No description available.

<p>The interactions between the brain and the immune system are extensive and each has a profound influence on the other. The hippocampus is a brain region that is strongly impacted by the immune system, especially considering its large population of microglia, the resident immune cells of the brain. Cytokines and chemokines, the signaling molecules from immune cells, signal within the central nervous system (CNS) as well, and they are critical in hippocampal function. The relationship between the immune system and the hippocampus may underlie its particular vulnerability to diseases and disorders of the nervous system and the periphery. Conversely, immune signaling within the hippocampus is affected by alterations in hippocampal resilience and flexibility, such that increased hippocampal plasticity reduces vulnerability to immune challenges. The balance between risk and resilience in the hippocampus is modulated by immune signaling, especially by microglia.</p><p> The hippocampus is vulnerable to immune challenges, disease and injury, but it is simultaneously a region capable of profound plasticity and flexibility. The following dissertation experiments were designed to assess the roles of microglia and their signaling molecules, cytokines and chemokines, during normal hippocampal processes, such as learning and memory and response to immune challenge. The first set of experiments examined the effects of a neonatal bacterial infection in rats on hippocampal-dependent learning and memory as well as neuronal and microglial signaling in adulthood. In the first experiment, neonatally infected rats have impaired memory during fear conditioning following an immune challenge in adulthood. The impairment is caused by the exaggerated expression of the pro-inflammatory cytokine, interleukin (IL)-1&#946;, within the hippocampus during learning. Hippocampal microglia are the primary source of IL-1&#946; and the microglia in neonatally infected rats are "primed" by the infection into adulthood. In the second experiment, neonatally infected rats are more accurate on a Morris Water maze task following minimal training in adulthood, but have significantly impaired memory for a reversal platform location. In addition to improved accuracy, they have lower neural activation as measured by Arc protein expression within the dentate gyrus (DG) of the hippocampus. The next set of experiments assessed the effects of increasing hippocampal plasticity on immune signaling within the hippocampus. Following 7 weeks of environmental enrichment (EE), enriched rats had an attenuated pro-inflammatory response within the hippocampus in response to an in vivo peripheral immune challenge. The reduced immune response was specific to a subset of cytokines and chemokines and occurred only within the hippocampus and not adjacent cortical regions. Enrichment increased glial antigen expression within the DG as well. In another group of enriched rats, an ex vivo stimulation of isolated hippocampal microglia from EE rats demonstrated that the reduced microglial reactivity observed in vivo requires influence of other neural cell types on microglia phenotype, such that microglia within the DG of EE rats are smaller than controls. Taken together, these experiments define cellular and molecular mechanisms of hippocampal vulnerability and resilience as a function of interactions between the brain and the immune system.</p> / Dissertation

No description available.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV8), has been been identified in all epidemiological forms of KS as well as in tissue obtained from primary effusion lymphoma (PEL) and Multicentric Castleman's disease (MCD). The KSHV genome contains several putative oncogenes, suggesting that viral infection may induce cellular transformation and tumorgenesis. Herpesviruses encode a number of different surface glycoproteins, which are involved in virus-host interactions. Studies have shown that the viral glycoproteins H and L form a complex that plays an essential role in viral attachment and cell to cell fusion. Both glycoproteins have been identified in KSHV and were expressed in mammalian cells. Expression studies revealed that KSHV gH and gL exhibit similar features to those seen in other herpesviruses. However, KSI-IV gL appears to traffic independently and may function in cell to cell fusion processes even when expressed alone. KSI-IV de novo infections are rare and the lack of a reliable cell culture system has delayed pathogenesis studies. As part of this thesis the hepatoma cell line HepG2 has been shown to allow limited KSHV infection, as judged by nested PCR. Studies have shown that infection leads to increased apoptosis, although viral replication could not be detected. Furthermore, Epstein Barr Virus (EBV) appeared to modulate the ability of KSHV to infect HepG2 cells. Finally, a microtitre plate assay has been established for the quantification of the KSHV genome. A comparison of plasma and serum samples obtained at the same time point showed that plasma is more reliable in testing for KSHV, the DNA copy number in serum samples being reduced up to 10 fold. In conclusion, this new assay is a potentially useful tool for both diagnostic proposes and research studies.

Herein we describe a novel APC population in mice, designated LAPCs. LAPCs are BM-derived myeloid leukocytes, distinctive from other immune cells. As APCs, LAPCs respond to various virus infections including VACV, CBV3 and influenza A virus. Notably, influenza virus-activated LAPCs capture Ag in the lungs, and migrate into the DLN and spleen with delayed kinetics compared to DCs. In the DLN, influenza virus-activated LAPCs co-localize with T cells and selectively induce Th2 effector cell polarization by cell-cell contact-mediated modulation of GATA-3 expression. In support of a role for LAPCs in anti-influenza T2 immunity, adoptive transfer experiments revealed that influenza virus-activated LAPCs selectively augmented Th2 effector T cell responses in the DLN, increased production of anti-influenza immunoglobulin (Ig) including IgE in peripheral blood and increased levels of IL-5 and eotaxin in BAL fluid in recipient influenza infected mice. LAPC recipient mice exhibited exacerbated pulmonary pathology, with delayed viral clearance and enhanced pulmonary eosinophilia. Collectively, these results highlight the importance of LAPCs as novel immuno-modulators of T2 immunity during influenza A virus infection, which is implicated in both immunoprotection and immunopathology. Subsequently, we examined the immuno-modulatory effect of type-I IFN, specifically IFN-on the immune response against pulmonary influenza virus infection. We have provided evidence that a single dose of IFN- (1×105U) augmented DC migration but inhibited LAPC migration into the DLN. mIFN- treatment skewed the immune balance toward T1 immunity, identified as enhanced T1 effector T cell responses (Th1 and CTL) but diminished T2 effector T cell responses (Th2) in influenza virus infected mice. Finally, IFN- treated mice showed accelerated viral clearance and diminished pulmonary eosinophilia in lung tissue compared to control mice. Taken together, these results suggest that anti-influenza T1 and T2 immunity may be modulated differently by DCs and LAPCs, respectively. Furthermore, these results support the therapeutic potential of type I IFNs, especially IFN-, as an alternative antiviral to control both viral replication and immunopathology induced by influenza A virus infection in humans.

The last decade has been witness to a number of seminal discoveries in the field of innate immunity. The discovery that microbial molecules and endogenous danger signals can be detected by germ-line encoded receptors has changed the way we study the immune system. Indeed, the characterization of Toll in Drosophila as a sensor of microbial products in 1997 then led to the discovery of a family of Toll Like Receptors (TLRs) in mammals. TLRs are critical for the induction of inflammatory responses and the generation of a successful adaptive immune response. The array of ligands that these transmembrane proteins recognized mediates defense against bacteria, viruses, fungus and parasites, as well as, possibly, cancerous cells. In addition to this membrane-bound family of recognition proteins, two families of pattern recognition receptors have been recently shown to respond to microbial and chemical ligands within the cytosol. These represent the Nod Like Receptors (NLRs) and RIGI-like helicase receptor (RLH) families. Nod1 and Nod2 are members of the NLR family of proteins, which are responsible for the recognition of components derived from the bacterial cell wall, more precisely, moieties of peptidoglycan. As such, Nod1 and Nod2 are implicated in the recognition and the defense against bacterial pathogens. Importantly, the genes encoding these two proteins have also been linked to the etiology of several inflammatory disorders such as Crohn’s disease and asthma. In this thesis, we show that recognition of Nod1 and Nod2 ligands generates a rapid and transient inflammatory response in vivo. When co-injected with a model protein, Nod1 and Nod2 ligands exhibit adjuvant properties that lead to the generation of an antigen-specific Th2 type adaptive immune response. Surprisingly, recognition of the Nod1 ligand in non-hematopoietic cells is critical for the generation of this immune response. In contrast, TLRs classically tip the balance towards a Th1 response and interestingly, co-injection of TLR and Nod ligands synergize to generate a more potent immune response characterized by the generation of Th1, Th2 and Th17 T cell respones. To study the role of Nod1 and Nod2 in the context of a bacterial infection in vivo, we used an intestinal mouse pathogen, Salmonella enterica serovar Typhimurium. We were able to show that Nod1-deficient mice, but not Nod2-deficient mice, are more susceptible to the strain of this bacterium, which enters the host through the active pickup in the intestinal lumen by underlying myeloid cells. This sampling mechanism is mediated by a subset of dendritic cells that populate the intestinal lamina propria. Accordingly, the defect seen in Nod1-deficient mice localizes to the mucosal barrier where these dendritic cells appear to have an impaired response towards the bacteria. Taken together, these results increase our knowledge on the general role of Nod1 and Nod2 in immunity and might generate new avenues of research and potential therapeutic targets.

[Abstract]Crown rot, caused by Fusarium pseudograminearum (Fpg), is an important soilborne disease of wheat and barley. The degree of crop damage depends on seasonal conditions. Typically, high moisture conditions early in the season encourage seedling infection from stubble residues. Moisture stress later in the season leads to the production of unfilled “whiteheads”. Current control relies on cultural practices and sowing of partially resistant varieties. In order to understand the nature of partial resistance, I have examined the patterns of disease symptom development and pathogen spread in susceptible and partially resistant tissues of both pot-grown wheat, barley and oat seedlings and field-grown inoculated wheat trials. Further research was conducted to determine whether differences in pathogenicity occur amongst a small subset of Australian Fpg isolates. Seedling experiments confirmed that differences in disease ratings between susceptible and partially resistant genotypes are detected in younger leaf sheaths of older seedlings. At later harvest times differences between these genotypes are not significant in older leaf sheaths. Re-isolation of Fpg from inoculated seedlings has shown that each tissue was infected later in partially resistant genotypes compared to susceptible ones with a significantly lower number of isolations recorded at each harvest time in 42 day old seedlings. Barley cultivars were rapidly infected by the pathogen and exhibited high levels of disease symptoms. By comparison levels of infection in oats were low compared to all other genotypes. No significant differences between genotypes were observed in coleoptile tissues, either in fungal colonisation or development of disease symptoms. Disease development in the subcrown internode varied between lines/cultivars but was not representative of the relative susceptibility of each genotype. The pathogen did not appear to invade plant tissue via the vascular system but rather spread directly across the stem from leaf sheath to leaf sheath. Field trials were designed to study disease symptom development and localisation of Fpg hyphae in all expanded tissues (excluding head and roots) in wheat genotypes of known susceptibility to crown rot. Plants were harvested at approximately fortnightly intervals throughout the growing season. The main effects and interactions of harvest, genotype and tiller on each plant part were examined with a detailed statistical analysis of differences seen in these factors between susceptible and partially resistant wheat genotypes, in two inoculated field trials. While differences between genotypes were mostly not significant at each harvest when disease rating or isolations from leaf sheath tissues were examined, important differences between susceptible and resistant genotypes were seen in disease developments and Fpg infections of stem tissue in field trials. Restriction of pathogen growth and symptom development was more pronounced in the tissues of 2-49 (possesses seedling resistance) than in the field resistant Sunco. At present, the mechanisms that lead to these resistance responses are unknown. The pathogenicity study aimed to determine whether 7 Fpg isolates and a mixed inoculum differed in ability to cause crown rot in 9 wheat genotypes ranging in susceptibility to this disease. Although a genotype*inoculum interaction was significant, there is no evidence of stable pathogenic races in the isolates examined in these experiments. The growth of all isolates was partially inhibited in a consistent manner on resistant genotypes when compared to very susceptible genotypes. These results confirm significant differences in the aggressiveness of Fpg isolates on wheat, evidenced by variation in mean disease severity between isolates growing on a range of host genotypes.

Errata sheet has been pasted onto the front end-paper. / Bibliography: leaves 208-217. / x, 217 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Outlines a systematic synthesis and testing of continual sterilization design. Principal aim is to evaluate and develop mathematical models for sterilization, undertake experimental studies for determining thermal inactivation effects on continuous processing of a liquid containing contaminant bacteria and compare the data obtained with those predicted by a selected model. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemical Engineering, 2001?

The purpose of this study was to examine the relationships between inflammation, infection and lung function in cystic fibrosis during the evolution of lung disease in childhood and early adolescence. The developmental stages of childhood and the progression of lung disease together affected the methods and techniques used in the study, with the consequence that the work for this thesis fell naturally into two parts. The first part concerned the study of early lung disease in infants and young children who were unable to expectorate or to cooperate with lung function testing. In the second part, the inflammatory processes in both stable lung disease and during clinical exacerbations in older children and adolescents were studied non-invasively using sputum. The absence of a recognised definition of pulmonary exacerbation lead to further investigation into clinical heterogeneity in the diagnosis and management of an exacerbation. In early lung disease, inflammation was not found to be independent of infection, with pathogens in the lower airways found to correlate with levels of inflammation, respiratory system compliance and degree of air trapping (a relationship not previously shown). This suggested that infection remains the key target to minimizing lung damage in cystic fibrosis. The relationship between sputum markers of inflammation and lung pathology in established disease was found to be less clear, with high inflammation levels in both stability and during exacerbation. Reduction in sputum inflammatory levels following treatment of an exacerbation was found to be greater in those with lower pre-treatment levels. The definition and management of an exacerbation was found to be an area lacking consensus among clinicians, with likely consequent heterogeneity of clinical care and therefore inhomogeneity of hospitalization as a surrogate measure of exacerbation in a research setting. The work from this thesis, and the ensuing publications, has contributed to the understanding of the interactions between the inflammatory and infectious processes involved in CF lungdisease, in both early and more established lung disease in childhood.

Baculoviruses are lytic insect viruses. Upon internalisation, the viral genome orchestrates a sequential expression process ultimately leading to lysis of the infected cell. Release of progeny capable of infecting other cells during the process completes the infection cycle. Studies of the infection cycle in cell culture are typically conducted by synchronous infection, i.e. near simultaneous infection of all cells, by means of high virus concentrations. The behaviour of the synchronously infected culture, such as the timing of onset of progeny release, is considered representative for the infection progression within individual cells. In reality, however, the synchronously infected culture only reflects the average behaviour of all infected cells. The infection progresses in individual cells display large variability; this is most obvious in the observation that within the same culture some cells undergo cell lysis at two days post infection while others remain viable up to four days post infection. Such variabilities or asynchronies observed in synchronously infected culture is the topic of this thesis. Using a simple phenomenological model, it is demonstrated that cell death and associated intracellular product release is adequately described assuming that the waiting time from infection to cell death follows a Gaussian distribution with a mean of 59 hours post infection (hpi) and a standard deviation of 15hpi. Unlike other deterministic model developed over the last decade (Licari and Bailey 1992; Nielsen 2000), this stochastic model does not make the biologically inconsistent assumption that cells continue to be metabolically active following loss of membrane integrity. While elegant in its simplicity, the model provides no explanation for the underlying stochasticity. Investigations into the cause of this dispersion of cell death highlighted further asynchronies in the specific recombinant protein yield, in viral DNA content, in virus budding rate, and in cell volume increase instead of clarifying the issue. A modelling framework developed by Licari & Bailey (1992) and later Hu & Bentley (2000) incorporates the number of infectious particles each individual cell receives as a possible source of the dispersions in the host cell responses. However, this was found NOT to be the cause of the observed asynchronies under non-substrate limiting conditions. The timing of cell death, cell volume increase, recombinant product yield, viral DNA content, and virus budding rate is identical in Sf9 cell cultures infected at multiplicities of infection of ~5, ~15, and ~45 infectious particles per cell. Cell cycle variation has previously been suggested as a possible cause for observed asynchronies in baculovirus infections (Brown and Faulkner, 1975). The cell cycle phase is indirectly linked to the cell volume, because a G2-phase cell prior to division is inherently twice the cell volume of a G1-phase cell after cell division. By the same logic, it is also apparent that a G2-phase cell possesses twice the number of ribosomes of a G1-phase cell and thus a doubled protein production capacity. The effect of the cell cycle or cell volume on the baculovirus infection was determined by splitting an exponentially growing Sf9 cell culture into 5 cell size dependent fractions by centrifugal elutriation. The subsequent infection of these fractions showed (1) no dependency of the timing of cell lysis and cell volume increase and (2) approximately twofold increase of a) recombinant protein yield, b) viral DNA concentration, and c) budded virus yield. The recombinant protein yield showed a strong proportionality to the initial cell volume and the total RNA concentration during the late phase of the infection. As argued in chapter 6, these proportionalities suggest that the observed differences in the responses of the cell fractions to the baculovirus infection are more likely caused by the difference in the protein production capacity than by cell cycle specific molecules. This investigation gave also reason to speculate that infected cells cannot progress beyond the G2/M phase, and cell cycle progression continues undisturbed until ~8hpi when all cellular DNA replication appears to cease. Resuspended, infected Sf9 cells synchronised by centrifugal elutriation showed an identical cell cycle distribution as the non-infected control cultures for the first ~8hpi; G1 and G2/M phase cell proportions remained unchanged, whereas S phase cells progress to G2/M phase. Subsequently, the non-infected control cells resumed normal cycling whereas all infected cells remained at the same cell cycle phase from 8 to 11hpi. The initial cell cycle arrests in G2/M phase in both infected and non-infected cultures were caused through medium exchange.