No description available.

Dendritic cells (DCs) are specialized members of the innate immune system that are responsible for the initiation of primary adaptive immune responses whose purpose is to resolve infection and inflammation. During most viral infections, mature dendritic cells present critical viral antigens to naïve T-cells within secondary lymphoid organs, resulting in the generation of an antigen-specific adaptive immune response and clearance of the virus. During infection with HIV-1 however, the virus is not cleared and a chronic systemic infection develops characterized by immune dysfunction, CD4+ T-cell depletion, systemic inflammation, and opportunistic infections. A growing body of evidence indicates that HIV-1 subversion of DCs contributes to both HIV-1 pathologies and viral dissemination. A number of similar effects by accessory HIV-1 peptides on DC physiology have also been reported. In vitro studies demonstrate that HIV-1 inhibits DC maturation and function. Ex vivo studies on the other hand describe partially mature, dysfunctional DCs collecting in secondary lymphoid organs. In vitro studies examining the effects of HIV-1-Tat and HIV-1-Vpr have described opposing effects on DC maturation. Therefore we undertook experiments to comprehensively describe the effects of HIV-1 and the Tat and Vpr accessory peptides on DC maturation and function. To understand the contributions of individual viral proteins to DC dysfunction we infected DCs with a dual tropic HIV-1 and examined phenotypic and functional changes after maturation with inflammatory cytokines. Following this we examined the influence of exogenous and endogenous HIV-1-Tat and HIV-1-Vpr on MDDC maturation and function using recombinant proteins and deletion mutant lab adapted HIV-1 strains. Live dual tropic HIV-1 was found to selectively inhibit aspects of phenotypic maturation as well as antigen capture and presentation functions. MDDC MAPK responsiveness to bacterial LPS remained intact however. Exogenous accessory HIV-1 Tat and Vpr did not affect MDDC phenotype but inhibited dextran endocytosis and viral peptide presentation. HIV-1-gp120 increased iMDDC maturation while blunting cytokine induced decreases in MDDC antigen capture abilities. The deletion of HIV-1-Tat did not affect MDDC phenotype, but was found to affect antigen capture decreases by R5 tropic HIV-1BaL. Deletion of HIV-1-Vpr likewise did not affect MDDC phenotype, however it was found to be influential in HIV-1 induced decreases in MDDC antigen presentation to autologous T-cells. These accumulated results indicate that HIV-1 subverts DC maturation and function through whole virus effects and individual accessory peptide influences. Understanding the mechanisms of DC dysfunction in HIV infection may provide some insight into infection prevention strategies and therapies leading to adaptive immune system activation and viral clearance.

<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

Despite ongoing advancement in detection and treatment, breast cancer remains a major clinical challenge worldwide. Cancer has traditionally been conceptualized as a ‘disease of the genes’ by virtue of the mutagenic events necessary for its inception. It is now clear, however, that complex interactions take place between cancer cells and the array of non-cancerous cells and molecules in their immediate surroundings, known generally as the tumour microenvironment. Cancer-microenvironment interactions are increasingly recognized as processes that critically influence the outcome of disease. Cells of the host immune system are major components of the breast tumour microenvironment. While their presence in tumours is thought to reflect an attempt at disease eradication or containment, cancer cells can exploit the immune system through a variety of means, including the recognition of leukocyte-derived cytokines. As such, intratumoral leukocytes and high cytokine content are frequently associated with aggressive subtypes of breast cancer and poor prognosis. This dissertation explores the influence of one such cytokine, oncostatin-M (OSM), on the behaviour of breast cancer cells. Our results collectively demonstrate that OSM can rapidly and potently induce aggressive features in well-characterized cell models of luminal, well-differentiated breast cancer. These features include suppression of the important biomarker estrogen receptor-α (the key molecular target of endocrine therapy), gain of the breast cancer oncogene S100A7, loss of luminal epithelial differentiation and gain of mesenchymal features, and induction of a phenotype consistent with breast cancer stem cells. Each of these changes can potentially influence treatment responsiveness, the metastatic process, or both. Along with high levels of intratumoural leukocytes, the OSM-induced features listed above are known to associate with one another in human breast cancer. Tumours that display such characteristics have a poor prognosis and present the greatest challenges for modern breast cancer therapy, both because they are inherently prone to rapid metastasis and because targeted therapies for such tumours are lacking. The etiology of these aggressive disease subsets is largely unknown, and resolution of this issue would represent a major advancement in our understanding of breast cancer. Importantly, we found that expression of OSM and/or its receptor OSMR was reproducibly associated with these features in multiple breast cancer cohorts, largely confirming our experimental results. OSMR, in particular, was associated with poor clinical outcome. OSM signalling may thus provide a novel mechanistic explanation for the development of aggressive forms of breast cancer. If our findings are validated and expanded upon in future studies, OSM signalling could serve as a novel therapeutic target and may be an important consideration in the design and deployment of breast cancer immunotherapies. / Graduate

T cells are a crucial component of the immune system and play an important role in responses to pathogens, tumours, and transplanted tissues. In many human cancers, elevated numbers of tumour-infiltrating CD8+ killer T cells are associated with favourable outcomes, suggesting that enhancing T-cell responses could provide major therapeutic benefit for cancer patients. Thus, identifying factors that can promote protective T-cell responses is of great clinical importance. The cytokine interleukin-2 (IL-2) is a major inducer of T-cell proliferation and differentiation, and is used clinically to treat melanoma and renal cell carcinoma. The first two chapters of this thesis focus on the biochemical mechanisms by which IL-2 induces T-cell proliferation. By using mutant and chimeric cytokine receptors expressed in lymphocyte cell lines, the interplay between Shc and STAT5, two major mitogenic signaling pathways activated by the IL-2 receptor, are investigated, revealing an essential synergy between the two pathways for optimal lymphocyte proliferation. The third chapter of this thesis describes work done to identify cytokines that promote T-cell responses within the ovarian cancer microenvironment. In human diseases such as HIV/AIDS and cancer, high numbers of “polyfunctional” T cells (i.e., T cells capable of multiple effector functions) are associated with favourable outcomes. Using clinical ovarian cancer samples in a novel ex vivo assay, it was found that the ovarian tumour environment inhibits polyfunctional T-cell responses to varying extents among patients. After surveying a large panel of cytokines, the cytokine combination of IL-2, IL-12, and IL-18 was found to overcome the immunosuppressive environment to potently enhance CD8+ T-cell proliferation and polyfunctionality in all patient samples. The polyfunctional profiles induced by these cytokines are associated with protective immunity in various human conditions. Thus, these findings suggest that given the right signals, T cells can become highly polyfunctional effectors in the ovarian cancer microenvironment, which offers promise for the development of effective T-cell based therapies for this clinically challenging disease. / Graduate / 0982

This thesis presents studies on the interaction between the human T cell lymphotropic virus type 1 (HTLV-1) Tax protein and the 20S proteasome and the role of the interaction in cellular processes and the cytotoxic T cell (CTL) response against HTLV-1. The rapid translocation of Tax into the nucleus is described. Tax accumulates in the nucleus and forms unique bodies involved in transcriptional activation. It was further found that Tax associated with assembled nuclear 20S proteasomes and stimulated the chymotryptic and tryptic activities of the 20S proteasome, independent of the induction of the LMP2 and LMP7 proteasome subunits. Confocal microscopy revealed a partial colocalisation of Tax with nuclear proteasomes. A panel of Tax mutants was generated and their subcellular localisation and association with the 20S proteasome analysed. This analysis revealed that both the N- and C-terminus of Tax play a role in proteasome binding of Tax and further showed that proteasome binding was not sufficient for nuclear localisation of Tax. Therefore, Tax probably translocates into the nucleus prior to and independent of proteasome association. Tax specific CTL clones were generated and characterised using tetrameric MHC class I/peptide complexes. These CTL clones were used to investigate the requirements for processing and presentation of Tax for recognition by CTL. It was found that Tax was a metabolically very stable protein and that the presentation of the immunodominant Tax 11-19 epitope was dependent on the transporter associated with antigen presentation (TAP), independent of the expression of LMP2 and LMP7 proteasome subunits and resistant to treatment with the proteasome inhibitor lactacystin. It is proposed that the interaction between Tax and the 20S proteasome plays a role in Tax mediated transcriptional activation, leading to cellular activation and proliferation, and may not determine the immunodominance of Tax in the CTL response against HTLV-1.

Type 1 diabetes is caused by immune destruction of pancreatic β cells. There is increasing evidence that genes outside the MHC region contribute to the pathogenesis of type 1 diabetes. Cytokines due to their role in immune regulation seem to play a crucial role in the pathogenesis of the disease. Three hundred and eight patients with type 1 diabetes and 150 normal controls were genotyped for polymorphism in the genes for IFN-γ, IL-4, IL-6, and TGF-β1. All assays employed in this study were PCR based. The IFN-γ CA repeats was an octa-allelic repeat and the 3 / 3 genotype showed a significant association with type 1 diabetes (p=0.0001). The IL-4 C (-590) T polymorphism did not show a significant association with type 1 diabetes. The GG genotype of G (-174) C of the IL-6 gene polymorphism showed a strong association with the susceptibility towards type 1 diabetes (p= 0.002). The TC genotype of the TGF-β1 T (+869) C polymorphism also showed a significant association with type 1 diabetes (p= 0.003). The association of the 3 I 3 genotype of the IFNG CA repeats and no association of IL-4 C (-590) T polymorphism may support the idea of dominance of the TH1 cytokine profile and type 1 diabetes suggesting a cell mediated disease. The IL-6 G (-174) C result attests an existing hypothesis of the important role of IL-6 in the onset of type 1 diabetes and its development. Immunosuppression of the TGF-β1 may have been initiated after deviation of the TH1/TH2 cytokine milieu. The GC of the IL-6 G (-174) C and the TC of the TGF-β1 T (+869) C showed strong association with diabetic nephropathy. Haplotype studies showed that cytokine function might be as a result of a cytokine network rather than individual cytokines. Further, the genetic susceptibility may be influenced not only by genetic composition but by the gender of patients as well as age at onset of type 1 diabetes. In conclusion these results suggest a contribution of the IFNG CA repeats, the TGF-β1 T (+869) C, and the IL-6 G (-174) C to the genetic susceptibility of type l diabetes and may have future therapeutical values.

Three bovine casein peptides, LLY, PGPIPN, and TTMPLW, have been reported to stimulate phagocytosis of sheep red blood cells by murine macrophages. TTMPLW also protected mice against Klebsiella pneumoniae infection. The purpose of this study was to investigate the effects of these three peptides on cytokine (TNF-$ alpha$ and IL-6) production and nitric oxide (NO) release by bone marrow macrophages (BMM). The peptides alone were incapable of stimulating cytokine production or NO release in naive or IFN-$ gamma$-primed BMM. However, when BMM were coincubated with the peptides (1.0 $ mu$M) and LPS (100 ng/ml), an augmentative effect on TNF-$ alpha,$ IL-6 and NO production was observed. The peptides increased the response of BMM to stimulation with LPS in a dose- and time-dependent manner. Of the three peptides, TTMPLW (0.01, or 1.0 $ mu$M) had the greatest augmentative effect on NO production by LPS-stimulated BMM. Tumor necrosis factor-$ alpha$ production peaked after 4 hr stimulation, and decreased rapidly thereafter. Among three peptides, TTMPLW induced the highest amount of TNF-$ alpha$ production at a concentration of 1.0 $ mu$M. When used at a concentration as low as 0.01 $ mu$M, TTMPLW and PGPIPN, but not LLY, potentiated TNF-$ alpha$ production. All the peptides (1.0 $ mu$M) stimulated IL-6 production by BMM, which plateaued after 12 hr. The auto/paracrine TNF-$ alpha$ produced by LPS-stimulated BMM was partially responsible for release of NO. After all the TNF-$ alpha$ was neutralized, release of NO was reduced by about 21% (P $<$ 0.01). However, neutralization of IL-1$ beta$ and IL-6 did not have any effect on NO production by LPS-stimulated BMM. These results demonstrate that bovine casein peptides can costimulate naive macrophages with LPS for proinflammatory cytokine production and NO release and may play a role in host defense against pathogens.

No description available.

Introduction: Alzheimer’s disease (AD) and dementias constitute a significant public health burden and it is estimated that one in 85 people may be living with AD by 2050. Dementias are a spectrum of diseases with common traits including amyloid protein growth, neurodegradation, neurofibrillary plaque and tangle formation, and which may be influenced by pro- and anti- inflammatory immune mechanisms. Even a modest delay in onset could result in significant reductions in the social and economic burdens of dementias. An important lifestyle factor identified in risk reduction is physical activity (PA). Although the association between dementia risk and PA has been established, the exact physiological mechanisms through which protection occurs are not known. This research consists of two experiments that were designed to explore the effects of physical activity on pro- and anti-inflammatory cytokines and apoptosis in the mouse hippocampus, a brain region implicated in learning, memory, and cognition. Methods: Study #1: Female C57BL/6 mice, 4-5 months of age, were divided into three groups: sedentary controls (NOTREAD) (n = 22), treadmill exercise with immediate sacrifice (TREAD-Imm) (n = 21), or treadmill exercise with sacrifice after 2 hours (TREAD-2h) (n = 20). TNF-α, IL-6, and IL-1β expression in the hippocampus and intestinal lymphocytes were measured by Western blot analysis. Percentages of hippocampal cells undergoing apoptosis (Annexin+) or necrosis (Propidium Iodide+) were determined through flow cytometry. Plasma levels of 8-isoprostane and corticosterone were measured using commercially available EIA kits. Study # 2: Female C57BL/6 mice, 3-4 weeks of age, were assigned to wheel running (WR; n = 20) or a control condition (No WR; n = 22) and sacrificed after the 16 weeks. Data collected included measures of training status (running volume, body weight, run-to-exhaustion time, and skeletal muscle cytochrome c oxidase activity), flow cytometric analysis of hippocampal cell phenotypes and apoptosis (CD45+, CD11b+, Annexin+, Annexin+/PI+, PI+), and cytokine concentrations (TNF-α, IL-1β, IL-12, IL-6, IL-1ra, and IL-10) in cell lysates. Results: Study #1: Acute treadmill exercise lead to significant decreases in TNF-α (p<0.05) and increases in IL-6 (p<0.05) expression in the hippocampus of healthy mice. No effects of acute exercise on the apoptotic status of hippocampal cells were observed. In intestinal lymphocytes, the exercise bout lead to significant increases in TNF-α (p<0.05), IL-6 (p<0.05), and IL-1β (p<0.05). Acute exercise was associated with a significant increase in both plasma 8-isoprostane (p<0.05) and corticosterone (p<0.05) levels. Study #2: WR mice had measurable training effects and significantly lower TNF-α (p<0.05) and higher IL-6 (p<0.05), IL-1ra (p<0.05) and IL-12 (p<0.05) expression in the hippocampus compared to controls. IL-1β, IL-10, and the percent of apoptotic, dead cells, and cell phenotypes did not change due to training. Conclusion: Exercise chronicity (acute vs. chronic), stress characteristics of the exercise (forced vs. voluntary) and tissue location (systemic vs. central) emerged as important variables with effects on both cytokine concentrations and plasma levels of stress hormones. Physical activity may protect the hippocampus against inflammatory damage caused by TNF-α, and the suppression of this cytokine may be due to increased glucocorticoid secretion during acute exercise. It is also proposed that elevated IL-6 expression (central and systemic) may mediate this protection by creating an anti-apoptotic environment in the hippocampus. Less apoptosis may also contribute to maintenance of cognitive function during acute and long-term physical activity.