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Submitted in part fulfillment for the Degree of Master of Technology: Biotechnology, Durban Institute of Technology, 2003. / Evidence suggests that the induction of tissue kallikrein, and the subsequently formed kinins, enhances proliferation of tumour cells because of their mitogenic property. Additionally, the kinin peptides are believed to promote the invasion of normal tissue by tumour cells. TGF-l is a potent inhibitor of the growth of renal epithelial cells, and is a classical anti-mitogen, which is central to many of its antiproliferative effects. No studies thus far have been performed, as to whether the proposed anti-mitogenesis ofTGF-1 has a regulatory effect on the cell proliferative action of kinins on renal epithelial and carcinoma cells. / M

Pneumonic pasteurellosis is a significant disease in beef production medicine. The most recent information suggests that this disease is a $700 million dollar per year economic burden in bovine food animal production The medical and pathological characteristics of this disease are well documented. Many pathological findings associated with pneumonic pasteurellosis may be explained by disruption of the pulmonary vascular adrenergic system. However, only a limited amount of research has addressed the adrenergic system and its relationship to the etiology and pathophysiology of this disease. In an attempt to further investigate the contributions of the vascular adrenergic receptor mechanism to the development of pneumonic pasteurellosis a series of six experiments have been completed. It is to be noted, that in 1999 the organism Pasteurella haemolytica was renamed Mannheimia haemolytica. The name change was based on the taxonomic features of the organism from other closely related organisms, in particular Pasteurella multocida.. The differences noted were identified and described by Dr. Mannheim in 1974. The familiarity of the past nomenclature and the lack of familiarity for the new nomenclature suggests that the more commonly recognized name of Pasteurella haemolytica should be used throughout this document. Scientific evidence suggests that the disruption of the normal homeostatic mechanisms of the pulmonary vasculature to beta adrenergic agents may be part of the etiology of pneumonic pasteurellosis. The dynamics and kinetics of the involvement of the beta receptors, following prophylactic vaccination and in the disease state, has yet to be fully investigated with respect to the events associated with pneumonic pasteurellosis. Evaluation of the time frame of the onset and duration of the events associated with the disruption of pulmonary vascular beta adrenergic receptor mechanisms revealed that an escalating level of dysfunction occurs over the first 24-48 hour period after exposure to parenteral Pasteurella haemolytica and lasts for at least 21 days. A component of P.haemolytica organism or contained in the vaccine using the organism is likely associated with the disruption of vascular beta adrenergic mechanism. This factor is, as yet, not specifically identified, however the likely culprit is the lipid A moiety of the endotoxin. Using the well defined and purified Escherichia coli endotoxin, trials were run to examine the effect of endotoxin on the pharmacological response of vascular associated beta adrenergic receptor mechanisms. The effects of Escherichia coli endotoxin, administered parenterally, on beta adrenergic receptor mechanisms were pharmacologically indistinguishable from those effects following parenterally administered Pasteurella haemolytica. The nature of the disruption in the beta adrenergic receptor remains a mystery. The receptor mechanism involves at least two second messengers to initiate vascular relaxation. Initial activation of the beta adrenergic receptor with a beta selective drug starts a cascade of events involving adenylylate cyclase and cyclic adenylylate monophosphate (cAMP) and nitric oxide. A disruption in the receptor mechanism, as a result of the parenteral administration of Pasteurella haemolytica, which is "upstream" of adenylyl cyclase, would result in a diminished amount of cAMP when compared to the unvaccinated negative controls. An investigation of cAMP accumulation, at the receptor level was inconclusive. The assessment of some previously used vaccines has demonstrated that there is an, as yet unidentified virulence factor, associated with these vaccines that results in the pharmacological disruption of beta adrenergic receptor mechanisms. Two newer vaccines, Once PMH® and One Shot® have been evaluated and there is evidence to suggest that these currently used vaccines also have the ability to disrupt beta adrenergic receptor mechanisms in rats. The effects of parenteral P. haemolytica on the alpha-2 adrenergic receptor mechanism, is described. The alpha-2 receptor mechanism, unlike the beta receptor mechanism appears to increase the amount of vasoconstriction. The possibility that the alpha-2 adrenergic receptor could also mediate vasorelaxation under certain conditions was investigated. The evidence suggests that in the presence of high alpha-1 mediated vascular tone, the alpha -2 receptor can cause vasorelaxation. Evidence, from other scientists active in this area of investigation, suggests that a vasorelaxation response may be mediated by nitric oxide. Elimination of the nitric oxide mediated relaxation may offer an explanation for the increased vasoconstriction noted with alpha-2 selective drugs after exposure to parenteral P. haemolytica. Finally, the importance of the beta adrenergic receptor to the disease process is addressed by elucidation of one of the mechanisms by which Micotil 300® (tilmicosin phosphate) acts to improve cattle with symptomatic pneumonic pasteurellosis. The rapid improvement of animals on Micotil 300®, with-in 24 hours suggests that there is a mechanism beyond the antimicrobial effect of the drug that mediates the clinical improvement. Evaluation of the effect of Micotil 300® demonstrates a pharmacologically measurable amount of beta adrenergic activity with respect to the bovine pulmonary artery and vein. Based on the conclusions drawn as a result of these experiments, the adrenergic system in general, and the beta adrenergic system in particular are important to the development of pneumonic pasteurellosis in cattle. The beta adrenergic system is affected by endotoxin. Further, these receptors maybe responsible for the mediation of the pathological and clinical signs associated with pneumonic pasteurellosis. In conclusion, these investigations have suggested, that it is likely that a disruption in the homeostatic mechanisms mediated by the beta and alpha-2 adrenergic receptors are intimately involved in the development of post vaccination receptor failure as well as the pathophysiology associated with pneumonic pasteurellosis in cattle. / Ph. D.

Introduction: Plasma kallikrein (PK) is a cofactor in blood coagulation and modulates inflammation through the release of bradykinin (BK). Previously it was believed that plasma prekallikrein (PPK), the precursor of PK and a member of the serine protease superfamily, was synthesised exclusively by hepatocytes and secreted into circulation. However, recent studies show that various human tissues contain PPK mRNA. In this study we sought to determine in which human tissues PK is expressed. Methods: Following approval by the Ethics Committee at the University of Natal, tissue samples from the spinal cord, 13 different regions of the brain, 7 different blood vessels and various other organs were collected at autopsy within 24h of death (n =10). Sections were probed using polyclonal antibodies specific for PK. PK concentrations in extracts of these tissues were measured by competitive EllSA. Results: A Western blot analysis demonstrated the monospecificity of the antibody for the PK protein. The presence of immunoreactive PK in cells of the pancreatic islets of Langerhans served as a positive control for each immunolabeling experiment. The hepatocytes, renal distal convoluted tubules and epithelial cells lining the bronchiole and pulmonary alveoli labeled positively for PK. In the gastrointestinal tract tissue, immunoreactive PK was visualised in the acinar cells of the salivary gland, in stromal and glandular duct cells of the oesophagus, and in some chief and glandular cells in the stomach. Some of the above-mentioned tissues contained a few inflammatory cells which stained intensely for PK. Immunoreactive PK was visualised in the endothelial cells and smooth muscle cells of the all the blood vessels examined, except the renal vein. Increased immunolabeling for PK in the endothelial cells, foam cells and macrophages was observed in arteries with atheromatous plaques. In neural tissue immunoreactive PK was observed in neurons, ependymal cells, fibre tracts, and in secretory cells of the anterior pituitary gland. Immunolabeling for PK was visualised in some neurons of the spinal cord and in different brain regions viz. hypothalamus, cerebral cortex, thalamus, brain stem and hippocampus. In sections of the hypothalamus and spinal cord, we observed immunolabeling for PK in ependymal cells lining the third ventricle and central canal respectively. Positive labeling for PK was evident in fibre tracts of the pons, medulla and hippocampus. No immunoreactive PK was visualised in the choroid plexus or cerebellum. High amounts of PK were measured by competitive ELlSA in extracts of the pancreas (12.94 ± 2.04 /-lg/ml), the pons (1.67 ± 1.46 /-lg/ml) and aorta (0.44 ± 0.14 /-lg/ml). The basilar artery (0.09 ± 0.07 /-lg/ml) and spinal cord (0.09 ± 0.04 /-lg/ml) had the least PK concentrations. Discussion and Conclusions: We have shown that the PPK mRNA demonstrated in various human tissues is most likely translated into protein by the immunolocalisation of PK within specific cells in the different tissues examined. The actions of PK within these tissues may be two fold, firstly by its kininogenase activity it may release BK from high molecular weight kininogen, or alternatively, PK may act as a proteolytic enzyme on other proteins. With respect to the latter) PK may be involved in the processing of protein precursors, for example precursors of the digestive enzymes found in saliva and in gastric secretion, insulin precursors in the pancreas, and hormonal precursors in the pituitary gland. The localisation of PK and B1 and B2 kinin receptors in the kidney, lung, stomach, blood vessels and brain suggests that the effects of PK in these tissues are mediated by BK-receptor interaction. These may include the regulation of glucose uptake in the pancreas, water and ion transport in the kidney, and local and systemic blood pressure in the cardiovascular system. The presence of immunoreactive PK in neurons suggests that BK-receptor mediated interaction may regulate neurophysiological processes such as synaptic transmission. Immunolabeling for PK in polymorphonuclear leukocytes observed in some of these tissue sections suggests the potential to mediate the inflammatory process. / Thesis (M.Med.Sc.)-University of Natal, Durban, 2000

Inflammation is the normal biological response to tissue injury, and is characterised by the interactive activation of multiple mediators and cell types. One response to tissue injury is the production of pain, not only by direct trauma to sensory fibres, but also through the release of mediators from sensory nerve terminals. One such mediator is kinin which is a vasoactive peptide considered to play a primary role in inflammation by causing constriction of venules, dilation of arterioles, increasing permeability of the capillary membrane, and interacting with sensory nerve terminal transmitters to evoke pain. The kinin forming enzymes (kallikreins) reach inflammation sites either on the surface of migrating neutrophils or by transudation from plasma. The kininogen molecule which contains the kinin moiety, has been localised on the external surface of the neutrophil, and provides the substrate from which kinins can be cleaved through enzymatic action. The cellular actions of kinins are mediated through B2 receptors, which are also located on the external surface of the neutrophils. In addition, the induced effects of kinins are regulated by B1 receptors. The formation of nitric oxide (NO) from arginine released from the kinin C terminus, and receptor membrane signal transduction by nitric oxide following kinin receptor activation is discussed. A molecular response to cell injury is the formation of chemotactic mediators that attract neutrophils to sites of inflammation. The question whether neutrophils contribute to circulating levels of kinins was examined in infections and inflammatory disorders. This novel hypothesis was tested using circulating neutrophils harvested from patients with tuberculosis meningitis and pneumonia. These neutrophils showed a distinct loss of only the kinin moiety from the kininogen molecule located on the external surface. The confocal images of fixed, permeabilised neutrophils provided multi-dimensional constructs, and the intensity of fluorescence reflected the relative amounts of the molecule present in both neutrophils harvested from healthy volunteers as well as patient blood. The immunocytochemical labelling experiments using colloidal gold as markers, confirmed, at the ultrastructural level, the presence or disappearance of the kinin moiety from the kininogen molecule on the neutrophil surface. The cell component of synovial fluid in rheumatoid athritis (RA) consists mainly of neutrophils. This study demonstrates the absence of the kinin moiety from circulating and synovial fluid neutrophils from patients with RA, as well as an increased signal from immunolabelled B2 receptors in synovial fluid neutrophils. These findings support the hypothesis that in RA, kinins are released during the inflammatory response in the joints, and suggests that there is an upregulation of the B2 receptor at the site of inflammation. Neutrophils chemotactically drawn to the site of inflammation become activated to release kinin from the kininogen molecule, and thereafter re-enter the circulation where they were harvested systemically. B2 receptors may be upregulated following activation by kinins or by other mediators present in the inflammatory milieu. Interleukin-1 has been shown to upregulate kinin receptors on human synovial cells. Anti-peptide antibodies to the loops of cloned B1 and B2 receptors have provided powerful probes for the cellular identification of the two kinin receptor families. Mapping of the B2 receptors showed upregulation on the neutrophils gathered from inflamed joints. However, no activation of the Br receptors was observed in normal blood neutrophils as well as those obtained from the different disease states. / Thesis (M.Med.)-University of Natal, 1996.

(1→3)-β-D-Glucans are carbohydrate polymers that are present in the cell wall of various fungi and bacteria; they are pathogen associated molecular patterns that circulate during infection and modulate immunity. Our laboratory has previously established the pharmacokinetics of intravenously and orally administered glucans; the present studies investigated the renal excretion of (1→3)-β-D-glucans following intravenous and oral administration. Three fluorescently-labeled glucans were administered to adult male rats in the presence or absence of toxic challenge. Urine specimens were collected and analyzed by fluorescence spectroscopy, size-exclusion chromatography and GPC/MALLS. 71 ± 3% of fluorescence remained in the >5K MWCO fraction; this fraction showed a minor peak with a molecular mass (171 ± 11K) corresponding to injected glucan (~150K). Most excreted glucans were of lower molecular mass (13 ± 8.5K), indicating most (1→3)-β-D-glucans are excreted by the kidneys as smaller polysaccharides. The presence of urinary glucans may be an important indicator of fungal infection.

Cannabidiol (CBD), a non-psychotropic phytocannabinoid with structural similarity to Δ 9 -tetrahydrocannabinol (THC), is currently being investigated as a therapeutic for its immunosuppressive effects. One disease for which CBD is extensively researched is multiple sclerosis (MS), a demyelinating, autoimmune disorder, and its murine model counterpart, experimental autoimmune encephalomyelitis (EAE). The focus of this dissertation aimed to analyze the transcriptomic brain pathways in EAE and its comparison to MS in addition to CBD’s immunosuppressive mechanisms in the innate and adaptive immune systems. Evidence presented here showed that transcriptomic signaling pathways in the EAE brain of mice with clinical symptoms were similar to the transcriptome of active lesions from MS patients. The transcriptomic analysis also presented two differentially expressed genes that were increased in CBD-treated, asymptomatic EAE mouse brains: oxytocin and vasopressin. Expression of these genes was also increased in naïve, CBD-treated mouse brains, which may indicate potential as efficacy biomarkers. Subsequently, as disease progression requires input from the innate and adaptive immune systems, the mechanisms of CBD were analyzed under naïve and stimulatory conditions in macrophages and splenocytes. In macrophages, CBD exerted an anti-inflammatory effect by dampening the M1 polarization phenotype, decreasing pro-inflammatory cytokines and chemokines, reducing TNF-α through intracellular TACE retention, and diminishing the translocation of RelA to the nucleus. Notably, similar impact of CBD on TACE was evident in naïve macrophages, suggesting that CBD exerted an effect under naïve conditions. In splenocytes, CBD exhibited a long-term effect on the percentage of various immune populations during naïve and splenic T cell activation (with anti-CD3/anti-CD28) conditions but only provided temporary relief and short-term from TNF-α and IFN-γ cytokine secretion. CBD also increased early mRNA expression of Tnfa in CBD in stimulated splenocytes. In naïve splenocytes, CBD impacted key immune mediators discovered from a transcriptomic re-analysis of human neuroblastoma cells, including decreased early expression of Noxo1 but increased expression of Ctsb. In summary, this dissertation presented evidence that CBD impacts the immune system from the transcriptional level in the brain, the innate and adaptive immune systems at the cellular level, and the overall EAE disease phenotype.