Thesis (M.D.)--University of Hong Kong, 1995. / Includes bibliographical references (leaf 172-203). Also available in print.

No description available.

>Magister Scientiae - MSc / Systemic lupus erythematosus is a chronic, inflammatory disease characterised by the production of autoantibodies which target particularly the nuclear components of multiple cell types throughout the body. MicroRNA’s have been well-established to regulate gene function by partial-, or complete binding to the 3’-UTR of the target genes, causing repression or complete degradation of the target gene. As a result, proteins normally produced by the targeted mRNA would exhibit a decrease in production.The aim of this study was to investigate the interactions between genes and microRNAs implicated in the pathogenesis of SLE. Objectives included curating lists of miRNAs and genes associated with lupus pathogenesis, to identify regulatory targets of miRNAs and genes targeted by miRNAs, and to find the intersections of these outputs. By examining the intersections of the resultant targets, we aimed to identify novel interactions using Pathway Analysis, which have not been previously reported in scientific literature, to be associated with the pathogenesis of SLE. Understanding the miRNA-gene target interactions in the progression of SLE may provide us with essential biomarkers and targets for disease diagnosis and therapy. / National Research Foundation (NRF) and DAAD

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production and deposition of nuclear self-antigen-containing immune complexes. Epigenetic factors, including altered microRNA (miRNA) expression, may contribute to aberrant immune cell function in SLE. miRNAs are small, noncoding RNAs that bind to the 3’ untranslated region of target mRNAs resulting in post-transcriptional gene modulation. IL-6, an inflammatory cytokine overproduced by mesangial cells in SLE, contains a potential binding site for miR-let-7a. In order to examine if alterations in miR-let-7a expression can influence inflammatory mediator production in SLE, we isolated mesangial cell miRNAs from 8 and 32- week-old female New Zealand Black/White (NZB/W) mice. We found miR-let-7a expression was significantly increased in the mesangial cells of pre-diseased and actively diseased NZB/W mice compared to age-matched female New Zealand White (NZW) controls. Overexpression of miR-let-7a in vitro increased IL-6 production in LPS/IFN-γ-stimulated mesangial cells compared to the stimulated control. Due to the crucial role of miR-let-7a in cell division and inflammation, we investigated miR-let-7a-mediated proliferation and NFκB activation in J774A.1 macrophages and MES 13 mesangial cells in vitro. Cell proliferation, retinoblastoma protein (Rb) phosphorylation, and NFκB activation were increased in cells transfected with miR-let-7a and stimulated with LPS/IFN-γ. Expression and production of the cell cycle inhibitor E2F5 was decreased in stimulated cells overexpressing miR-let-7a. We found that the cell cycle promoter E2F2 and NFκB target the miR-let-7a promoter. Next we sought to determine alterations in iii specific disease-associated miRNAs in female NZB/W mice treated with hydroxychloroquine (HCQ) or prednisone (PRED) for 12 weeks beginning at 20 weeks-of-age. We found that treatment with HCQ or PRED induced unique changes to miRNA expression in multiple tissues. In order to identify specific miRNAs as disease-modifying agents and not merely disease correlates, further in vitro analyses confirmed HCQ or PRED-mediated inhibition of miRNAs is critical to alter the inflammatory response. Taken together, our results suggest that overexpression of miR-let-7a may contribute to hyperplasia and the proinflammatory response in SLE. Our studies indicate that lupus therapeutics may work, in part, by altering the expression of disease-associated miRNAs in immune cells and the urine. / Ph. D.

The major pathologic feature of the human autoimmune disease Systemic Lupus Erythematosus (SLE) and its murine counterpart, murine lupus, is the production of autoantibodies to nucleic acid antigens. In this study, a panel of six murine monoclonal anti-DNA autoantibodies was characterized at both the cellular and molecular levels in order to determine their possible role in the etiology of autoimmune disease. At the cellular level the autoantibodies were found to be highly cross-reactive, binding to three different antigenic forms of DNA as well as to the cell surface of various lymphoid cell lines. Furthermore, the fact that this autoantibody binding could be abrogated by pretreating the cells with either Proteinase K or DNase supports the hypothesis that a DNA binding protein may exist on the cell surface and that DNA bound to this receptor may serve as the target for the anti-DNA autoantibody. At the molecular level, the immunoglobulin (Ig) gene segments (V(H), D, J(H)) used to encode the variable region of the heavy chain of an anti-DNA autoantibody were sequenced. All three gene segments could be identified as members of established Ig gene segment families. In fact, the heavy chain of an antibody directed against the hapten L-glutamine₆₀-L-alanine₃₀-L-tyrosine₁₀ polymer (GAT) was found to utilize the same combination of V(H), D, and J(H) gene segments as the anti-DNA autoantibody. These results clearly indicate that autoantibodies are encoded by gene segments from the same Ig gene families used to encode antibodies to exogenous antigens. However, the discovery that this anti-DNA autoantibody is encoded by the same V(H) gene segment which encodes another anti-DNA autoantibody, derived from a different autoimmune mouse strain, supports the idea that certain V(H) gene segments may, in fact, be preferentially used to encode autoantibodies.

No description available.

ABSTRACT OBJECTIVE: To determine the prevalence and clinical correlates of anti-phospholipid antibodies (aPL), including anti-cardiolipin antibodies (aCL), lupus anti-coagulant (LA), anti- β2-glycoprotein 1 (aβ2GP1) and anti-prothrombin (aPT) antibodies, in Black South African patients with systemic lupus erythematosus (SLE) METHODS: A cross-sectional study of 100 SLE patients in whom clinical characteristics, including features of the anti-phospholipid syndrome (APS), disease activity, and damage were documented, and sera tested for aCL, aβ2GP, and aPT of all isotypes, and LA. RESULTS: Positive aCL, aβ2GP1 and aPT and LA were found in 53, 84, 20, and 2 patients, respectively. Immunoglobulin (Ig)A aCL and IgG aβ2GP1 were the commonest aCL (49.1%) and aβ2GP1 (47%) isotypes, respectively. IgA aβ2GP1 were associated with both a history of thrombosis alone (p<0.05) and a history of any clinical feature, thrombosis and/or spontaneous abortion of the APS (p<0.05); IgA aCL were associated with a history of any clinical APS event (p<0.05); and aβ2GPI of any isotype were associated with a history of arthritis (p<0.001). CONCLUSION: My findings provide further evidence that the screening for aβ2GP1 and IgA aCL isotype may improve the risk assessment for APS in SLE patients of African extraction. Further prospective studies are warranted to determine the clinical utility of these tests and to elucidate the genetic basis for increased IgA aPL response in SLE patients of African extraction.

Wong, Tsz Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 140-159). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abbreviations --- p.III / Abstract --- p.V / 摘要 --- p.VIII / Publications --- p.XI / Table of Contents --- p.XII / Chapter Chapter 1: --- General Information / Chapter 1.1 --- Characteristics and Prevalence of SLE --- p.1 / Chapter 1.2 --- Diagnosis of SLE --- p.2 / Chapter 1.3 --- Assessment of Disease Activity --- p.4 / Chapter 1.4 --- Causes of SLE --- p.4 / Chapter 1.4.1 --- Genetic Factors --- p.4 / Chapter 1.4.2 --- Hormonal Factors --- p.6 / Chapter 1.4.3 --- Environment Factors --- p.7 / Chapter 1.5 --- Drug Treatments of SLE --- p.8 / Chapter 1.6 --- Immunological Dysregulation in SLE --- p.9 / Chapter 1.6.1 --- Types and Properties of Lymphocytes --- p.9 / Chapter 1.6.2 --- Cytokines and Chemokines --- p.14 / Chapter 1.6.3 --- Toll-like Receptors --- p.17 / Chapter 1.6.4 --- Intracellular Signal Transduction Pathways --- p.21 / Chapter 1.7 --- Objectives of Our study --- p.25 / Chapter Chapter 2: --- Material and Methods / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- SLE Patients and Control Subjects --- p.27 / Chapter 2.1.2 --- Reagents for cell culture --- p.28 / Chapter 2.1.3 --- Reagents for Flow Cytometry --- p.30 / Chapter 2.1.4 --- Reagents for Phosphorylation State Analysis --- p.33 / Chapter 2.1.5 --- Reagents for Total RNA Extraction --- p.35 / Chapter 2.1.6 --- Reagents for Polymerase Chain Reaction (PCR) --- p.36 / Chapter 2.1.7 --- Reagents for Gel Electrophoresis --- p.38 / Chapter 2.1.8 --- Reagents for Real-Time Polymerase Chain Reaction --- p.39 / Chapter 2.1.9 --- Other Reagents --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Preparation of Plasma and Purification of Peripheral Blood Mononuclear Cells (PBMC) from EDTA-Blood --- p.41 / Chapter 2.2.2 --- Immunophenotyping of Cell Surface Molecules by Flow Cytometry --- p.42 / Chapter 2.2.3 --- Immunophenotyping of Intracellular Molecules by Flow Cytometry --- p.43 / Chapter 2.2.4 --- Phosphorylation State Analysis of Intracellular Signaling Molecules --- p.43 / Chapter 2.2.5 --- Cytometric Bead Array of Cytokines and Chemokines --- p.44 / Chapter 2.2.6 --- Total RNA Extraction from PBMC --- p.46 / Chapter 2.2.7 --- Reverse Transcription of the Extracted Total RNA --- p.46 / Chapter 2.2.8 --- Real-time Polymerase Chain Reaction --- p.46 / Chapter 2.2.9 --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.48 / Chapter 2.2.10 --- Enzyme-Linked Immunosorbent Spot (ELISPOT) --- p.48 / Chapter 2.2.11 --- Statistical Analysis --- p.49 / Chapter Chapter 3: --- B Cell Chemokine CXCL13 in SLE / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Methods --- p.52 / Chapter 3.3 --- Results --- p.52 / Chapter 3.3.1 --- Characteristics of SLE Patients and Control Subjects --- p.52 / Chapter 3.3.2 --- Expression of Plasma CXCL13 --- p.53 / Chapter 3.3.3 --- Gene Expression of TNF-α in PBMC --- p.53 / Chapter 3.3.4 --- Expression of sTNFRl in Plasma --- p.53 / Chapter 3.4 --- Discussion --- p.57 / Chapter Chapter 4: --- T lymphocyte Co-stimulatory Molecule CD26 in SLE / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Methods --- p.64 / Chapter 4.3 --- Results --- p.66 / Chapter 4.3.1 --- Characteristic of SLE Patients and Control Subjects --- p.66 / Chapter 4.3.2 --- Expression of Human CD26 in Plasma --- p.66 / Chapter 4.3.3 --- "Cell Surface Expression of CD26 on Monocytes, Th, Tc plus Ts, B and iNKT Lymphocytes" --- p.66 / Chapter 4.3.4 --- Circulating Number of iNKT Lymphocytes --- p.67 / Chapter 4.4 --- Discussion --- p.71 / Chapter Chapter 5: --- Thl7 Lymphocytes and Expression of IL-17 in SLE / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.2 --- Methods --- p.78 / Chapter 5.3 --- Results --- p.79 / Chapter 5.3.1 --- Characteristics of SLE Patients and Control Subjects --- p.79 / Chapter 5.3.2 --- Ex vivo production of IL-17A from PBMC --- p.79 / Chapter 5.3.3 --- Circulating Number of Thl7 Lymphocytes --- p.79 / Chapter 5.4 --- Discussion --- p.82 / Chapter Chapter 6: --- Intracellular Mitogen Activated Protein Kinases in SLE / Chapter 6.1 --- Introduction --- p.87 / Chapter 6.2 --- Methods --- p.89 / Chapter 6.3 --- Results --- p.91 / Chapter 6.3.1 --- Characteristics of SLE Patients and Control Subjects --- p.91 / Chapter 6.3.2 --- Expression of Phospho-p38 MAPK in PBMC --- p.91 / Chapter 6.3.3 --- Expression of Phospho-ERK in PBMC --- p.92 / Chapter 6.3.4 --- Expression of Phospho-JNK in PBMC --- p.92 / Chapter 6.3.5 --- "Relative Percentage Increase of Phosphorylated p38 MAPK, ERK and JNK upon IL-18 Activation" --- p.93 / Chapter 6.4 --- Discussion --- p.104 / Chapter Chapter 7: --- Toll-like Receptors in SLE / Chapter 7.1 --- Introduction --- p.111 / Chapter 7.2 --- Methods --- p.113 / Chapter 7.3 --- Results --- p.115 / Chapter 7.3.1 --- Characteristics of SLE Patients and Control Subjects --- p.115 / Chapter 7.3.2 --- Expression of TLRl to TLR9 of PBMC --- p.115 / Chapter 7.3.3 --- Preliminary Results of Cytokine and Chemokine Expression Upon TLR Lignad Activation --- p.116 / Chapter 7.4 --- Discussion --- p.126 / Chapter Chapter 8: --- Conclusion and Future Perspectives --- p.133 / Appendix --- p.138 / References --- p.140

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with a strong and complex genetic basis. To dissect the function of the lupus susceptibility loci on New Zealand black (NZB) mouse chromosome 1, the lab had previously generated congenic mice with an introgressed homozygous NZB chromosome 1 intervals extending from ~35 or ~82 to 106 cM on the C57BL/6 background. Although both mouse strains made IgG anti-nuclear antibodies (ANAs), ANA titres and cellular activation were significantly higher in mice with the longer interval. These studies suggest the presence of two susceptibility genes. In this thesis I have sought to further characterize the cellular abnormalities and underlying genetic polymorphisms that produce them in these mice. Using mixed hematopoietic chimeric mice, with a mixture of tagged-B6 and congenic bone marrow I demonstrate that there are intrinsic B and T cell functional defects in chromosome 1 congenic mice. I further show that an intrinsic B cell defect is required for efficient recruitment of B cells into the spontaneous germinal centres and differentiation of autoantibody producing cells in these mice. To more precisely localize the susceptibility loci, I produced and characterized a number of additional subcongenic mouse strains. This revealed surprising genetic complexity with the presence of at least four lupus susceptibility loci and a suppressor locus on chromosome 1, several of which appeared to impact on T cell function. Finally, I generated bicongenic mice carrying both NZB chromosome 1 and 13 intervals, hypothesizing that since these were two of the major intervals associated with autoimmune disease in NZB mice they would fully recapitulate the autoimmune phenotypes. Although this hypothesis was incorrect, several novel phenotypes developed including marked expansion of the plasmacytoid and myeloid dendritic cell compartments and increased BAFF and IgA autoantibody production. Although this expansion was associated with TLR hyper-responsiveness, disease severity remained mild, possibly due to the lack of IFN- production, which appeared to be inhibited in these mice. Thus, lupus arises from immune defects affecting several cellular populations, which are the product of multiple genetic polymorphisms that interact in a complex fashion to produce the autoimmune phenotype.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with a strong and complex genetic basis. To dissect the function of the lupus susceptibility loci on New Zealand black (NZB) mouse chromosome 1, the lab had previously generated congenic mice with an introgressed homozygous NZB chromosome 1 intervals extending from ~35 or ~82 to 106 cM on the C57BL/6 background. Although both mouse strains made IgG anti-nuclear antibodies (ANAs), ANA titres and cellular activation were significantly higher in mice with the longer interval. These studies suggest the presence of two susceptibility genes. In this thesis I have sought to further characterize the cellular abnormalities and underlying genetic polymorphisms that produce them in these mice. Using mixed hematopoietic chimeric mice, with a mixture of tagged-B6 and congenic bone marrow I demonstrate that there are intrinsic B and T cell functional defects in chromosome 1 congenic mice. I further show that an intrinsic B cell defect is required for efficient recruitment of B cells into the spontaneous germinal centres and differentiation of autoantibody producing cells in these mice. To more precisely localize the susceptibility loci, I produced and characterized a number of additional subcongenic mouse strains. This revealed surprising genetic complexity with the presence of at least four lupus susceptibility loci and a suppressor locus on chromosome 1, several of which appeared to impact on T cell function. Finally, I generated bicongenic mice carrying both NZB chromosome 1 and 13 intervals, hypothesizing that since these were two of the major intervals associated with autoimmune disease in NZB mice they would fully recapitulate the autoimmune phenotypes. Although this hypothesis was incorrect, several novel phenotypes developed including marked expansion of the plasmacytoid and myeloid dendritic cell compartments and increased BAFF and IgA autoantibody production. Although this expansion was associated with TLR hyper-responsiveness, disease severity remained mild, possibly due to the lack of IFN- production, which appeared to be inhibited in these mice. Thus, lupus arises from immune defects affecting several cellular populations, which are the product of multiple genetic polymorphisms that interact in a complex fashion to produce the autoimmune phenotype.