One of the major challenges facing clinicians is how to effectively manage excessive host immune response to pathogenic insults resulting in sepsis. This is demonstrated by the fact that despite over half-century research efforts, sepsis and its spectrum of diseases (severe sepsis and septic shock) are still associated with poor clinical outcome. Currently, sepsis is a leading cause of death in intensive care units. The immune system protects the host against pathogens and is therefore armed with an arsenal of deadly ammunitions (including chemicals, cells and proteins) necessary for the elimination of microbes. It is therefore paramount that the immune system must develop mechanisms necessary to prevent destruction of the host it is designed to protect. A good example of such a mechanism is found in the subset of lymphocytes known as regulatory T cells (Tregs). There is unequivocal experimental evidence of the role of Tregs in the maintenance of immune homeostasis and self tolerance and aberrant Treg function has been linked with several inflammatory diseases. Since sepsis is a disease marked by a hyper-inflammatory state, I investigated the possible role of Tregs in dampening sepsis-induced excessive inflammation. Using a murine model of lipopolysaccharide (LPS) infusion and bacterial infection, I show that Tregs are essential for survival during sepsis because their depletion leads to acute death to an otherwise non-lethal dose of LPS. This enhanced susceptibility to LPS following Treg depletion was also observed using live E. coli infection. Next, I probed the mechanism by which Tregs protect against LPS challenge. I found that defective Treg function leads to exaggerated activity of two immune cells – CD4+ effector T cells and neutrophils in response to LPS, leading to severe inflammatory response. Hence, this work successfully illustrates the critical role of Tregs in regulating other immune cells and the catastrophic consequences of defective Treg function during an immune response. Overall, this work highlights the significant role of Tregs in the regulation of bacteria associated inflammatory processes. The findings hold implications for the successful management of sepsis and have potential for use in development of adequate therapeutic intervention for sepsis. / October 2016

Lfc is a Rho specific guanine nucleotide exchange factor (GEF) that is bound and inhibited by the microtubule (MT) cytoskeleton. In epithelial cells, Lfc promotes actomyosin contractility in response to MT depolymerization; however, its role in leukocytes has not been assessed. Through genetic ablation, we generated an Lfc knockout mouse (Lfc-/-) and tested biochemical and cell biological responses to MT depolymerization in bone marrow derived cells. Lfc was necessary for characteristic actomyosin based contractile behaviours of neutrophils and macrophages, in response to MT depolymerization. Gout is a painful arthritic inflammatory disease, caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a MT-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, blocks neutrophil infiltration to sites of MSU crystal-induced inflammation. We found that Lfc was necessary for the ability of colchicine to inhibit MSU-induced neutrophil infiltration in two in vivo models of gout-like inflammation. Efficient recruitment of leukocytes from the vasculature is a critical step in the immune response to infection. Leukocyte extravasation, which includes rolling, crawling, and diapedesis across the endothelial barrier, is enhanced by fluid shear stress. Through comparison of Lfc+/+ and Lfc-/- mice, we found that Lfc was necessary for in vivo leukocyte crawling and emigration out of the vasculature. Lfc-/- mice also showed defective neutrophil infiltration in response to acute inflammatory insults, and increased mortality in response to polymicrobial infection. In vitro, we found that Lfc was necessary for neutrophil responses to shear stress.

Lfc is a Rho specific guanine nucleotide exchange factor (GEF) that is bound and inhibited by the microtubule (MT) cytoskeleton. In epithelial cells, Lfc promotes actomyosin contractility in response to MT depolymerization; however, its role in leukocytes has not been assessed. Through genetic ablation, we generated an Lfc knockout mouse (Lfc-/-) and tested biochemical and cell biological responses to MT depolymerization in bone marrow derived cells. Lfc was necessary for characteristic actomyosin based contractile behaviours of neutrophils and macrophages, in response to MT depolymerization. Gout is a painful arthritic inflammatory disease, caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a MT-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, blocks neutrophil infiltration to sites of MSU crystal-induced inflammation. We found that Lfc was necessary for the ability of colchicine to inhibit MSU-induced neutrophil infiltration in two in vivo models of gout-like inflammation. Efficient recruitment of leukocytes from the vasculature is a critical step in the immune response to infection. Leukocyte extravasation, which includes rolling, crawling, and diapedesis across the endothelial barrier, is enhanced by fluid shear stress. Through comparison of Lfc+/+ and Lfc-/- mice, we found that Lfc was necessary for in vivo leukocyte crawling and emigration out of the vasculature. Lfc-/- mice also showed defective neutrophil infiltration in response to acute inflammatory insults, and increased mortality in response to polymicrobial infection. In vitro, we found that Lfc was necessary for neutrophil responses to shear stress.

No description available.

No description available.

The purpose of this study was first to describe the clinical findings, case management and short-term outcome of horses with septic synovial structures over the last 25 years, and to identify risk factors and treatment modalities associated with specific short-term outcomes. Secondly, we wanted to evaluate a broad range real time polymerase chain reaction (RT PCR) assay for the diagnosis of septic synovitis from synovial fluid (SF) samples of horses, and compare its performance to currently available diagnostic methods. For the retrospective study, 367 cases met the inclusion criteria. Lavage of the synovial structure and endoscopic surgery were associated with an increased likelihood of discharge from hospital, however, none of the local antimicrobial delivery modalities were associated with a significant change in outcome. No significant improvement in hospital outcome of horses with septic synovitis was identified over the past 25 years. For the RT PCR study, 48 SF samples from horses with suspected septic synovitis were included, and RT PCR and microbial culture was performed on all samples. One additional RT PCR assay was performed on samples with discordant results or identification of dissimilar organisms. Thirty-eight percent of SF samples had positive culture results, and 42% of SF samples had positive RT PCR results. Sensitivity and specificity for the RT PCR assay relative to agreement of observers on the likelihood of infection were 87% and 72%, respectively, whereas for culture they were 56% and 86%, respectively (P=0.001). The combination of culture and RT PCR assay resulted in sensitivity and specificity of 85% and 81%, respectively. The broad range RT PCR assay was more sensitive than culture for identification of horses with septic synovitis. Further refinement of the RT PCR assay technique may facilitate use in a clinical setting. / Equine Guelph, University of Guelph

This study identified striking increase in plasma of plasminogen activator inhibitor 1(PAI-I), a major inhibitor of fibrinolysis levels in septic patients who are non-neutropenic. Neutropenic patients show less striking changes. Where shock occurs both groups of patients show very high levels of PAI-1. These observations suggest a role for leucocytes in PAI production. In the second section neutrophils are identified as containing PAI-1 in normal subjects, the levels rising significantly in sepsis. Monocytes contain no PAI-1 but do contain Plasminogen activator inhibitor 2(PAI-2) levels of which inhibitor also rise in sepsis. Normal neutrophils contained no PAI-2 but neutrophils from septic patients contained significant quantities of this inhibitor. In the third section mononuclear cells from septic patients are identified as enhancing PAI-1 production in cultured endothelial cell (EC). Septic neutrophils have a more complex effect on EC. Mononuclear cells and neutrophils therefore, both contribute to the fibrinolytic inhibition of septic disorders but by different mechanisms. Each cell type contains one of the major inhibitor of plasminogen activator and levels of these rise in sepsis. Both cell types from septic patients promote greater release of PAI-1 from endothelial cells than do cells from normal individuals. Inhibition of fibrinolysis by leucocytes may contribute to fibrin persistence in sepsis. This may be useful in localizing infection. If generalized, it may contribute to vascular occlusive complications of sepsis such as shock lung, acute renal failure or digital gangrene. Absence of leucocytes may account for the apparent reduction of vascular occlusive complications in leucopenic septic patients.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves : 188-225). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / "May 1996" Typescript. Vita. Includes bibliographical references (l. 188-225). Also available on the Internet.