The host cell probably plays an important role in neutralization but previous work on influenza virus neutralization used dedifferentiated cultured cells derived from tissues which are not the natural target of the virus. The aim of this investigation was to study the mechanisms of neutralization of influenza virus by different antibody isotypes using fully differentiated, ciliated epithelial cells of tracheal organ cultures. Tracheal organ cultures derived from the mouse were used together with mouse pathogenic strains (A/FPV/Rostock/34 (adpFPV/R) and A/PR/8) and mouse antibodies, so that all the participating components came from the same species. Parallel experiments were performed using cultured BHK cells and erythrocytes to enable comparison between different cell types. As previous work had used only a single saturating amount of antibody I decided to use a range of antibody concentrations with a constant amount of virus. Three potential mechanisms of neutralization were investigated: aggregation of virus, inhibition of virus attachment to cells and inhibition of virus internalization by cells. Each isotype (IgG, IgM and IgA) aggregated influenza virus (by EM determination) when used in sub-saturating amounts. Increasing the amount of antibody reduced aggregation and at higher antibody concentrations, the virus was monodisperse. Sub-saturating amounts of IgG monoclonal antibody partially inhibited the attachment of adpFPV/R to tracheal organ culture cells, BHK cells and erythrocytes. Together, aggregation and inhibition of virus attachment by IgG accounted for more than a 90% loss of infectivity, although this was usually less than the amount of neutralization observed. It is argued that these represent potentially important mechanisms of neutralization. Increasing the IgG concentration increased neutralization and increased attachment of virus to tracheal organ culture and BHK cells but not to erythrocytes. Therefore when there is a high ratio of IgG to virus, neither aggregation or inhibition of virus attachment accounted for any loss of infectivity. Under these conditions IgG did not prevent internalization of virus that had attached to tracheal organ culture or BHK cells and it was concluded that inhibition of an intracellular stage of the virus infectious pathway was the major mechanism of neutralization. Polyclonal IgM partially inhibited attachment of virus to organ culture cells, BHK cells and erythrocytes. Increasing the amount of IgM did not increase virus attachment to any of the cell types. Internalization of IgM-neutralized virus that had attached to tracheal organ culture and BHK cells was blocked, confirming earlier observations when a single saturating concentration of IgM was used. Neutralization of A/PR/8 by sub-saturating amounts of monoclonal polymeric IgA also inhibited virus attachment to BHK cells and erythrocytes. There was no rise in attachment when the amounts of IgA were increased and internalization of attached virus was inhibited. Sub-saturating amounts of IgA also greatly reduced virus attachment to tracheal organ culture cells. However increasing the relative IgA concentration resulted in increased attachment. The highest concentration of IgA used enhanced virus attachment by more than five-fold compared to infectious virus but this was accompanied by no increase in infectivity. Enhanced attachment was neuraminidase-resistant and was possibly mediated via an IgA-specific Fc receptor.

Cultures of astrocytes prepared from the brains of newborn mice, G26-24 oligodendroglioma cells and C1300 neuroblastoma cells were treated with Interferon (IFN) and the effect on major histocompatibility complex (MHC) antigen expression assessed by indirect immunofluorescence. IFN-αβ increased class I, but not class II, MHC antigen expression on astrocytes, G26-24 cells and C1300 cells. IFN-β1, increased class I, but not class II, MHC antigen expression on astrocytes. IFN-γ increased both class I and class II MHC antigen expression on astrocytes and G26-24 cells. IFN-γ increased class I, but not class II, MHC antigen expression on C1300 cells. IFN-αβ and IFN-β were additive with IFN-γ in the induction of class I MHC antigen expression on astrocytes, but inhibited the ability of IFN-γ to induce class II MHC antigen expression. IFN-αβ and IFN-γ increased the susceptibility of astrocytes, C26-24 cells and C1300 cells to lysis by alloreactive cytotoxic T-lymphocytes (CTL) indicating that IFNs increased the ability of the cells to participate in class I MHC restricted T-cell immune reactions. Astrocytes treated with IFN-αβ or IFN-γ, and G26-24 cells and C1300 cells treated with IFN-γ, prior to infection with Semliki Forest virus (SFV), showed a similar or increased susceptibility to SFV-specific CTL lysis, despite a reduction of SFV antigen display on the cells, as assessed by indirect immunofluorescence and susceptibility to lysis by anti-SFV antibody plus complement. It is concluded that even when SFV antigen expression is reduced by IFN treatment, in the context of enhanced class I MHC antigen expression cells remain susceptible to SFV-specific CTL lysis. IFN-αβ and IFN-γ treatment of astrocytes, and IFN-γ treatment of G26-24 cells, prior to treatment with a β-propiolactone inactivated preparation of SFV, increased the ability of the cells to stimulate SFV-specific T-cell release of IFN-γ. This increased ability correlated with an increase in MHC antigen expression on the cells. IFN-γ released by SFV-specific T-cells increased class I and class II MHC antigen expression on astrocytes and G26-24 cells indicating that a positive feedback mechanism could operate. SFV-infected newborn and adult mice possessed high levels of IFN-αβ in the brain. Brain extracts prepared from SFV-infected newborn and adult mice increased class I, but not class II, MHC antigen expression on astrocytes in vitro. Class I and class II MHC antigen expression was slightly elevated in the brains of SFV-infected newborn mice. To study the role of endogenous IFN-γ, R4-6A2 anti-IFN-γ monoclonal antibody was administered to adult mice, prior to infection with SFV, and the effect on the clinical course of SFV-disease monitored. R4- 6A2 antibody had no effect and preliminary experiments indicated that the antibody may not neutralise all IFN-γ activity in vivo under the conditions used.

A modified assay to detect interferon production from individual cells has been designed which is more accurate than those already described. Use of this modified assay has demonstrated that the difference between cell lines that can be induced to produce high yields of interferon, and those which are only capable of producing low yields of interferon, resides in the number of individual cells able to produce interferon in that culture. Thus the apparent homogeneous response of a cell culture to an interferon inducing agent, masks the heterogeneous response of the individual cells which make up that culture. This modified assay is probably sensitive enough to detect all cells capable of producing interferon within a given cell population; and the data presented in section one suggests that this assay can be used with confidence to assay interferon production in cell systems which only produce small amounts of interferon. Cloned 'nullipotent' embryonal carcinoma (ec) cells, like the pluri- potent ec cells described by Burke et al (1978), do not possess an active interferon system, and it is proposed that such cells lack the ability to produce interferon mRNA in response to an interferon-inducing agent. When these 'nullipotent' ec cells are treated with retinoic acid they show an activation of the interferon system which extends for approximately ten to fifteen days. The extent of activation seen varied between different embryonal carcinoma cell lines. In these differentiating cultures there is a parallel increase, both in the percentage of individual cells able to produce interferon, and in the yield of interferon per producer cell. The percentage of single cells able to produce interferon always remained small compared to the non-producer cell3 in the culture. The pattern of development of interferon inducibility and sensitivity does not distinguish between the different types of endoderm-like cell generated by the various differentiating teratocarcinoma cell lines, nor can the amount of interferon produced by different cell lines be used to quantitate the extent of differentiation which has occurred. However, the activation of the interferon system; because it coincides with changes in morphology and in protein synthesis, can be used as an additional positive marker for the production of differentiated cells in this system. The data presented in section three demonstrates that during the first third of pregnancy the embryo is unable to produce interferon in response to a virus infection, and furthermore suggests that the antiviral action of interferon may be non-specifically inhibited by the tissues of the reproductive system from the adult female mbuse. A functional interferon system develops during the seventh day of embryonic development, and the embryonic ectoderm and the visceral extra- embryonic endoderm are the last tissues to show a lack of interferon inducibility. Thus, the mouse embryo can be seen to become capable of mounting an interferon-based antiviral response during a period when it is unable to mount a humoral and cell mediated antiviral immune response. This factor may be of importance in the reduced susceptibility to the pathogenic effects of virus infections, which is a feature of the mid to late term mammalian embryo.

Background: Obesity is not a new disease, with roots that can be traced back to 400 BC. However, with the staggering increase in individuals that are overweight and obese since the 1980s, now over a quarter of individuals in Europe and the Americas are classed as obese. This presents a global health problem that needs to be addressed with novel therapies. It is now well accepted that obesity is a chronic, low-grade inflammatory condition that could predispose individuals to a number of comorbidities. Obesity is associated with cardiovascular diseases (CVDs) and type 2 diabetes (T2D) as part of “the metabolic syndrome,” and as first identified by Dr Vauge, central distribution of white adipose tissue (WAT) is an important risk factor in the development of these diseases. Subsequently, visceral WAT (vWAT) was shown to be an important factor in this association with CVDs and T2D, and increasing inflammation. As the obese WAT expands, mainly through hypertrophy, there is an increase in inflammation that recruits numerous immune cells to the tissue that further exacerbate this inflammation, causing local and systemic inflammatory and metabolic effects. One of the main types of immune cell involved in this pathogenic process is pro-inflammatory M1 adipose tissue macrophages (ATMs). MicroRNAs (miRNAs) are a species of small RNAs that post-transcriptionally regulate gene expression by targeting gene mRNA, causing its degradation or translational repression. These miRNAs are promiscuous, regulating numerous genes and pathways involved in a disease, making them useful therapeutic targets, but also difficult to study. miR-34a has been shown to increase in the serum, liver, pancreas, and subcutaneous (sc)WAT of patients with obesity, non- alcoholic fatty liver disease (NAFLD) and T2D. Additionally, miR-34a has been shown to regulate a number of metabolic and inflammatory genes in numerous cell types, including those in macrophages. However, the role of miR-34a in regulating vWAT metabolism and inflammation is poorly understood. Hypothesis: miR-34a is dysregulated in the adipose tissue during obesity, causing dysregulation of metabolic and inflammatory pathways in adipocytes and ATMs that contribute to adipose inflammation and obesity’s comorbidities, particularly T2D. Method/Results: The role of miR-34a in adipose inflammation was investigated using a murine miR-34a-/- diet-induced obesity model, and primary in vitro models of adipocyte differentiation and inflammatory bone marrow-derived macrophages (BMDMs). miR-34a was shown to be ubiquitously expressed throughout the murine epididymal (e)WAT of obese high-fat diet (HFD)-fed WT mice and ob/ob mice, as well as omental WAT from patients with obesity. Additionally, miR-34a transcripts were increased in the liver and brown adipose tissue (BAT) of ob/ob and HFD-fed WT mice, compared to WT controls. When miR-34a-/- mice were fed HFD ad libitum for 24 weeks they were significantly heavier than their WT counterparts by the end of the study. Ex vivo examinations showed that miR-34a-/- eWAT had a smaller adipocyte area on chow, which significantly increased to WT levels during HFD-feeding. Additionally, miR-34a-/- eWAT showed basal increases in cholesterol and fatty acid metabolism genes Cd36, Hmgcr, Lxrα, Pgc1α, and Fasn. miR-34a-/- iBAT showed basal reductions in Cebpα and Cebpβ, with increased Pgc1α expression during HFD- feeding. The miR-34a-/- liver additionally showed increased basal transcript expression of Pgc1α, suggesting miR-34a may broadly regulate PGC1α. Accompanying the ex vivo changes in cholesterol and fatty acid metabolism genes, in vitro miR-34a-/- white adipocytes showed increased lipid content. An F4/80high macrophage population was identified in HFD-fed miR-34a-/- eWAT, with increased Il-10 transcripts and serum IL-5 protein. Following these ex vivo observations, BMDMs from WT mice upregulated miR-34a expression in response to TNFα stimulation. Additionally, miR-34a-/- BMDMs showed an ablated CXCL1 response to TNFα. Conclusion: These findings suggest miR-34a has a multi-factorial role in controlling a susceptibility to obesity, by regulating inflammatory and metabolic pathways, potentially through regulation of PGC1α.

The food-borne pathogene Listeria monocytogenes has been considered a significant threat to human health worldwide. It mainly infects individuals suffering insuffecint immunity such as pregnant women. During pregnancy, L. monocytogenes is capable of causing a serious damage to the mother and the fetus. It can spread to different organs including the placenta via adaptation to interacellular lifestyle. To maintain pregnancy, the levels of the hormones progesterone and β-estradiol increase and reduction in hormone levels was proposed to be associated with fetal death and abortion. The objectives of this project therefore were to investigate the role of pregnancy hormones on the growth and virulence of L. monocytogenes, and to identify bacterial genes with possible roles in binding to pregnancy hormones. It was obsereved that the growth of L. monocytogenes in the presence of progesterone under anaerobic condition was affected by the action of the hormone and the effect was dose/time-dependent of exposure as increasing concentrations showed greater effect on the bacterial growth. Interestingly, bacterial growth was restored within 24 h of exposure to the hormone. In parallel, a Tn917-LTV3 insertion library was constructed and a number of mutants isolated that had reduced growth in the presence of β-estradiol were identified. However, reduction in growth was not microbiologically significant. Furthermore, bioinformatics analysis was performed to identify listerial genes with possible role in hormones degradation. It was observed that L. monocytogenes encodes for a protein that is possibly involved in steroid degradation; therefore, gene expression and a clear-deletion mutant were performed to test this hypothesis. This revealed no significant role of this protein in the growth restoration observed in the presence of progesterone. Also, the deleted gene was investigated of its ability to reduce NADPH in the presence of a possible substrate (progesterone, β-estradiol). This showed that this gene could possess an enzymatic activity toward pregnancy hormones. An attempt to purify this protein for further investigation was performed and protein expression in a soluble form was unsuccessful. The findings presented in this thesis represent an important view when considering the relation between pregnancy hormones and L. monocytogenes; however, further investigations of hormone-degrading proteins from L. monocytogenes are needed. This knowledge may form the basis of a therapy to protect pregnant individuals.

Murine models of graft-versus-host disease (GvHD) provide important information relevant to clinical bone marrow transplantation (BMT), as well as to other types of T cell-mediated pathology. The nature of the GvHD which develops in (C57BL/6 X DBA/2)F1 (BDF1) mice injected with parental lymphocytes is dependent on whether C57B1/6 (B6) or DBA/2 parental donor cells are used. BDF1 mice injected with B6 donor cells (B6 => BDF1) develop an acute GvHD with early lymphoid hyperplasia and NK cell activation, followed by immunosuppression, activation of anti-host cytotoxic T lymphocytes (CTL), weight loss and early death. In contrast, BDF1 mice given DBA/2 donor cells (DBA/2 => BDF1) exhibit a chronic, stimulatory GvHD, characterised by B cell hyperreactivity, autoantibody production and immune complex-mediated glomerulonephritis (ICGN). Previous studies have shown that the distinct forms of GvHD in BDF1 recipient mice are associated with different patterns of cytokine production. Whereas acute GvHD is characterised by production of high levels of Th1 cytokines, chronic GvHD is associated with a preferential Th2 response. Therefore, it was suggested that the two forms of GvHD may reflect differential activation of distinct subsets of CD4+ T helper (Th) cells. However, when and why such T cell polarisation should occur has remained unclear. A number of recent studies have demonstrated that cytokines produced by cells of the non-specific immune system during the early phase of an immune response can strongly influence the type of specific response which develops subsequently. The main aim of this thesis was to explore the role of these early immune mediators in determining the outcome of the GvHD in BDF1 mice. These studies of the cellular and molecular interactions involved in murine GvHD have implications for understanding the pathogenesis of clinical GvHD and the development of specific therapy following BMT. In addition, they provide an important insight into the regulation of immune responses during other immunologically-mediated diseases.

No description available.

The concept of microglial priming has developed through in vivo studies and is operationally defined as an exaggerated microglial production of soluble mediators (NO and cytokines e.g. IL-1β, TNF-α, IL-6) following a pro-inflammatory activation event (e.g. LPS-treatment). In practice microglial priming predisposes the brain to degeneration through the promotion of inflammatory mechanisms. In vivo studies of Crry (a major murine cell-surface C3-regulator) KO mice previously identified a novel role for C in the induction of the primed microglial phenotype, implicating iC3b ligation of microglial CR3. The purpose of this study was to further investigate C-dependent microglial priming and its mechanism(s) through study of microglia in isolation in vitro. Experiments using purified fluid-phase human iC3b failed to demonstrate any phenotypic effects of ligand exposure. Given the results of previous investigations concerning CR3 ligands, combined with the results of binding studies and sequence comparisons, it appears likely that, while still able to engage the cell-borne CR3, fluid-phase iC3b is incapable of exerting significant effects on the microglial phenotype. Studies using Zymsoan and C-fixing mAb-sensitised TC plastic as a means to generate ligands to investigate the consequences of microglial CR3 engagement by iC3b were confounded by the stimulatory effects of the C-activating agents (i.e. zymsoan or mAb) which prevented attempts to dissect the effects of the isolated interaction. Nonetheless, specific effects were attributable to the C3-derived CR3 ligands generated, which dramatically and significantly reduced the pro-inflammatory responses evoked by the C-activating agents. Investigations using C3-activation fragments immobilised on native (i.e. non-sensitised) TC plastic demonstrated phenotypic effects of microglial iC3b-CR3 ligation consistent with the previously reported mechanism of C-dependent microglial priming. Experiments using cultured Crry KO microglia demonstrated increased sensitivity to autologous C activation. Phenotyping experiments, however, failed to show any consequence of Crry expression status, even when the intrinsic sensitivity of Crry KO cells to C3 activation and deposition was effected, thus mimicking the in vivo scenario (including the potential for iC3b ligation of CR3). Data gathered from the several systems designed to ligate CR3 of microglial cells with C3-derived ligands highlight the broad range of potential cellular responses mediated by CR3 and emphasise the importance of context for the consequence of this interaction. In so doing, these data also further evidence that under certain circumstances, iC3b-CR3 binding can induce a primed microglial phenotype.

No description available.

No description available.