Hyperbaric oxygen (HBO) therapy is the intermittent inhalation of 100% oxygen at a pressure greater than one atmosphere absolute. It is an effective treatment for various inflammatory conditions, including chronic wounds which are characterized by an excessive influx of neutrophils and their prolonged persistence at the wound site. Neutrophil apoptosis and clearance have been shown to be required for resolution of inflammation. The mechanisms by which HBO aids wound healing are well documented, but its effects on cellular inflammatory response are not well understood particularly with respect to neutrophils. The hypothesis presented in this thesis is that increased oxygenation via HBO assists chronic wound healing by enhancing non-inflammatory neutrophil defences and cell death through apoptosis. An investigation was carried out into the effects of HBO on neutrophil antimicrobial function and apoptosis using differentiated HL-60 cells as an in vitro neutrophil model. The data clearly showed that a single HBO treatment for 90 min caused an increase in the oxidative burst activity of neutrophil-like cells as shown by increased NBT staining, superoxide (cytochrome c reduction) and H2O2 production (Kruskal-Wallis, P < 0.05), and phagocytosis of Staphylococcus aureus. HBO treatment displayed a pro-apoptotic effect, enhancing caspase 3/7 activity both in the presence and absence of a TNF-α stimulus (Kruskal-Wallis, P < 0.05) and causing morphological changes (observed using Giemsa and SYBR® Safe staining) associated with apoptosis. Although no consistent pattern was observed, both hyperoxia and pressure alone seemed to contribute to both the increase in antimicrobial activity and the increase in apoptosis induced by HBO in these neutrophil-like cells (Chapters 4 and 5). HBO-enhanced neutrophil clearance by macrophages was investigated using bovine neutrophils and monocyte-derived macrophages (MDMФ). A single 90 min HBO exposure significantly increased the clearance of fresh and 22 h-aged neutrophils by MDMФ (two-way ANOVA, P < 0.05), suggesting an increase in phosphatidylserine (PS) exposure in apoptotic neutrophils after HBO treatment (Chapter 6). Importantly, a long-term repetitive exposure to HBO in patients with chronic wounds caused a significant decrease in the antioxidant enzyme defence system (one-way repeated measures ANOVA, P < 0.05), plasma TNF-α and IL-1β after 30 HBO sessions, with down regulation of expression of the anti-apoptotic factors, NF-B and Bcl-2 (Chapter 7). These findings may go some way towards explaining the effectiveness of HBO treatment not only for chronic wounds but also for other inflammatory conditions that may be affected by this treatment.

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein, is involved in neutrophil recruitment into peritoneum. Because mechanisms of excessive migration of activated neutrophils into inflamed lungs, credited with tissue damage, are not fully understood, we explored the hitherto unknown expression and role of LSP1 in neutrophil migration in a mouse model of acute lung inflammation. We induced acute lung inflammation through intranasal E. coli lipopolysacharide (LPS) (80μg) in wild-type 129/SvJ (WT) and LSP1 deficient (LSP1-/-) mice. WT (n=10) and LSP1-/- (n=11) mice showed significant neutrophilia and more neutrophils in bronchoalveolar lavage (BAL) at 9 hour post-LPS challenge compared to respective saline-treated controls (WT=7; LSP1-/-=10). LPS treatment induced more BAL neutrophils (P<0.001), myeloperoxidase concentrations and Gr-1+ neutrophils in lung tissues in WT mice compared to LSP1-/- mice. Lung myeloperoxidase and Gr-1+ (P<0.05) were higher in LPS-treated WT compared to the LSP1-/- mice. Lung tissue and BAL fluid KC, MCP-1, MIP-1α and MIP-1β concentration and vascular permeability were not different between LPS-treated WT and LSP1-/- mice but TNF-α concentration was higher in LPS-treated WT mice. Hematoxylin and eosin staining showed more septal congestion in LPS-treated WT mice compared to LSP1-/- mice. LSP1 expression was increased in lungs from LPS-treated mice compared to saline control. The autopsied lungs from septic humans, compared to their respective controls, showed increased expression of LSP1. These data show that LSP1 expression is modulated in acute lung inflammation and that LSP1 deficiency reduces neutrophil migration into acute lung inflammation.

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.

Prenatal stressors have been reported to affect postnatal cognitive, metabolic, reproductive and immune functions. This study examined immune indices and function in Brahman calves prenatally stressed by transportation of their dams on d 60, 80, 100, 120 and 140 ± 5 d of gestation. Based on assessment of cow’s temperament and their reactions to repeated transportation it was evident that temperamental cows displayed greater pre-transport cortisol (P < 0.0001) and glucose (P < 0.03) concentrations, and habituated slower to the stressor compared to cows of calm and intermediate temperament. Serum concentration of cortisol at birth was greater (P < 0.03) in prenatally stressed versus control calves. Total and differential white blood cell counts and serum cortisol concentration in calves from birth through the age of weaning were determined. We identified a sexual dimorphism in neutrophil cell counts at birth (P = 0.0506) and cortisol concentration (P < 0.02) beginning at 14 d of age, with females having greater amounts of both. Whether weaning stress differentially affected cell counts, cortisol concentrations and neutrophil function of prenatally stressed and control male calves was examined. At 2 d post weaning, all calves had increased cortisol concentration (P < 0.0001) and neutrophil cell counts (P < 0.0001). However, in vitro production of reactive oxidative species by neutrophils was decreased (P = 0.0002) 2 d post weaning. Moreover, prenatally stressed calves demonstrated a larger (P = 0.0203) decrease in their immune function relative to control calves at 2 d post-weaning. Importantly, prenatally stressed calves took longer than controls to recover from the weaning stress. Additional studies are needed to clarify if prenatally stressed calves are more susceptible than control calves to pathogens during the post weaning period. Management practices to improve animal welfare and livestock production may need modification if follow-up studies demonstrate that prenatal stress also affects reproductive development, growth, performance and meat quality.

Calcium (Ca$ sp{2+}$) and protein kinase C (PKC) are believed to act as intracellular signals triggering the activation of NADPH oxidase in neutrophils leading to superoxide generation. This was tested on bovine neutrophils by chelating extracellular and/or intracellular free Ca$ sp{2+}$ and by measuring PKC activity when the cells were stimulated by phorbol myristate acetate (PMA) or opsonized zymosan (OZ). Chelation of extracellular Ca$ sp{2+}$ with EGTA did not alter O$ sb2 sp{-}$ production from PMA stimulated cells. However, it did cause a 64% decrease in O$ sb2 sp{-}$ production in the neutrophils when stimulated with OZ. When intracellular Ca$ sp{2+}$ was chelated with BAPTA/AM, there was a significant decrease in O$ sb2 sp{-}$ generation following PMA activation. Yet, OZ activated cells, pre-treated with BAPTA/AM, showed an increase in the respiratory burst proportional to the chelator's concentration. Moreover, although OZ was previously shown to increase O$ sb2 sp{-}$ generation by neutrophils, no significant changes in PKC activity were observed. PMA stimulation led to an increase in PKC activity at the membrane level. Furthermore, treating the cells with calphostin C, a PKC activity inhibitor, caused a 69% decrease in O$ sb2 sp{-}$ production demonstrating the involvement of PKC in PMA-stimulated cells. However, no differences were observed between the OZ activated cells incubated with the inhibitor and the control cells. These data provide evidence that activation of NADPH oxidase can be achieved by either a PKC-dependent or a PKC-independent pathway depending on the stimulatory agent.

Neutrophil potential phagocytic capacity (NPPC), measured on 25 AI Canadian Holstein bulls, was investigated for evidence of association with production and type traits, SCC, and survival in dairy cows. Bulls were ranked based on different degrees of NPPC (Uptakes of 0, 1, 2, and 3 or more latex beads), using the solutions coming from an animal model. A total of 42,103 first lactation records, collected from 1985 through 1993 in 2,919 Quebec dairy herds, were used to obtain EBV's for SCC and for log SCC (LogSCC) for 697 sires. Correlations between NPPC measurements and somatic cell EBV's were null. Canadian official ETA's for type traits related with mammary system had a tendency of being positively correlated with higher NPPC and negatively with Uptake-0. Canadian official ETA's for production traits were negatively correlated with higher NPPC and positively with Uptake-0. A total of 17,202 first lactation records of daughters of the 25 AI bulls were used to study the effect of NPPC and log SCC on survival in dairy cows. Survival after first lactation was more closely related to sires' NPPC-EBV's than to LogSCC-EBV.

The test for antineutrophil cytoplasmic antibodies (ANCA) was introduced into the author's laboratory in 1987. An improved indirect immunofluorescent method was developed, using a system which allows 16 instead of one serum sample to be screened on each microscope slide. The known disease associations of ANCA that have been explored include systemic vasculitis, renal limited vasculitis, chronic inflammatory bowel disease and HIV disease. In general the findings are similar to those which are emerging from other centres and confirm the value not only of the positivity but also the relevance of the intracellular disposition of the neutrophil cytoplasmic fluorescence in diagnosis. In this study 85% of patients with Wegener's granulomatosis were found to have C-ANCA. C, P and X-ANCA staining patterns were found in 57% of patients with ulcerative colitis. Forty one per cent of patients with symptomatic HIV have ANCA. Certain histological features such as neutrophil and vascular damage in invasive amoebiasis, and the established lytic effect of amoebae on neutrophils prompted the investigation of the possibility that ANCA may be generated in this disease. Seventy eight amoebiasis sera were screened and 98,70/0 gave a positive ANCA test with a pattern of fluorescence resembling that found in Wegener's granulomatosis. An ELISA test for specificity confirmed that, as in Wegener's granulomatosis, this amoebiasis-associated ANCA had proteinase 3 specificity. Of practical clinical importance is the fact that both HIV and amoebiasis are associated with a high level of ANCA positivity. These findings will need to be considered when ANCA tests are used in clinical decision making in an area where HIV disease and amoebiasis are endemic. A large number of normal volunteer blood donors have been tested and the false positivity rate of 0,5% confirms the specificity of the test. / Thesis (M.Med.)-University of Natal, Durban, 1993.

Diapedesis, phagocytosis and microbicidal activity are important parameters of neutrophil functionality and thus outcome of mastitis. An in vitro model of an "alveolar pavement" using the MAC-T3 bovine mammary epithelial cell line was developed to assess neutrophil diapedesis. Features of this biologically-meaningful barrier include: characteristic transepithelial resistance, tight junction complexes and polarity. Continuous transepithelial resistance measurements showed no significant changes throughout the assay period. Neither a Staphylococcus aureus challenge ($1 times10 sp7$ and $2 times10 sp9$ cfu/ml), or the presence of neutrophils, both resting and challenged had any deleterious effects on monolayer integrity over a short term (1-2 h) exposure. Neutrophils, both resting and challenged gave no indication of causing damage to the epithelium over the short term. Neutrophils isolated from proven sires and evaluated for phagocytic activity were found to differ significantly (p $<$ 0.05) in activity, rate and capacity to uptake particles. Correlations between phagocytic parameters and production traits were negative and small in magnitude. Microbicidal activity of neutrophils isolated from proven sires showed a highly significant variation between animals due to test day (p $<$ 0.001), however variation due to source of cells (i.e. animal) was not significant. in vitro analysis of diapedesis and phagocytosis is promising as a tool for the assessment of resistance or susceptibility to mastitis.

Neutrophils are among the first white blood cells recruited to the site of infection once microbial pathogens enter the host organism. At site, they perform a well-orchestrated chain of processes that aims to kill the microbial invader. Most prominent, neutrophils engulf microbes to inactivate them intracellularly, a process called phagocytosis. Alternatively, neutrophils can release neutrophil extracellular traps (NETs). NETs consist of chromatin decorated with antimicrobial effector proteins – a structure that can entangle bacteria and fungi. Neutrophils are crucial during fungal infections. This is reflected in the increased risk of fungal infections resulting of neutropenia. The concept of nutritional immunity describes every infection as a battle for resources. Those are mostly metal trace elements. For a long time, neutrophils were seen as powerful, but “mindless”, killers with a limited set of actions and no transcriptional capacity, but this view is in the flux. In the presented thesis, it was my goal to gain new insights into the interplay of neutrophils and fungi – with special attention to metal-nutritional aspects. We compared human neutrophils lacking the ability to undergo NETosis, due to a non-functional NADPH complex, and neutrophils from the same person that were “cured” by gene therapy. We investigated those NETs and found that their inhibitory activity towards the mold A. nidulans depends on calprotectin, a known zinc-chelator. Considering the high influx of neutrophils, we wanted to unravel the neutrophils’ contribution to the metal milieu at the site of infection and trace element changes resulting from NETosis. By combining synchrotron radiation XRF and ICP-MS, we analyzed the neutrophil metallome and the spatial element distribution in activated neutrophils and NETs. Most strikingly, we found neutrophils to be exceptionally high in Fe and the process of NETosis to be reducing available Zn in the surrounding and the early phagosome, possibly by the formation of Zn-rich vesicles. Using RNA-sequencing, we analyzed the interplay of the C. albicans and neutrophils face-to-face. We dissected their transcriptional profile and revealed a manifold response in neutrophils that include cytokine induction and cellular rearrangement. We further were the firsts to explore the transcriptional response of C. albicans to NETs. Our data indicates a distinct response compared to intact neutrophils or other known stress triggers. Metal homeostasis was affected in Candida in both set-ups. In summary, this thesis provides new insights into the interaction of fungal pathogens with neutrophils and emphasizes the impact of nutritional aspects on this interplay. A deeper understanding of the nutritional immunity during fungal infection might open up new strategies to tackle fungal infections – a growing threat worldwide.

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.