Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 94-101). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 94-101). Also available on the Internet.

No description available.

abstract / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

A more detailed understanding of the immune mechanisms involved in anti-inflammatory events in fish is required for effective disease control in aquaculture. Significant expansion of immune genes, often resulting from whole genome duplication events in teleost fish, adds complexity to their potentially anti-inflammatory pathways. This thesis contributes to a more complete insight into the suppressor of cytokine signalling (SOCS) and transforming growth factor-β1 (TGF-β1) gene system in fish by the discovery of paralogues and reporting of ways in which these potential negative regulators of immune responses can be induced in the the commercially valuable rainbow trout (Oncorhynchus mykiss). Gene expression analysis, revealed that a novel trout TGF-β1 paralogue (TGF-β1b) was more inducible by immune stimulants than the known TGF-β1a and likely represents an important intrinsic factor in macrophages. Both trout TGF-β type I and type II receptor (TGFBR1 and TGFBR2) genes identified encode highly conserved serine/threonine kinases and they are modulated during immune responses. Counter-regulation of the two receptor transcripts in immune stimulated macrophages may indicate regulatory mechanisms operating at the receptor level. Thorough phylogenetic and synteny analysis of the lower vertebrate SOCS system led to further paralogue discovery and uncovered that the proposed teleost-specific SOCS-8 and SOCS-9 members are more likely paralogues of cytokine inducible SH2-containing protein (CISH) and SOCS-5. The trout SOCS-2 subfamily was expanded to three members by cloning SOCS-2b and SOCS-2bRel in addition to SOCS-2a. I revealed for the first time that four paralogues in a SOCS subfamily exist in a teleost by cloning trout CISHa2, CISHb1 and CISHb2 in addition to the known CISHa1. In vivo and in vitro expression of the CISH paralogues suggest possible subfunctionalisation in immunity and development. Attempts to produce trout recombinant proteins of potentially anti-inflammatory cytokines in transiently transfected mammalian cells primed exploitation of these molecules in functional studies.

The aims of the work reported in this thesis were threefold; firstly, to investigate the effect of experimental exposure to polycyclic aromatic hydrocarbons (PAHs) on the immune response of dab (<I>Limanda limanda</I>), a sentinel species of flat fish for pollution studies, and rainbow trout (<I>Oncorhynhus mykiss</I>); secondly, to then go into the field to investigate the effects of exposure to PAHs on dab immunity through a major oil spill and thirdly, to increase current knowledge on the cellular immune functions of dab so as to provide better assays for biomonitoring purposes. Chapter 1 of this thesis is a general introduction to immunotoxicology, subspeciality of toxicology, which described brief accounts for both immunology and toxicology. Some brief accounts for the use of fish (lower vertebrates) in a pollution monitoring programme and the toxicology aspects of polycyclic aromatic hydrocarbons, were also given. In chapter 2, a laboratory exposure of animals to oil-contaminated sediment was established where four doses of diesel oil-based drilling mud used, i.e. 4, 8 (seen 500m from oil exploration platforms in the North Sea), 12 and 16% (w/w), with two exposure times employed, i.e. 2 and 4 weeks. Haematocrits and lymphocyte numbers tend to increase with low diesel oil doses, whereas high doses (particularly with longer exposure) gave significantly decreased values. Serum lysozyme levels were decreased, but serum bactericidal and anti-protease activities tended to increase following exposure. Kidney phagocyte respiratory burst and neutrophil migration activity also showed a trend to lower levels relative to control fish, whereas the number of antibody-secreting cells were increased with high drilling mud concentration. Lastly, there was a dose-dependent hepatomegaly. In chapter 3, rainbow trout were injected intra-peritoneally with an extract obtained from diesel oil-based drilling mud. Fish were exposed to different doses of extracts (made up to 2.4 mL/kg with olive oil), i.e. 0, 0.6, 1.2 and 2.4 mL/kg body weight (B.W.), for 6 weeks in the dose effect experiment.

The aim of this study was to determine whether breast milk had a stimulating effect on the development of immune responses in infants. Breast- and bottle- fed infants were studied from birth to 9 months of age. Several significant differences in immune responses were found between the groups. These differences were related to age and fell into two main time periods. In the early neonatal period, lymphocytes from breast-fed infants showed significantly greater spontaneous proliferation and proliferative responses to the T cell mitogen PHA to vitro than cells from bottle-fed infants. This may be due to a stimulatory effect to vivo of the growth factors and lymphokines in human milk acting on T cells and/or their precursors. Serum immunoglobulin levels did not however, reflect this increased lymphocyte responsiveness and although salivary IgM and IgA levels were significantly increased in the breast-fed infants at 6 days of age, this may have been due to residual milk immunoglobulins. In contrast, by 3 to 9 months of age, the cells from the bottle-fed group showed significantly greater to vitro proliferation of all classes of lymphocytes than the cells from the breast- fed infants. Salivary IgM and IgA levels and serum IgM antibodies to commensal gut organisms were also significantly higher in bottle-fed infants at this time. This indicated a higher rate of to vivo stimulation of the immune system in bottle-fed infants. It is suggested that this is due to an increased exposure of bottle-fed 'infants to antigenic material at mucosal surfaces and a greater uptake of these local antigens into the systemic circulation. Breast-feeding may therefore have contrasting effects on the development of immune responses, a stimulating effect by growth factors, particularly in the early immature period, and a suppressive effect resulting indirectly from exclusion of antigens (including those of a potentially harmful nature).

Research Doctorate - Doctor of Philosophy (PhD) / Asthma involves chronic inflammation of the airways that is heterogeneous in nature. Eosinophilic airway responses are well described in asthma, however non-eosinophilic subtypes of asthma have been recently reported, and can involve the influx of neutrophils into the airways (neutrophilic asthma). Neutrophils are important effector cells of the innate immune system. These cells are the first to migrate to inflammatory sites, where they contain and eliminate pathogenic microorganisms. Neutrophils also release cytokines and chemokines that initiate and amplify inflammatory responses. The mechanisms of neutrophilic asthma remain largely unknown; however activation of the innate immune response is implicated, particularly increased levels of proinflammatory cytokines Interleukin (IL)-8 and IL-1beta and gene expression of Toll Like Receptor (TLR)-4 and TLR2 have been demonstrated in induced sputum samples. This thesis examines innate immune responses of airway and circulating neutrophils, with a focus on neutrophilic asthma. Innate immune neutrophil activation occurs in response to exposure to Lipopolysaccharide (LPS), which activates TLR4. The activation response consists of the release of preformed granule associated mediators such as Matrix Metalloproteinase (MMP)-9 and Oncostatin M (OSM), new gene transcription and release of inflammatory cytokines such as IL-8, IL-1beta and Tumor Necrosis Factor (TNF)-alpha, and new gene transcription of TLR2 & TLR4 which serve to amplify neutrophil responses. In addition, this thesis examines whole genome gene expression profiles of circulating neutrophils in neutrophilic and eosinophilic asthma. The aims of this thesis are based on the hypothesis that dysregulation of innate immune neutrophil responses occurs with ageing and airway disease, particularly neutrophilic asthma and chronic obstructive pulmonary disease (COPD). With advancing age, there were alterations in the innate immune responses of neutrophils, which were characterised by enhanced spontaneous activation of both airway and circulating neutrophils, and a decreased response of circulating neutrophils to LPS. There was a decreased activation of airway neutrophils in airway disease that was most pronounced in neutrophilic asthma and COPD, with decreased production and release of proinflammatory cytokines most likely due to a downregulation of TLR4. TLR2 was downregulated in resting and LPS stimulated circulating neutrophils in asthma, particularly neutrophilic asthma. Circulating neutrophils had a decreased spontaneous release of total MMP-9, and downregulation of OSM, TLR2 and TLR4 at rest in COPD. However when stimulated with LPS, subjects with COPD had an enhanced proinflammatory cytokine release, with increases in IL-8 and TNF-alpha compared to subjects with asthma or healthy controls. Analysis of whole genome gene expression of circulating neutrophils in asthma revealed distinct gene profiles relating to asthma subtype. There was upregulation of genes relating to cell motility, inhibition of apoptosis and the NF-kB in neutrophilic asthma, which would contribute to their accumulation in the airways. The innate immune response is critical in controlling infections by bacteria and viruses. The reduced innate immune response of airway neutrophils in airway disease could contribute to impaired local defense, which may lead to an increased susceptibility to infection by invading pathogens. Systemically, the molecular mechanisms of neutrophilic asthma are distinct from eosinophilic asthma and may involve the enhancement of neutrophil chemotaxis and survival, contributing to their accumulation in the airways.

Aims of the research:To characterise the tissue histology and tissue distribution patterns of C. albicans during systemic murine candidiasis.To develop a reliable, reproducible and sensitive SQ-RT-PCR for the quantitation of in vivo cytokine gene transcription.To use this technique to determine the in vivo pattern of tissue specific cytokine gene expression during systemic candidiasis.To determine if cytokine gene expression patterns vary between resistant BALB/c and sensitive CBA/CaH mice during primary systemic candidiasis.To determine if differences in tissue distribution of C. albicans in infected mice is matched by differences in tissue responses to infection.To determine if cytokine mRNA expression patterns during secondary systemic candidiasis, are different to those during primary systemic candidiasis.To determine if cytokine gene expression patterns vary between resistant BALB/c and sensitive CBA/CaH mice during secondary systemic candidiasis.