"October 2001" Amendments (4 leaves) inserted inside back cover. Includes bibliographical references (leaves 215-266)

An animal model of respiratory infection was used to determine the effect of various factors, thought to influence the ability of the host to clear bacteria, on the host?s innate response to an NTHi lung infection. Mucosal immunisation with NTHi has previously been shown to enhance the clearance of NTHi from the lung in an animal model of infection through the increased recruitment of phagocytes. Comparisons of cytokine and chemokine kinetic profiles were made in order to determine differences between innate and acquired immune response and the way in which mucosal immunisation controls the innate immune response to NTHi. Increased production of proinflammatory cytokines and chemokines in the early stages of NTHi lung infection enhanced the ability to clear bacteria from the rat lung in the immune animals through the increased recruitment of phagocytes to the site. Mucosal immunisation was found to alter the cytokine and chemokine mRNA profiles of CD4+ and CD8+ cells, with increased levels of MCP-1 protein being detected in both types of immune cells. An antecedent viral infection has been shown to increase the chance of developing a respiratory bacterial infection. The NTHi model of respiratory infection was used to characterise the effect that a viral infection had on the host response to the host?s innate response to a bacterial infection and the ability to clear the bacteria. The host?s ability to clear NTHi from the rat lung was enhanced by an antecedent viral infection through alterations to the innate immune response and the cytokine and chemokine kinetic profiles. The use of a mutant strain of NTHi deficient in a component of Lipooligosaccharide (LOS), Phosphorylcholine (ChoP), was utilised as a tool to characterise the innate immune response to LOS. Animals challenged with the LOS mutant strain had a reduced inflammatory response to NTHi through the decreased production of pro-inflammatory cytokines and chemokines and the reduced recruitment of phagocytes to the site of infection. This thesis has contributed valuable information to enable a better understanding of the host?s innate immune response to respiratory infection. This study has identified the role of cytokines and chemokines in the innate response to a respiratory bacterial infection and the enhanced ability of the host to clear NTHi from the lung.

The suppressor of cytokine signaling ( SOCS ) proteins have been identified as important regulators of cytokine signaling. SOCS - 3 has been identified as being essential for normal fetal growth and survival, with the null mutation of the socs - 3 gene resulting in embryo death. The specific role of SOCS - 3 in fetal development, however, has yet to be characterized. Therefore, the overall aim of this thesis was to identify and quantify SOCS - 3 mRNA in a range of fetal tissues in the sheep. After identification of SOCS - 3 expression in fetal tissues, we then aimed to determine the ontogenic profile of SOCS - 3 in three key fetal tissues ; the liver, adipose tissue and adrenal gland, and whether SOCS - 3 expression in these tissues was altered after withdrawal and stimulation of prolactin ( PRL ). SOCS - 3 mRNA was found to be differentially expressed in a range of fetal tissues in late gestation and was higher in the fetal liver than in the pancreas, spleen and kidney. SOCS - 3 expression increased throughout gestation in the fetal liver, however, its expression decreased in the fetal adipose tissue and adrenal in late gestation. The pituitary hormone PRL has previously been implicated as a fetal growth factor. In the sheep fetus, PRL receptors are expressed in the fetal liver, adipose tissue and adrenal. We aimed to determine whether PRL plays a role in the maintenance of SOCS - 3 expression in the liver, adipose tissue and adrenal gland in late gestation, and whether SOCS - 3 expression can be regulated by acute PRL stimulation. We have demonstrated that PRL withdrawal suppressed SOCS - 3 expression in the liver, whereas acute PRL stimulation upregulated SOCS - 3 expression in the adrenal. Neither PRL withdrawal nor stimulation had an effect on SOCS - 3 expression in the adipose tissue. In summary, the data presented in this thesis would suggest that SOCS - 3 has tissue specific functions in late gestation. Furthermore, its expression is regulated in a tissue specific manner in response to the withdrawal or acute stimulation by PRL This provides the first evidence to suggest that the fetal liver and adrenal are both sensitive to either chronic or acute changes in plasma PRL concentrations, measured as the suppression or upregulation of SOCS - 3. We speculate that changes in SOCS - 3 mRNA expression relates to the regulation of growth and functional maturation of fetal tissues throughout gestation, and that PRL may represent an important factor which acts to alter SOCS - 3 expression in key fetal tissues. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2006.

Normal tissue homeostasis and appropriate responses to injury and infection are dependent on cellular communication mediated by cell surface receptors that respond to extrinsic stimuli. The GM-CSF receptor was the major focus of this project. This receptor shares a common signalling subunit, β [subscript c], with the IL-3 and IL-5 receptors. The unique GM-CSF receptor α-subunit ( GMRα ) confers ligand binding specificity to the complex and is essential for GM-CSF receptor signalling, although the full complement of signalling events mediated by GMRα remains elusive. Through cloning of candidate interacting proteins, expression and co-immunoprecipitation studies, we have confirmed interactions for two proteins previously reported to interact with the GMRα, p85 and IKKβ. Additionally, we identified the Src family kinase, Lyn, as a novel direct interacting partner of GMRα and provide insights into possible roles of this kinase in initiating signalling from the GM-CSF receptor. In addition to GMRα associated events we aimed to further characterise the role of the common β [subscript c] subunit in GM-CSF mediated signalling. We utilised two classes of consitutively active β [subscript c] mutants ( extracellular or transmembrane ) which transform the bi-potential myeloid FDB1 cell line to either factor-independent growth and survival, or granulocyte-macrophage differentiation, respectively. Here we report a comprehensive biochemical analysis of signalling by these two classes of mutants in this cell line. The two activated GMR mutants displayed distinct and non-overlapping signalling capacity. In particular, expression of a mutant with a substitution in the transmembrane domain ( V449E ) selectively activated JAK / STAT5 and MAPK pathways resulting in a high level of sensitivity to JAK and MEK inhibitors. In contrast, expression of a mutant with a 37 amino acid duplication in its extracellular domain ( FI Δ ) selectively activates the PI3K / AKT and IKKβ / NFkB pathways. Cells responding to this mutant display a relative high level of sensitivity to two independent PI3K inhibitors and relative resistance to inhibition of MEK and JAK2. The non-overlapping nature of signalling by these two activated mutants suggests that there are alternative modes of receptor activation that differentially dependent on JAK2 and that act synergistically in the mature liganded cytokine receptor complex. Further detailed analysis of these mutants will facilitate the dissection of the signalling pathways involved in the GM-CSF response that mediate proliferation, survival and differentiation. / Thesis (Ph.D.)--School of Medicine, 2007.

Leukocyte adhesion to cerebral endothelial cells plays a critical role in the inflammatory response following transient global cerebral ischemia but its contribution to delayed neuronal cell death is not completely understood. We compared ischemic mice treated with a monoclonal antibody to β2-integrin adhesion receptors (anti-CD18) or a non-binding control antibody following ischemia. Inflammation was characterized by increased CD18 expression on leukocytes and inflammatory mediators in the peripheral blood and brain tissue. Notably, interleukin-1β, which has been shown to mediate cell death in neurons, was elevated in the blood and brain. Anti-CD18 blocked leukocyte adhesion as well as the inflammatory responses, including interleukin-1β expression in neurons. Blocking leukocyte adhesion protected the structural integrity of the hippocampus, cerebral cortex and thalamus, and preserved spatial. Leukocytes adhesion to endothelial cells plays an important role in the evolution of neurological deficit in global cerebral ischemia despite the lack of transmigration of leukocytes across blood-brain-barrier.

Two experiments were designed to study endometritis in postpartum dairy cows. In the first experiment, 30 cows 28 to 41 days in milk (DIM) and without evidence of clinical endometritis were sampled using cytobrush cytology. Cytobrush sampling provided sufficient endometrial material to prepare cytologic specimens and to extract endometrial mRNA. Pro-inflammatory cytokines were analyzed in harvested endometrial tissue taken from cows with and without endometritis. Cytokine expression varied between experimental groups with 30-fold higher IL-6 expression levels (P=0.01), greater than 50-fold higher IL-8 expression levels (P=0.0001), and 20-fold higher TNF-α expression levels (P=0.001) in endometritis-positive versus negative cows. Regression analysis of cytokine expression levels (Ct) and the percentage of PMNs in subclinical endometritis-positive cows showed that for each additional threshold cycle required for IL-8 detection, which corresponded to two-fold less mRNA, the percentage of PMN decreased by 3.3% (P=0.00001). Similarly, for each additional threshold cycle required to detect IL-6 and TNF-α, the percentage of PMNs in endometritis-positive cows decreased by 2.3% (P=0.015) and 2.4% (P=0.054), respectively. Cows with > 18% PMNs required significantly fewer amplification cycles to detect IL-6 (P = 0.01), IL-8 (P =0.0001) and TNF-α (P=0.053) mRNA than cows with <18% PMNs (endometritis-negative). There was a highly significant positive correlation between the expression of individual pro-inflammatory cytokines when comparing IL-8 and IL-6 (P=0.0001), IL-8 and TNF-α (P=0.00001), and finally IL-6 and TNF-α (P=0.0002). In the second experiment, 340 cows 28 to 41 days in milk were examined using cytobrush cytology and transrectal ultrasonography of the uterus and ovaries. One-half of the cows were treated with benzathine cephapirin uterine infusion to determine the lowest PMN percentage where a significant improvement in reproductive performance occurred. Subclinical endometritispositive (>15%) cows in this study were defined as those with the lowest percentage of PMNs that was associated with a significant positive treatment effect. Treated cows with >15% PMNs required 31 fewer days (P=0.041) to become pregnant and had 2.5 times fewer services per conception (P=0.0001) than untreated cows with >15% PMNs. The likelihood of there being CLs at the time of examination in cows with >15% PMNs in endometrial cytobrush cytology was 2.3 times significantly higher (P=0.04). The treatment of cows with ultrasonographically detectable fluid in the uterine lumen with benzathine cephapirin had no effect on days open compared to treatment of cows without fluid in the uterus (P=0.39). Cervical diameter and endometrial thicknesses did not differ between groups of cows with >, < 15%PMNs (P=0.46, P=0.36, respectively). In summary, based on the response to a single treatment with benzathine cephapirin, and the analysis of pro-inflammatory cytokine gene expression, we recommend that a threshold of >18% PMNs be used to define endometritis-positive disease status in cows 28 to 41 DIM. Cervical diameter, ultrasonographic evidence of uterine fluid and ultrasonographic measurement of endometrial thickness were not useful for diagnosing benzathine cephapirin responsive endometritis.

Two experiments were designed to study endometritis in postpartum dairy cows. In the first experiment, 30 cows 28 to 41 days in milk (DIM) and without evidence of clinical endometritis were sampled using cytobrush cytology. Cytobrush sampling provided sufficient endometrial material to prepare cytologic specimens and to extract endometrial mRNA. Pro-inflammatory cytokines were analyzed in harvested endometrial tissue taken from cows with and without endometritis. Cytokine expression varied between experimental groups with 30-fold higher IL-6 expression levels (P=0.01), greater than 50-fold higher IL-8 expression levels (P=0.0001), and 20-fold higher TNF-α expression levels (P=0.001) in endometritis-positive versus negative cows. Regression analysis of cytokine expression levels (Ct) and the percentage of PMNs in subclinical endometritis-positive cows showed that for each additional threshold cycle required for IL-8 detection, which corresponded to two-fold less mRNA, the percentage of PMN decreased by 3.3% (P=0.00001). Similarly, for each additional threshold cycle required to detect IL-6 and TNF-α, the percentage of PMNs in endometritis-positive cows decreased by 2.3% (P=0.015) and 2.4% (P=0.054), respectively. Cows with > 18% PMNs required significantly fewer amplification cycles to detect IL-6 (P = 0.01), IL-8 (P =0.0001) and TNF-α (P=0.053) mRNA than cows with <18% PMNs (endometritis-negative). There was a highly significant positive correlation between the expression of individual pro-inflammatory cytokines when comparing IL-8 and IL-6 (P=0.0001), IL-8 and TNF-α (P=0.00001), and finally IL-6 and TNF-α (P=0.0002). In the second experiment, 340 cows 28 to 41 days in milk were examined using cytobrush cytology and transrectal ultrasonography of the uterus and ovaries. One-half of the cows were treated with benzathine cephapirin uterine infusion to determine the lowest PMN percentage where a significant improvement in reproductive performance occurred. Subclinical endometritispositive (>15%) cows in this study were defined as those with the lowest percentage of PMNs that was associated with a significant positive treatment effect. Treated cows with >15% PMNs required 31 fewer days (P=0.041) to become pregnant and had 2.5 times fewer services per conception (P=0.0001) than untreated cows with >15% PMNs. The likelihood of there being CLs at the time of examination in cows with >15% PMNs in endometrial cytobrush cytology was 2.3 times significantly higher (P=0.04). The treatment of cows with ultrasonographically detectable fluid in the uterine lumen with benzathine cephapirin had no effect on days open compared to treatment of cows without fluid in the uterus (P=0.39). Cervical diameter and endometrial thicknesses did not differ between groups of cows with >, < 15%PMNs (P=0.46, P=0.36, respectively). In summary, based on the response to a single treatment with benzathine cephapirin, and the analysis of pro-inflammatory cytokine gene expression, we recommend that a threshold of >18% PMNs be used to define endometritis-positive disease status in cows 28 to 41 DIM. Cervical diameter, ultrasonographic evidence of uterine fluid and ultrasonographic measurement of endometrial thickness were not useful for diagnosing benzathine cephapirin responsive endometritis.

Leukocyte adhesion to cerebral endothelial cells plays a critical role in the inflammatory response following transient global cerebral ischemia but its contribution to delayed neuronal cell death is not completely understood. We compared ischemic mice treated with a monoclonal antibody to β2-integrin adhesion receptors (anti-CD18) or a non-binding control antibody following ischemia. Inflammation was characterized by increased CD18 expression on leukocytes and inflammatory mediators in the peripheral blood and brain tissue. Notably, interleukin-1β, which has been shown to mediate cell death in neurons, was elevated in the blood and brain. Anti-CD18 blocked leukocyte adhesion as well as the inflammatory responses, including interleukin-1β expression in neurons. Blocking leukocyte adhesion protected the structural integrity of the hippocampus, cerebral cortex and thalamus, and preserved spatial. Leukocytes adhesion to endothelial cells plays an important role in the evolution of neurological deficit in global cerebral ischemia despite the lack of transmigration of leukocytes across blood-brain-barrier.

Introduction: Alzheimer’s disease (AD) and dementias constitute a significant public health burden and it is estimated that one in 85 people may be living with AD by 2050. Dementias are a spectrum of diseases with common traits including amyloid protein growth, neurodegradation, neurofibrillary plaque and tangle formation, and which may be influenced by pro- and anti- inflammatory immune mechanisms. Even a modest delay in onset could result in significant reductions in the social and economic burdens of dementias. An important lifestyle factor identified in risk reduction is physical activity (PA). Although the association between dementia risk and PA has been established, the exact physiological mechanisms through which protection occurs are not known. This research consists of two experiments that were designed to explore the effects of physical activity on pro- and anti-inflammatory cytokines and apoptosis in the mouse hippocampus, a brain region implicated in learning, memory, and cognition. Methods: Study #1: Female C57BL/6 mice, 4-5 months of age, were divided into three groups: sedentary controls (NOTREAD) (n = 22), treadmill exercise with immediate sacrifice (TREAD-Imm) (n = 21), or treadmill exercise with sacrifice after 2 hours (TREAD-2h) (n = 20). TNF-α, IL-6, and IL-1β expression in the hippocampus and intestinal lymphocytes were measured by Western blot analysis. Percentages of hippocampal cells undergoing apoptosis (Annexin+) or necrosis (Propidium Iodide+) were determined through flow cytometry. Plasma levels of 8-isoprostane and corticosterone were measured using commercially available EIA kits. Study # 2: Female C57BL/6 mice, 3-4 weeks of age, were assigned to wheel running (WR; n = 20) or a control condition (No WR; n = 22) and sacrificed after the 16 weeks. Data collected included measures of training status (running volume, body weight, run-to-exhaustion time, and skeletal muscle cytochrome c oxidase activity), flow cytometric analysis of hippocampal cell phenotypes and apoptosis (CD45+, CD11b+, Annexin+, Annexin+/PI+, PI+), and cytokine concentrations (TNF-α, IL-1β, IL-12, IL-6, IL-1ra, and IL-10) in cell lysates. Results: Study #1: Acute treadmill exercise lead to significant decreases in TNF-α (p<0.05) and increases in IL-6 (p<0.05) expression in the hippocampus of healthy mice. No effects of acute exercise on the apoptotic status of hippocampal cells were observed. In intestinal lymphocytes, the exercise bout lead to significant increases in TNF-α (p<0.05), IL-6 (p<0.05), and IL-1β (p<0.05). Acute exercise was associated with a significant increase in both plasma 8-isoprostane (p<0.05) and corticosterone (p<0.05) levels. Study #2: WR mice had measurable training effects and significantly lower TNF-α (p<0.05) and higher IL-6 (p<0.05), IL-1ra (p<0.05) and IL-12 (p<0.05) expression in the hippocampus compared to controls. IL-1β, IL-10, and the percent of apoptotic, dead cells, and cell phenotypes did not change due to training. Conclusion: Exercise chronicity (acute vs. chronic), stress characteristics of the exercise (forced vs. voluntary) and tissue location (systemic vs. central) emerged as important variables with effects on both cytokine concentrations and plasma levels of stress hormones. Physical activity may protect the hippocampus against inflammatory damage caused by TNF-α, and the suppression of this cytokine may be due to increased glucocorticoid secretion during acute exercise. It is also proposed that elevated IL-6 expression (central and systemic) may mediate this protection by creating an anti-apoptotic environment in the hippocampus. Less apoptosis may also contribute to maintenance of cognitive function during acute and long-term physical activity.

Tuberculosis is the second leading cause of morbidity and mortality worldwide due to an infectious disease. Development of a new tuberculosis (TB) vaccine would be facilitated by a better understanding of the mechanisms of protection induced by the current TB vaccine, Mycobacterium bovis BCG. Recombinant guinea pig (rgp)CCL5 and anti-rgpCCL5 were developed and characterized. The biological activity of rgpCCL5 was determined in a chemotaxis assay using T lymphocytes and pleural exudate cells. The specificity of rabbit anti-rgpCCL5 polyclonal antibody was confirmed by Western blot. RgpCCL5 was used to stimulate alveolar and peritoneal macrophages in vitro. and cytokine/chemokine gene expression was evaluated using real-time PCR. RgpCCL5 stimulated TNF&#945;, IL-1&#946;, CCL2, and CXCL8 mRNA expression and TNF&#945; protein production (as assessed in the L929 cell bioassay) in macrophages. The effect of BCG-vaccination on CCL5 expression and production in leukocytes infected with M. tuberculosis was examined in vitro and in vivo. Polyclonal anti-rgpCCL5 was used to develop an ELISA assay to quantify gpCCL5 protein levels, and real-time PCR was used to detect CCL5 mRNA. Leukocytes isolated from BCG-vaccinated guinea pigs and infected in vitro with virulent M. tuberculosis demonstrated significantly elevated gpCCL5 mRNA and protein compared to cells from naive animals. The response of gpCCL5 to M. tuberculosis in vivo was studied in tuberculous pleural effusions, where peak levels of CCL5 mRNA and protein were reached at day 4 post-induction. Disease severity, cellular differentiation, histology, and cytokine/chemokine mRNA levels in pleural cells and granulomas were analyzed on day 4 in guinea pigs induced with tuberculous pleurisy and treated with either rgpCCL5 or anti-rgpCCL5 by direct intra-pleural injection. In these studies, neutralizing CCL5 resulted in reduced macrophage accumulation, diminished levels of IFN&#947;, TNF&#945;, and CCL5 mRNA in pleural effusion cells, and reduced spontaneous lymphocyte proliferation. Together these studies suggest an important role for gpCCL5 in activating leukocytes during M. tuberculosis infection.