No description available.

No description available.

No description available.

No description available.

No description available.

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease with a mortality rate of over 40%, and is a major public health concern in the endemic regions of Thailand and northern Australia. Bp is a resilient pathogen capable of surviving in diverse environments including soil, fresh and seawater, and plant and animal tissues for extended lengths of time. Bp is intrinsically resistant to antibiotics, which contributes to persistence and relapse in over 25% of melioidosis patients, and there is currently no vaccine. Our lab has previously shown that immunization with Bp outer membrane vesicles (OMVs) derived from Bp grown in Luria Burtani broth provides significant protection against melioidosis in mice. However, this protection was limited to the acute phase of infection and animals immunized with OMVs were unable to clear the bacteria. In this work, we show that by manipulating the growth media to simulate various bacterial niches, including the natural hypertonic soil environment (NaCl-supplemented), the limited-nutrient host macrophage intracellular environment (M9CG minimal media), and quorum sensing conditions (QS-molecule supplemented), OMV protein content can be modified to include those proteins potentially important for Bp survival and may contribute to protection against chronic or persistent infection. Here, we characterize the composition of selectively enriched Bp OMVs and demonstrate that enriched OMVs are non-toxic and well-tolerated both in vitro and in vivo. Immunization of BALB/c mice with QS OMVs elicits significantly greater OMV-, CPS-, and LPS- serum IgG along with cell-mediated immune responses compared to mice immunized with LB OMVs. LB, M9CG, and QS OMV immunization provided equal protection against aerosolized Bp through the acute phase of infection, and M9CG OMV-immunized mice demonstrated fewer signs of morbidity and less weight-loss over the course of infection, indicating potential control of the bacteria. These results suggest that immunizing with OMVs selectively enriched with intracellular proteins may elicit the necessary immune responses to protect against persistent melioidosis. / Nicole L Kikendall

Paraquat (PQ) is a cationic non-selective bipyridyl herbicide widely used in agriculture to control weeds and grasses. Epidemiologic studies indicate that exposure to pesticides can be a risk factor in the incidence of Parkinson`s disease (PD). A strong correlation has been reported between exposure to paraquat and PD incidence in Canada, Taiwan, and United States. This correlation is supported by animal studies showing that paraquat produces toxicity in dopaminergic neurons of the rat and mouse brain. However, it is unclear how paraquat triggers toxicity in dopaminergic neurons. Based on the previous reports, it was hypothesized that paraquat may induce oxidative stress and proteasomal dysfunction-mediated toxicity in dopaminergic neurons. To explore this possibility, dopaminergic SH-SY5Y human neuroblastoma cells were treated with paraquat, and several biomarkers of oxidative stress or proteasomal dysfunction were investigated. First, a specific dopamine transporter inhibitor GBR12909 significantly protected SY5Y cells against the toxicity of paraquat, indicating that paraquat exerts its toxicity by a mechanism involving the dopamine transporter (DAT). Second, paraquat increased the levels of reactive oxygen species (ROS) in SY5Y cells, but decreased the levels of glutathione. Third, paraquat inhibited glutathione peroxidase activity, but did not affect glutathione reductase activity. On the other hand, paraquat increased GST activity by 24 hr, after which GST activity returned to the control value at 48 hr. Fourth, paraquat decreased mitochondrial transmembrane potential (MTP). Fifth, paraquat produced the increases in malondialdehyde (MDA) and protein carbonyls, as well as DNA fragmentation, indicating oxidative damage to major cellular components. Sixth, paraquat decreased proteasomal activity, the activities of mitochondrial complex I and V, and intracellular ATP levels, but increased the activities of caspase 3 and 9, indicating that proteasomal inhibition is linked to mitochondrial dysfunction accompanied by the activation of apoptotic signaling pathway. Seventh, paraquat increased the protein levels of heme oxygenase-1 (HO-1), p53, Bax, α-synuclein and ubiquitinated proteins. Eighth, paraquat induced nuclear condensation. Taken together, these findings support the hypothesis that paraquat produces oxidative stress and proteasomal dysfunctionmediated toxicity in SY5Y cells. Thus, current findings suggest that paraquat may induce the pathogenesis of dopaminergic neurons through oxidative stress and proteasomal dysfunction.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

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