Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 202-219). Also available in print.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 178-199). Available also in a digital version from Dissertation Abstracts.

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references.

Metabolic Syndrome (MetS), the clustering of obesity, high blood pressure, and disordered glucose and lipid/lipoprotein metabolism within a single individual, is associated with poorer cognitive function and dementia in later life. It has been hypothesized that cognitive impairment in MetS occurs primarily within the context of inflammation. MetS risk factors are also associated with thinning of the cerebral cortex. However, the mechanisms by which MetS and inflammation affect the brain are poorly understood. The present study used statistical mediation to examine the relationship between MetS risk factors, cortical thickness in a priori regions of interest (ROIs) and inflammation. ROIs in the inferior frontal, superior temporal, middle frontal, supra marginal, anterior cingulate and middle occipital regions were chosen from the previous literature. Serum levels of pro-inflammatory markers (interleukin 1, interleukin 2, interleukin 6 and C-Reactive Protein) were measured using enzyme-linked immunosorbent assays. Forty-three adults between the ages of 40 and 60 years underwent a health screen, neuropsychological testing and structural magnetic resonance imaging. A higher number of MetS risk factors was associated with thinning in the inferior frontal ROI (β=-0.35, p = 0.019). A higher number of MetS risk factors was also associated with higher levels of serum interleukin 2 (β=0.31, p=0.04). A higher level of serum interleukin 2 was also associated with reduced thickness in the inferior frontal ROI (β=-0.41, p=0.013). After accounting for the effects of interleukin 2, the number of MetS risk factors was no longer associated with cortical thickness in the inferior frontal ROI indicating successful statistical mediation and pointing towards a potentially important role for imflammation in linking MetS to cortical thinning and cognitive vunlerability. / text

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第19001号 / 工博第4043号 / 新制||工||1622 / 31952 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 白川 昌宏, 教授 今堀 博, 教授 森 泰生 / 学位規則第4条第1項該当

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

Coccidioidomycosis is a growing problem in the United States. There is still an incomplete understanding of the immunological response associated with human coccidioido-mycosis, although previous studies have indicated that cell-mediated immunity (CMI) is critical in the control of this disease. We have examined necrotizing pulmonary coccidioidal granulomata using immunohistochemical staining for lymphocyte subsets and for the cytokines interleukin-10 (IL-10) and gamma interferon (IFN-?). Discrete perigranulomatous lymphocytic clusters were identified containing roughly equal numbers of T (CD3+) and B (CD20+) lymphocytes. While the number of cells expressing IL-10 was similar in the mantle and in the perigranulomatous clusters, there were significantly more cells expressing IFN-? in the mantle compared to the clusters (P = 0.037). Confocal microscopy revealed that CD4+ T lymphocytes and B lymphocytes are associated with IL-10 production. CD4+CD25+ T lymphocytes were also identified in the perigranulomatous clusters but were not associated with IL-10 production. This is the first report noting perigranulomatous lymphocyte clusters and IL-10 in association with human coccidioidal granulomata, suggesting a down-regulation of cellular immune response.IL-12 is critical in driving a T helper type 1 (Th1) immune response. To understand the mechanism associated with hyporesponsiveness to IL-12 stimulation seen in some anergic patients with disseminated coccidioidomycosis, IL-12 receptor expression and function were determined in PBMCs from immune and nonimmune healthy donors. By using a relative quantitative RT-PCR technique, we found that IL-12R?1 is constitutively expressed in PBMCs and is equally up-regulated by coccidioidal antigen preparation T27K in both immune and nonimmune donors. On the other hand, the IL-12R?2 expression level is increased by T27K only in PBMCs from immune but not nonimmune donors. In addition, the increased IL-12R?2 expression in immune PBMCs is correlated with Stat4 activation and the induction of IFN-? production by IL-12. These data suggest that the IL-12R?2 expression and signal transduction is functional in host immunity against Coccidioides infection. Subsequent research, which involved the stimulation of lymphocytes with autologous dendritic cells (DCs) pulsed with T27K, revealed that the up-regulation of IL-12R?2 expression and signal transduction is associated with the induction of IFN-? production by DCs pulsed with T27K.

Interleukin (IL)-7 is an essential non-redundant cytokine and throughout the life-span of a T cell signaling via the IL-7 receptor influences cell survival, proliferation and function. It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. In this study I establish IL-7 down regulates CD127 gene transcription and surface protein expression in primary human CD8 T cells through two mechanisms. Upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at the critical tyrosine residue Y449. Concurrent activation of the JAK/STAT5 pathway stimulates expression of CIS, a member of the SOCS family of proteins. CIS protein already expressed at basal levels and induced by IL-7 bind directly to CD127 as demonstrated by Coimmunoprecipitation assays and colocalize with both CD127 and the early endosomal marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase. Through siRNA-mediated knockdowns I confirm CIS plays a predominant role in the IL-7 mediated degradation of CD127. The mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells was also examined. Through qPCR and nuclear run-on assays I illustrate that IL-7 suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7 mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably, cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the de novo synthesis of a transcriptional repressor of the CD127 gene. Through PCR arrays, qPCR and Western blot analysis the IL-7 inducible transcription factor c-Myb was identified as a candidate repressor. The region within the CD127 gene promoter required for IL-7 mediated transcriptional suppression was identified through progressive truncations using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of the TATA box and contains three putative c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, I illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells. A thorough understanding of the mechanisms by which IL-7 regulates CD127 expression is imperative and may reveal novel insights into the contribution of abnormal IL-7 signaling to diseases affecting immune function.

Inflammatory bone diseases like rheumatoid arthritis and periodontal disease lead to increased bone loss in the inflamed areas. The multinucleated bone resorbing cells, the osteoclasts, present in these diseases are larger than normal, and these larger osteoclasts (10+ nuclei) resorb more bone and more often than smaller osteoclasts (2-5 nuclei). Thus, the focus of this thesis was to determine if there are differences in mechanisms of osteoclast activation between large and small osteoclasts. Experiments using authentic rabbit osteoclasts and RAW 264.7-derived osteoclasts revealed differences in the expression of a number of activating factors; with large osteoclasts expressing higher levels of activating receptors (RANK, IL-1RI, TNFR1 and integrins αv and β3), as well as enzymes involved in cellular resorption, while small osteoclasts expressed higher levels of an alleged fusion receptor and the inhibitory receptor, IL-1RII. Further studies revealed that large osteoclasts more readily responded to stimulation by IL-1 compared to small osteoclasts and at lower concentrations suggesting this is a result of their higher expression of activating receptors. Differences in responses to the IL-1 isoforms, IL-1α and IL-1β, were also seen in large osteoclasts: IL-1α generated more large osteoclasts over the course of differentiation, while IL-1β induced changes in cell morphology and in the induction of integrin β3 phosphorylation. These observations suggested that differences in osteoclast responses are induced by IL-1α and IL-1β and it led to the hypothesis that there are differences in signaling between large and small osteoclasts. To elucidate differences in signaling mechanisms a signaling pathway microarray was used which revealed higher expression of Vegfa in large compared to small osteoclasts. Osteoclast differentiation with RANKL increased Vegfa gene expression in a time-dependent manner and VEGF-A secretion was elevated in populations enriched for large osteoclasts. Furthermore, mechanistic studies with inhibitors of transcription factors involved in differentiation revealed that RANKL-mediated Vegfa expression in large osteoclasts was regulated by the NF-κB pathway via induction of Hif1α. These results support the hypothesis that signaling differences exist between large and small osteoclasts and implicates VEGF-A in osteoclast hyperactivity in inflammatory conditions.