Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from rupture of a blood vessel in the brain. Tissue inside the hematoma is irreversibly damaged soon after ICH onset and when this thesis research began, there was a dearth of information regarding pathological changes outside the hematoma. Inflammation is often proposed as a mechanism of injury, but very little information was available to show that inflammatory cells were in the right place at the right time to cause secondary brain injury. Using the collagenase-induced model of ICH, this work sought to better define spatial and temporal relationships between secondary brain injury and the inflammatory response after ICH. To test the hypothesis that reducing inflammation can protect the brain from secondary injury, minocycline, an antibiotic with established anti-inflammatory effects, was administered 6 hours after ICH onset. A small number of neurons die in the parenchyma bordering the hematoma between 6 hours and 3 days after ICH onset. This area was not associated with neutrophil infiltration, and most activated microglia/macrophages did not accumulate until after most neuron death had occurred. Despite a pronounced microglial response and prolonged increase in expression of many inflammatory genes, including complement receptor-3, interleukin-1 beta, interleukin-6, and interleukin-1 converting enzyme, no dying neurons were observed further outside the hematoma at any time. Interestingly, less early neuron death was observed in aged than in young animals, without a concomitant difference in the amount of tissue lost at 28 days. However, aged animals had less early microglial activation and a larger residual lesion, which might have resulted from impaired phagocytosis by activated microglia/macrophages. Minocycline was less effective in reducing microglial activation in aged animals, and did not reduce neuron death in either young or aged animals. Edema and BBB disruption was associated with degradation of the basal lamina protein, collagen type IV, and that damaged vessels are associated with tumor necrosis factor-alpha (TNFα)-positive neutrophils and active matrix metalloprotease-12 (MMP-12), all of which were reduced by delayed minocycline treatment. In contrast to ischemic stroke, there is a limited ‘penumbra’ outside the hematoma. Nevertheless, BBB damage in this region appears to be a potential target for protection. Furthermore, the prominent inflammatory response that continues for days after ICH does not appear to be associated with damage to other areas of the brain. Minocycline appears to protect the BBB by reducing neutrophil infiltration and the MMP-12 mediated basal lamina degradation. Future studies should investigate other targets for protection (i.e., white matter injury), and seek drugs that modulate the inflammatory response in aged animals and promote lesion resolution.

Bovine mastitis due to bacterial infection is one of the most costly diseases affecting the dairy industry. The polymorphonuclear neutrophils (PMNs) present in milk have a central protective role against invading pathogens, However, the manner by which PMNs traverse the secretory epithelia and the relationship between PMN diapedesis and the epithelial damage are unclear. This in vitro study investigated the process and rate of bovine PMN transepithelial migration. The bovine mammary epithelial cell line, MAC-T, formed a confluent monolayer with characteristic tight junctions, polarity and functional barrier to the dye trypan blue. In the first series of experiments, neutrophils were added into the upper compartment of the culture insert and stimulated to migrate across the epithelium in an apical-to-basal direction by the addition of Staphylococcus aureus to the lower compartment. Light and transmission electron microscopy revealed the following series of events for PMN transmigration: (1) adherence of PMNs to the surface of the epithelium; (2) projection of pseudopods toward the intercellular junction; (3) migration between adjacent epithelial cells; and (4) re-approximation of epithelial cell membranes and reformation.

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mainly mediated by the Fc receptor family, including IgE receptors. Recently, we have shown that human PMNs from allergic asthmatic subjects express the high affinity receptor, FceRI. In this study, we have examined the regulation of FceRI by human PMNs in vitro and in vivo during the allergic pollen season. First we studied the pattern of expression of FceRI in PMNs during the pollen allergic and outside the pollen season. Peripheral blood neutrophils were isolated from adult atopic asthmatics (AA) (n=17), allergic non asthmatics (ANA) (n=15) and healthy donors (n=16) by dextran, ficoll gradient centrifugation and magnetic cell sorting (MACS). Surface, total protein and mRNA expression of FceRI were investigated in the three groups by FACS, immunocytochemistry (ICC) and fluorescent in situ hybridization (FISH) respectively. Secondly, we investigated the effect of Th-2 cytokines which are known to regulate IgE receptor expression. PMNs from atopic asthmatic subjects were stimulated in vitro with Th-2 cytokines (IL-4, IL-9, GM-CSF) and Th-1 cytokine IFN-gamma. Finally we determined whether the expression of FceRIbeta chain correlated with the surface expression of FceRIalpha chain in PMNs. Irrespective of the season, PMNs from atopic asthmatic subjects showed increased expression of FceRIalpha chain in surface, total protein and mRNA compared to atopic non asthmatics and healthy donors (n=20). Interestingly, FceRIalpha chain surface and mRNA expression increased significantly during pollen season compared to non pollen season (P=0.001) in PMNs isolated from AA (n=9) in contrast to healthy donors and ANA (n=8). Furthermore similar pattern of FceRI expression were observed in vitro when PMNs were stimulated with Th2 cytokines. IL-4, IL-9 and GM-CSF showed increased protein and mRNA expression of FceRIalpha chain at 6 and 18hrs (n=6) whereas IFN-gamma down regulated the mRNA expression of FceRIalpha chain at 6hrs. Also, irrespective of season AA (n=11) subjects showed increased expression of FceRI beta chain when compared to ANA (n=10) and healthy donors (n=9). Western blot analysis showed increased FceRI beta protein in atopic asthmatic subjects (n=4). Interestingly irrespective of the groups, there was a positive correlation r = 0.8054 between total protein expression of beta chain with surface expression of alpha chain of FceRI in neutrophils. Our data suggest that the expression of FceRI in neutrophils of atopic asthmatic patients is highly regulated. Our in vitro studies provide evidence that Th-2 cytokines such as IL-9, IL-4 and GM-CSF up-regulate the expression of FceRI. Furthermore we show evidence of increased expression of FceRIbeta chain in neutrophils of atopic asthmatic subjects. Collectively these results suggest that FceRI mediated neutrophil dependent activation may play a key role in allergic diseases.

Circulating neutrophil activation occurs during cardiac surgery with extracorporeal circulation (ECC) and is implicated in the pathophysiology of inflammatory tissue injury and peri-operative organ dysfunction. However, neutrophil directed antiinflammatory strategies have failed to demonstrate consistent therapeutic benefit indicating that the nature and significance of peri-operative circulating neutrophil activation remains incompletely defined. In particular, conformational activation of the b2 integrin Mac-1 (CD11b/CD18), which is required for neutrophil adhesion competence and facilitation of effector functions, has not previously been investigated during cardiac surgery, and the relative contribution of cellular activation and bone marrow neutrophil recruitment to peri-operative changes in circulating neutrophil phenotype and function is unknown. A novel whole blood flow cytometric technique was used to analyze circulating neutrophil phenotype (total Mac-1, conformationally-active CD11b, CD10, CD16, L-selectin and P-selectin glycoprotein ligand-1) and function in cardiac surgery patients to characterize the nature of changes in Mac-1 expression and activation status, and the effects of relative neutrophil immaturity on circulating neutrophil phenotype and function. The effect of heparin, a known CD11b ligand, on Mac-1 epitope expression was also investigated. Circulating neutrophil numbers observed during ECC were mathematically modeled to determine the acute response of the bone marrow neutrophil reserve to an inflammatory stimulus. Plasma cytokine, chemokine and acute phase mediators were measured in cardiac and lung surgery patients to determine potential regulators of systemic neutrophil recruitment. Neutrophils newlyemergent from the bone marrow were characterized as CD10-/CD16low and exhibited distinct changes in cell surface markers and enhanced functional responses, relative to their more mature CD10+ counterparts. Conformational activation of CD11b occurred peri-operatively and provided a more sensitive measure of circulating neutrophil activation status than changes in total Mac-1 or L-selectin expression, although detection of Mac-1 epitopes was reduced in the presence of heparin. Modeling of circulating neutrophil numbers predicted that post-mitotic maturation time was acutely abbreviated by 8.4 hours during 71 minutes of ECC. Systemic chemokine release occurred with cardiac but not non-cardiac thoracic surgery indicating some specificity of the acute inflammatory response. These findings expand the understanding of peri-operative circulating neutrophil activation and recruitment, and identify potential therapeutic targets to limit neutrophil injurious potential during cardiac surgery with ECC.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Michigan State University. Dept. of Biochemistry and Molecular Biology, 2008. / Title from PDF t.p. (viewed on Mar. 30, 2009). Includes bibliographical references (p. 204-227). Also issued in print.

Thesis (MPharm.--Faculty of Health Sciences)-University of Pretoria, 2007. / Includes bibliographical references.