Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 61-64).

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2007. / Committee Chair: Julia Babensee ; Committee Members: Barbara Boyan, John Brash, Andres Garcia, and Niren Murthy. Part of the SMARTech Electronic Thesis and Dissertation Collection.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse manifestations affecting multiple organs. Current understanding of its pathogenesis remains largely inadequate despite recent progress in SLE research on the characterization of immune system dysfunction and its link with heritable and environmental factors. Dendritic cells (DCs) are immune cells that express pattern recognition receptors (PRRs) for the recognition of pathogen associated molecular patterns (PAMPs). Aberrant functions of DCs have been reported in SLE including recognition of self-nucleic acids, presentation of self-antigens and strong induction of interferon response, depending on their expression of PRRs. Dectin-1 is a non-toll like receptor PRR that is highly expressed in DCs for the recognition of pathogenic carbohydrates found mostly in fungi. Recognition of fungal carbohydrates by dectin-1 promotes DC maturation and production of pro-inflammatory cytokines that preferentially skew towards a T helper-17 (Th17) response which has been found to be engaged in the pathogenesis of SLE and other autoimmune diseases. Therefore, the current investigation aimed to study the expression of dectin-1 and the effect of dectin-1 activation in DCs from SLE patients. Dectin-1 expression on CD14+ monocytes from peripheral blood of SLE patients and healthy controls was measured by flow cytometry. SLE patients (mean+/-SD = 92.05+/-3.471%) were found to have higher dectin-1 expression on CD14+ monocytes compared to controls (mean+/-SD = 83.7+/-14.64%) (p=0.02). Monocyte derived DCs (MoDCs) were then derived from CD14+ monocytes in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. Pre-treated moDCs with curdlan, a dectin-1 specific ligand, showed increased expression of costimulatory molecules including CD83 and CD86 and enhanced production of IL-1β compared with healthy controls (all p<0.05). Curdlan treated moDCs were further co-cultured with CD4+ na?ve T cells and polarization into Th17 cells was subsequently evaluated by measuring the percentage of Th17 per moDCs-na?ve T cells and expression of intracellular IL-17. Curdlan treated moDCs from SLE patients were found to have enhanced Th17 polarization capacity (mean+/-SEM = 28.33+/-5.64%) compared with controls (mean+/-SEM = 12.77+/-2.56%) (p<0.05). To address the mechanism of Th17 polarization, expression of caspase-1 which promotes the production of IL-1β was measured. Curdlan treated moDCs in SLE patients expressed higher levels of caspase-1 detected in the cell lysate as measured using Western Blot compared with healthy controls (p<0.05). In conclusion, compared with healthy individuals, SLE moDCs were more matured and activated in response to β-glucan as shown by their higher expression of co-stimulatory molecules, enhanced production of IL-1β and stronger Th17 polarizing effect. These findings suggest functional dysregulation of dectin-1 expressing DCs in patients with SLE which may be involved in the pathogenesis of this condition. / published_or_final_version / Medicine / Master / Master of Philosophy

Systemic lupus erythematosus (SLE) is a chronic multi-organ autoimmune disease that is characterised by diverse clinical manifestations. Immunologically, SLE features a prominent “interferon (IFN) signature” which is marked by an elevated expression of type I IFN-regulated genes in blood and tissue cells of patients with this condition. Plasmacytoid dendritic cells (pDCs), also known as the most potent type I IFN-producing cells, are therefore considered the major culprit in SLE pathogenesis. Previous studies from our group have demonstrated abnormalities in circulating and bone marrow (BM)-derived pDCs from SLE patients. In the light of this, the present study was undertaken to further evaluate the role of pDCs in SLE development and to seek for key mediator(s) that might lead to functional aberrations of pDCs in this condition. Recently, a growing attention has been drawn to microRNAs (miRNAs) for their critical role in regulating immune cell function and strong association with autoimmune diseases. Therefore, the current study hypothesised that microRNAs played an important role in modulating pDC response(s) to toll-like receptor (TLR) stimulation, and that dysregulated microRNA expression induction was responsible for pDC abnormalities in SLE pathogenesis. The spontaneous lupus mouse model, F1 hybrid of New Zealand Black and White strains (NZB/W F1), was used in this study. The disease profile of NZB/W F1 was characterised based on the development of serum antinuclear antibodies and proteinuria. Specifically, the development of lupus in these mice (symptomatic mice) was illustrated by high titres of serum antinuclear antibodies, persistent proteinuria, glomerular immune complex deposition and elevated expression of pro-inflammatory cytokine genes in the kidney. Young NZB/W F1 (pre-symptomatic) as well as age- and sex-matched non-lupus maternal NZW mice were used as controls. While the development of pDCs appeared to be unaffected by lupus, elevated upregulation of MHC class II and co-stimulatory molecules, and induction of IFN-stimulated gene Ifitm3 in TLR7-stimulated lupus pDCs suggested phenotypic and functional hypersensitivity of these cells. Furthermore, analysis of the expression profile of miRNAs in pDCs upon TLR7 activation identified six differentially regulated targets. Among these, miR-155 was the most highly induced and its induction was consistently higher in pDCs from symptomatic NZB/W F1 mice. Nevertheless, transfection of miR-155 mimics into pre-symptomatic pDCs resulted in a reduced expression of Ifitm3, suggesting that miR-155 has a negative regulatory role in IFN production in pDCs. The finding of upregulated induction of miR-155 in lupus pDCs reported in this thesis is in line with previous studies, which showed increased expression of miR-155 in splenic lymphocytes of lupus NZB/W F1 mice. Results obtained from the transfection experiments are also in accordance with other previous studies, which showed miR-155 functioned as a negative feedback regulator of IFN production in pDCs. However, the mechanism of the association between miR-155 expression and increased IFN response in SLE requires further investigations. It is hoped that findings from this study contribute to a better understanding of SLE pathogenesis and ignite future interests in evaluating the molecular layer of regulation in autoimmunity. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is characterized by auto-reactive T and B lymphocytes and abundant auto-antibodies against nuclear components that form immune-complexes and lead to inflammation, organ dysfunction and failure. The current treatment for SLE includes corticosteroid and immunosuppressant agents which are associated with side effects. Immunotherapy such as tolerogenic dendritic cells (DCs) and regulatory T cells have potential therapeutic implications in autoimmune diseases. DCs are professional antigen presenting cells with important role in promoting immune response and maintaining peripheral tolerance. Alternatively activated DC (aaDC) derived from treating monocyte-derived DCs with vitamin D3 and dexamethasone has demonstrated tolerogenicity and suppressed activation and proliferation of allogeneic T cells in in vitro human studies. As circulating DCs in SLE patients were reported to be hyperactive with increased expression of co-stimulatory molecules and hyper-responsiveness to immunostimulatory stimuli. Therefore, this study aims to examine if aaDCs derived from SLE patients possess tolerogenic properties, to delineate the underlying mechanisms and to examine for therapeutic effect by adoptive transfer of tolerogenic DCs in lupus mouse model. We found that lupus aaDCs derived in vitro displayed semi-mature phenotype with lower expression of co-stimulatory molecules compared with mature DCs. The tolerogenic phenotype remained stable despite challenge by CD40L, CpG-DNA and SLE serum. Lupus aaDCs showed comparable tolerogenic properties as aaDCs from healthy subjects with suppressive effect on allogeneic T cell activation and proliferation. In addition, lupus and normal aaDCs were shown to polarize normal and lupus naïve T cells into IL-10+ suppressive T cells that showed antigen-nonspecific suppressive effect on allogeneic third-party T cells. On the other hand, lupus and normal aaDCs skewed memory T cells to less inflammatory phenotype with reduced expression of IFN-ɤ and IL-17. Although aaDCs displayed a cytokine profile of IL-12loIL-10hi, addition of neutralizing anti-IL-10 and exogenous IL-12 did not reverse the suppressive effect of aaDCs on allogeneic T cells, suggesting their tolerogenicity was not related to cytokine imbalance between IL-12 and IL-10. Furthermore, aaDCs were found to express reduced level of RelB, a transcription factor regulating DC differentiation and maturation. As RelB can be a potential target to induce stable tolerogenic DCs, we constructed RelB shRNA to silence RelB in bone marrow derived DCs (BMDCs) from MRL/MPJ mice. The RelB shRNA transduced BMDCs showed lower level of RelB compared with scramble control shRNA, and displayed tolerogenic phenotype with decreased co-stimulatory molecules, but had no effect on the expression of chemokine receptors. When co-cultured with allogenic CD4+ T cells, RelB shRNA modified BMDCs showed suppressive function on T cell activation and proliferation and increased the production of IL-10 by T cells. However, in vivo study based on 5 mice per treatment group did not show significant effect of RelB shRNA modified BMDCs on disease progress of lupus mice compared to control mice. In conclusion, lupus aaDCs demonstrated tolerogenic properties with induction of IL-10 producing T cells with regulatory functions. RelB shRNA modified BMDCs showed tolerogenic properties in vitro but their in vivo effect on alleviation of murine lupus disease needs further study. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Most currently defined hematopoietic progenitor pools are heterogeneous, contributing to uncertainty regarding the development of certain blood cells. The origins of plasmacytoid dendritic cells, for instance, have long been controversial and progenitors exclusively committed to this lineage have never been described. We show here that the fate of hematopoietic progenitors is determined in part by their surface levels of 9-O-acetyl sialic acid. Pro-plasmacytoid dendritic cells were identified as lineage negative 9-O-acetyl sialic acid low progenitors that lack myeloid and lymphoid potential but differentiate into pre-plasmacytoid dendritic cells. The latter cells are also lineage negative, 9-O-acetyl sialic acid low cells but are exclusively committed to the plasmacytoid dendritic cell lineage. Levels of 9-O-acetyl sialic acid provide a distinct way to define progenitors and thus facilitate the study of hematopoietic differentiation.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The search for anticancer drugs that are tumor specific and cause minimal side effects and the development of effective cancer vaccines are focal points of cancer therapy today. Dendritic cells (DC) are considered potential candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC-based vaccines have exhibited minimal effectiveness in abrogating established tumors in mice and human cancer patients. The use of appropriate adjuvants can enhance the efficacy of DC-based cancer vaccines in treating established tumors.The studies in this dissertation describe a chemo-immunotherapeutic strategy, which combines a Vitamin E analog, a-tocopheryl succinate (a-TOS) that is selectively toxic to tumor cells with non-antigen pulsed, non-matured dendritic cells (nmDC) to treat established murine lung and breast tumors. The results demonstrate that a-TOS synergizes with nmDC to inhibit the growth of established tumors and significantly reduce residual lung metastasis when therapy is initiated after surgical removal of primary tumors. This outcome was correlated with increased IFN-g and IL-4 production by splenic and draining lymph node lymphocytes. In trying to understand the mechanism of action of the combination treatment we observed that a-TOS treated tumor cells factors cause DC maturation in vitro. This effect is mediated in part by heat shock proteins 60, 70 and 90 induced during a-TOS-mediated killing of tumor cells. This study demonstrates the potential usefulness of a-tocopheryl succinate, an agent non-toxic to normal cell types, as an adjuvant to augment the effectiveness of DC-based vaccines in treating cancer.