção periférica apresentaram porcentagem significantemente maior de células com capacidade replicativa, fase S do ciclo (22,9% ± 11,6), em relação à porção central (4,7% ± 2,9) e à pele normal (6,8% ± 4,9), e maior velocidade de divisão celular, fase G2/M (18,6 ± 12,0), em relação à porção central (35,6 ± 7,0) e pele normal (32,3 ± 6,9). Verificamos que a porção central apresentou maior porcentagem de células em apoptose (7,0% ± 2,1), comparado à porção periférica (4,9% ± 1,9) e pele normal (2,0% ± 0,86). Esses dados indicam que as células da porção periférica do quelóide parecem ser responsáveis pela elevada taxa de proliferação, justificando o crescimento expansivo a partir das margens da cicatriz queloideana, com desenvolvimento de lesão semelhante a tumor, bem como a porção central ser responsável pela fibrose, contendo células quiescentes e apoptóticas. Esses resultados sugerem modulação diferencial das reações celulares através das vias de sinalização para proliferação ou morte celular programada. Neste sentido, a baixa expressão dos receptores da IL-8, CXCR1 e principalmente de CXCR2, nos fibroblastos do quelóide sugere capacidade reduzida da IL-8 em promover cicatrização acelerada. A baixa atividade da IL-8 sobre os fibroblastos queloideanos estaria promovendo desregulação da resposta inflamatória e com isso atraindo novas células inflamatórias para o local e produzindo sinais alterados, como grande produção da citocina TGFβ. Essa desregulação do processo de cicatrização, com alteração de citocinas e da matriz extracelular, poderia ser responsável pelas duas populações de fibroblastos, uma proliferativa na periferia e outra quiescente e apoptótica na porção central. Finalizando, podemos concluir que nossos resultados correspondem às alterações histológicas e clínicas do quelóide que se expande nos limites da lesão. / A keloid is a benign fibrous tumor that occurs during wound healing in genetically predisposed individuals. Healing is a complex biological process and depends on the interaction of different tissue structures and a great number of resident and infiltrative cell types. The interleukin-8 (IL-8), a proinflammatory chemokine, showed higher expression in fibroblasts during the development of the granulation tissue, promoting more rapid tissue maturation. Since keloids result from abnormal wound healing, the objective of the present study was to determine the expression of CXCR1 and CXCR2, IL-8 receptors, and the proliferation capacity, throughout the cell cycle, of the keloid fibroblasts extracted ex vivo and those submitted to in vitro cultivation. Normal skin and keloid scar fibroblasts were obtained from 21 African-Brazilian patients, aged from 10 to 40 years, whose lesions had evolved for no longer than 2 years. Expression of receptors and the cell cycle was assessed by flow cytometry. We showed lower expression of the CXCR1 (35,7% ± 11,2) and CXCR2 (27,8%±11,3) in keloid fibroblasts, when compared with normal skin (44,1 ± 16,2 e 46,3 ± 27,1 respectively), but the difference was not significant for the CXCR1 receptor. This lower expression of IL-8 receptors in keloid fibroblasts could be due to the action of metalloproteinases, which regulate the surface protein enzymatically, or fibroblastic cytoskeleton conditions, which influence receptor internalization and recycling. The distribution assessment of cell cycle phases of fibroblasts cultivated from keloid scars and normal skin did not show significant difference in replication capacity and apoptosis. The keloid fibroblasts presented a significantly higher proportion of cells in the G2/M phase, suggesting higher rate of cell division. To confirm these results we studied the cell cycle of fibroblasts extracted ex vivo, now separated by central and peripheral portions of keloid and normal skin. The peripheral fibroblasts showed significant high cell proportions in phase S (22,9% ± 11,6), compared with the central portion (4,7% ± 2,9) and normal skin (6,8% ± 4,9), and higher cells in division phase G2/M (18,6% ± 12,0), compared with the central portion (35,6% ± 7,0) and normal skin (32,3% ± 6,9). The central portion showed higher proportion of apoptosis (7,0% ± 2,1), compared with the peripheral portion (4,9% ± 1,9) and normal skin (2,0% ± 0,86). These results suggest that the keloid peripheral cells could be responsible for the proliferation rate, justifying the expansive keloid growth at the borders of the keloid scar, in a similar fashion to tumor development and the central portion being responsible for fibrosis, with quiescent and apoptotic cells. These results suggest a differentiated modulation of cell reactions by signal pathways for programmed cellular proliferation or death. In this sense, the low expression of the IL-8 receptors CXCR1 and CXCR2 in keloid fibroblasts suggests a diminished capacity of IL-8 to promote accelerated healing. This low expression of IL-8 receptors in keloid fibroblasts could promote the dysregulation of the inflammatory response and thus attract more inflammatory cells to the site, producing different signals, such as a high production of the TGFβ cytokine. This dysregulation of the healing process, with changed cytokine and extracellular matrix expression, could be responsible for two different cell populations of fibroblasts, one proliferation at the periphery and the other fibrotic at the center of the lesion, with apoptotic and quiescent cells. Finally, we conclude that our results correspond to the histological and clinical changes of keloids that grow beyond the wound boundaries.

by Chuen Ka Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 134-166). / Abstracts in English and Chinese. / ACKNOWLDEGEMENT --- p.ii / PUBLICATIONS --- p.iii-iv / ABBREVIATIONS --- p.v-viii / INDEX FOR FIGURES --- p.ix xii / INDEX FOR TABLES --- p.xiii / ABSTRACT (Chinese and English) --- p.xiv-xvi / TABLE OF CONTENT --- p.xvii / Chapter 1. --- INTRODUCTION --- p.1-37 / Chapter 2. --- OBJECTIVES --- p.38-40 / Chapter 3. --- METHODS AND MATERIALS --- p.41 -70 / Chapter 4. --- RESULTS AND DISCUSSION --- p.71-130 / Chapter 5. --- GENERAL DISCUSSION AND CONCLUSION --- p.131-133 / BIBLIOGRAPHY --- p.134-166

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 102-112).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 77-87).

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 4 uppsatser.