Lupus nephritis is a severe organ manifestation of systemic lupus erythematosus (SLE), and is characterized by the production of anti-dsDNA antibodies. It is an important cause of renal failure. The mechanism through which anti-dsDNA antibodies bind to tissues and mediate kidney injury remains to be fully elucidated. Emerging evidence suggests that anti-dsDNA antibodies can bind to cells and extracellular antigens directly through cross-reactivity, independent of bridging chromatin material. Mesangial cells play an important role in normal kidney structure and functions, and its pathophysiology. Mesangial abnormalities in lupus nephritis precede more severe injuries such as lesions in the glomerular capillary loop. We previously demonstrated that the binding of human anti-dsDNA antibodies to mesangial cells (HMC) correlated with disease activity and induced inflammatory as well as fibrotic pathways. The aim of this project is to identify the cross-reactive antigen(s) on the mesangial cell surface that mediates anti-dsDNA antibody binding and the alterations in cell functions that result from this interaction. HMC plasma membrane proteins were purified. Using proteomic and biochemical approaches, we identified annexin II as the predominant cross-reactive antigen on the HMC surface that mediated human polyclonal anti-dsDNA antibody binding. Following this interaction, anti-dsDNA antibodies were internalized in a time- and temperature-dependent manner, and translocated to both the cytoplasm and nucleus within 30 min. This resulted in induction of annexin II synthesis, IL-6 secretion and cell proliferation, which was mediated through the activation of p38 MAPK, JNK and AKT. The binding activity to annexin II in the serum immunoglobulin fraction correlated with the titre of anti-dsDNA antibody. Binding activity of anti-dsDNA antibodies to annexin II correlated with clinical disease activity and circulating anti-dsDNA antibody levels. These correlations were more prominent in male patients with lupus nephritis. Glomerular annexin II expression was increased in patients with active lupus nephritis and co-localized with IgG and C3 deposition. Gene silencing of annexin II in HMC reduced anti-dsDNA antibody binding, which was accompanied by reduced IL-6 secretion and cell proliferation. Using female NZB/W F1 mice, an established murine model of lupus nephritis, we demonstrated that intra-glomerular annexin II expression increased with disease progression and was accompanied by an increase in the expression of p11, its cellular protein ligand. Our data suggest that annexin II may exist in the kidney as a heterotetramer and is involved in disease pathogenesis. At the ultrastructural level, annexin II was detected in the mesangial matrix, amongst electron dense deposits in the glomerular basement membrane, on the foot processes in podocytes and within the Bowman’s capsule. In conclusion, our data demonstrated that annexin II is the major cell surface antigen on HMC that mediates the cross-reactive binding of human anti-DNA antibodies. Through this interaction, cellular processes are triggered that contribute to the pathogenesis of lupus nephritis. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

abstract / Anatomy / Doctoral / Doctor of Philosophy

Implantation is a critical step in reproduction. It is complicated and well-coordinated consisting of apposition, attachment and invasion of embryo into the endometrium. The mechanism of implantation is unclear. Our previous proteomic study showed an increase of annexin A2 in the endometrium during the implantation window of mice, consistent with the increased annexin A2 expression in the receptive human endometrium. The hypothesis of this project was that annexin A2 mediatedthe embryo-endometrium attachment. The first objective was to study the spatio-temporal expression of endometrial annexin A2 immunoreactivities in humans and mice. The cyclical change in annexin A2 expression in the mouse and human reproductive cycle suggested the involvement of a steroid regulatory mechanism. Interestingly, annexinA2 was transiently expressed on the membrane between the mouse uterine luminal epithelium and the implanting embryos from Day 4 (pre-implantation) to Day 5 (post-implantation) of pregnancy. No such signal change was observed at the inter-implantation sites, showing that the implanting embryos partially regulated annexin A2 expression. These observations and the high expression of the molecule in the luminal epithelium of human endometrium in the mid-and late luteal phase were consistent with a role of annexin A2 in implantation. The second objective was to verify the action of steroids on annexin A2 expression. It was found that a combination of 6675 pmol/L of estrogen and 429.8nmol/L of progesterone increased the total and apical surface expression of annexin A2. In mice, estrogen but not progesterone, increased annexin A2 expression in the uterine luminal epithelium of ovariectomized mice. The third objective was to study the function of annexin A2 in embryo-endometrium attachment using an Ishikawa (endometrial epithelial cells)-JEG-3 trophoblast spheroids (embryo surrogate) coculture model. Knockdown of the expression of annexin A2 in either or both cell lines significantly decreased the attachment rate of the spheroids onto the endometrial cells. The suppressive action on the two cell lines was additive. The attachment was also suppressed in the presence of anti-annexin A2 antibody during coculture. Annexin A2 was also involved in mouse implantation as demonstrated by a significant decrease in implantation sites after injection of anti-annexin A2 antibody into the mouse uterine horn. The final objective was to study the action of annexin A2 as an adhesive molecule in embryo attachment. It was found that loss of P11, the binding partner of annexin A2, reduced the attachment rate of the JEG-3 spheroids probably by decreasing the translocation of annexin A2 to the surface of the endometrial cells. Recombinant P11 and annexin A2 protein failed to bind significantly to the Ishikawa cells and the JEG-3 cells. In summary, this study demonstrates the involvement of annexin A2 as an adherent molecule in the embryo-endometrium interaction. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

No description available.

No description available.

published_or_final_version / abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1990. / Includes bibliographical references (leaves 68-76) / Microfiche. / xi, 76 leaves, bound ill. 29 cm

Thesis (M.S.)--University of Missouri-Columbia, 2008. / "May 2008" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.