Le virus influenza a (via) est responsable d'epidemies annuelles et de pandemies sporadiques. un element clef, impactant a la fois la replication du virus et les symptomes de l'hote, est la reponse immunitaire innee. le via perturbe les voies metaboliques des cellules infectees, notamment la macroautophagie. la macroautophagie, par la suite appelee simplement autophagie, est une voie du catabolisme cellulaire. l'autophagie est constitutive dans les cellules nucleees et participe au maintien de l'homeostasie cellulaire. en reponse a un stress cellulaire, l'autophagie peut etre stimulee. un grand nombre de virus perturbe l'autophagie, soit en la stimulant, soit en l'inhibant. le via induit l'autophagie mais inhibe sa phase finale, un mecanisme impliquant sa proteine de matrice 2 (m2). les impacts de cette perturbation de l'autophagie sur la replication virale et sur la reponse de la cellule hote sont encore peu compris. au cours de ma these, j'ai developpe des modeles cellulaires dans lesquels la capacite d'autophagie peut etre specifiquement restauree dans des lignees cellulaires autrement incapables d'autophagie. l'utilisation de ces modeles m'a permis de montrer que l'autophagie ne change ni l'infectiosite du virus ni si capacite de replication intracellulaire mais inhibe l'induction de l'interferon-β et des genes induits par celui-ci. j'ai mis en evidence que m2 n'inhibe la phase finale de l'autophagie que dans le cadre de l'infection et de l'activation de l'apoptose. une meilleure comprehension des perturbations de l'autophagie pourrait permettre de developper des molecules antivirales et de nouveau virus attenues induisant une plus forte reponse immunitaire. / Influenza a virus (iav) is responsible for yearly epidemics and sporadic pandemics. understanding the mechanism by which the inflammatory response is mounted and controlled is key to manage the disease. iav perturbs a variety of metabolic pathways including macroautophagy. macroautophagy, hereafter referred to as autophagy, is a catabolic pathway that is active in all nucleated cells. in stress condition, autophagic activity can be increased. a variety of viruses perturb autophagy. iav has been described to both induce autophagy and block its completion mainly through its matrix protein 2 (m2). however, the impact of such perturbation on viral replication and host cell response to infection is still unknown. i developed cellular models in which autophagy capacity can be specifically restored in cell lines that are otherwise autophagy-incompetent. using these models, i showed that autophagy does not impact iav infection and replication but inhibits interferon-β induction at early stages post infection, leading to dampened induction of interferon-stimulated genes. i showed that m2 does not prevent autophagy completion by itself but only in the context of iav in a caspase-activation dependent fashion. in summary, my thesis work, using these novel autophagy models, revealed that early autophagy induction post-iav infection inhibits ifn-β, leading to a global decrease in interferon stimulated gene expression. indeed, sustained autophagy perturbation through m2 may allow iav to limit the ifn-β response throughout its life cycle. preventing m2-mediated autophagy perturbation may allow us to develop new antiviral strategies as well as new live attenuated iav vaccines.

Doctor of Philosophy / Department of Anatomy and Physiology / Deryl L. Troyer / Umbilical cord matrix stem (UCMS) cells are derived from Wharton's jelly and have been shown to express genes characteristic of primitive stem cells. They can be isolated in large numbers in a short time and thus potentially represent an abundant source of cells for therapeutic use. We investigated the migratory nature of human UCMS cells towards MDA 231 human breast carcinoma cells in an in vitro model of cell migration; UCMS cells cultured with or without MDA 231 cells for 24 hours. Next, we evaluated the effect of chemokines, stromal derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) on human UCMS cells by treating with increasing doses of SDF-1 and VEGF. UCMS cells were found to migrate towards MDA 231 cells in a dose dependent manner. Both SDF-1 and VEGF induced migration of UCMS cells in a dose dependent manner. These results suggest that MDA 231 cells might be releasing chemokine factors, such as SDF-1 and VEGF, which promote UCMS cell migration towards the tumor cells in vitro. Stem cells that migrate to tumors may allow targeted delivery of therapeutic agents that otherwise may have severe side effects. To evaluate the selective engraftment and therapeutic efficiency of human UCMS cells that were engineered to express interferon beta (UCMS-IFN-beta) MDA 231 cells (2,000,000) were intravenously injected into severe combined immune deficient (SCID) mice, followed by three weekly intravenous injections of fluorescently labeled UCMS-IFN-beta cells (500,000). To evaluate the synergistic effect of 5-Fluorouracil (5-FU) and IFN-beta, MDA 231 cells were intravenously injected into SCID mice, followed by three weekly intravenous injections of fluorescently labeled UCMS-IFN-beta cells and three weekly intra peritoneal injections of 5-FU. In both of the above experiments, mice were euthanized one week after the last UCMS cell transplant and lung weights were compared to the controls to determine the differences in tumor burden. After transplantation of UCMS-IFN-beta cells into MDA 231 tumor-bearing mice, UCMS cells were found near or within metastatic lung tumors but not in other tissues, and in these animals, the lung weight was significantly less than MDA 231 tumor-bearing animals that received saline injections. Histologically, there was significant reduction in the tumor area in MDA 231 tumor bearing lungs after UCMS-IFN-beta treatment. When 5-FU was given along with UCMS-IFN-beta cells, there was further reduction in tumor area. These results indicate that UCMS cells can potentially be used for targeted delivery of cancer therapeutics.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Breast cancer (BC) is one of the most common malignancies in women. Death results from treatment failure and metastatic disease. Thousands of lives might be saved if it was possible to detect and eliminate occult metastatic cells before they become clinically evident. Therefore, there is a critical need to identify new markers to improve treatment options for these patients. Genomic instability is the earliest indication of breast cancer and the use of genomic methodologies is a progress towards early detection and treatment, through the identification of biomarkers that can be translated into novel therapy targets. The interferon regulatory factor-1(IRF-1) gene, localized on chromosome 5q31.1, is believed to act as a tumor suppressor gene in breast cancer. The IRF-1 was found to be inactivated by single nucleotide polymorphism (SNP) in breast cancer suggesting that the loss of its function might be critical to the development of the disease. The phosphatidylinositol 3-kinase (PIK3) signaling pathway mediates key cellular functions and alterations of genes in this pathway, including PIK3CA, serine-threonine protein kinases (AKT1and AKT2), phosphatase and tensin homolog (PTEN), fibroblast growth factor receptor 2 (FGFR2) and ERBB2, whose expression have been demonstrated to be altered in breast cancer patients. In addition, these genes are linked to treatment resistance. vi In this study, we have investigated allelic loss of IRF-1 gene in primary tumors obtained from patients undergoing mastectomy at Groote Schuur hospital (Cape Town, South Africa). These samples were then further analyzed for the DNA copy number changes of specific genes involved in the PIK3/AKT signaling pathway. Statistical analysis has been performed in order to correlate genomic findings with clinical-histopathological and follow up information from the patients and to establish whether these genes can predict prognosis. Our data analysis has indicated that 46 cases (45.5%) out of 101 cases were informative for the IRF-1 dinucleotide marker used for LOH analysis (Figure 3.1). LOH was detected in 23 of these informative cases (23/46; 50%). No statistical significance was found between LOH at the IRF-1 locus and age (≤50 years or >50 years) (P value = 1.0000) and earlier stage (Stages I and II) (P value= 0.4982) based on Fisher’s exact test. Patients presented a high level of DNA copy number changes in genes involved in the PIK3/AKT pathway. The most frequent changes were observed in the PIK3CA and PTEN genes. PIK3CA presented high copy number in 36.8% of the cases. PTEN was observed with low copy number in 47.5% of the cases. This dissertation shows the effectiveness of genomic methodologies as means for the detection of early breast cancer progression in South African women. The PIK 3/AKT genes can validate the usefulness of breast cancer therapies.

Macrophages are professional phagocytes that efficiently clear microbes, dying cells, and debris. Nonetheless, some pathogenic bacteria and parasites can subvert the macrophage phagosome into a vacuolar replicative niche. Exogenous macrophage activation by the cytokine interferon gamma (IFNγ) tips the equilibrium toward pathogen restriction, host survival, and subsequent adaptive immune responses. The relevance of IFNγ-induced immunity to human health has been demonstrated in patients with genetic defects in IFNγ signaling, who are profoundly susceptible to vacuolar pathogens such as Mycobacterium tuberculosis. Still, much remains to be discovered about IFNγ effector functions, and about their co-regulation by signaling downstream of the many innate immune sensors in macrophages. First, we asked whether IFNγ-induced vesicle trafficking mechanisms affect the maturation of phagosomes containing the bacterium Legionella pneumophila, the causative agent of Legionnaire's disease. We used functional genetic screening to discover candidate genes involved. From 380 genes in a curated vesicle trafficking-related set, 15 were selected as candidate IFNγ pathway members by RNAi screening in cell line and primary mouse macrophages. Functional validation of top candidates was inconclusive, but revealed potential roles for membrane tetraspanins and the AP3 complex in IFNγ-induced microbial restriction. Our second goal was to determine whether innate immune sensing affects IFNγ-induced bacterial restriction. Using macrophages from mice deficient in key elements of innate immune sensing pathways, we discovered that the antiviral transcription factor IRF3, which functions downstream of many nucleic acid sensing pathways, suppresses IFNγ-induced restriction of L. pneumophila and the protozoan parasite Trypanosoma cruzi. While activated IRF3 localizes to the nuclei in resting macrophages infected with L. pneumophila, it is mostly excluded from nuclei in macrophages activated with IFNγ prior to infection. This suggests a cascade of suppression in which IFNγ responses inhibit IRF3 activation, but residual IRF3 activity antagonizes IFNγ effectors. IRF3-mediated inhibition of IFNγ-inducible nitric oxide synthase was partially, but incompletely responsible for the phenotype observed; further candidate effectors were identified by gene expression profiling. We speculate that antagonism between IFNγ and IRF3-mediated mechanisms may facilitate a balance of vacuolar pathogen immunity with viral defense, or with protection of tissue damage by nitric oxide and other IFNγ-dependent responses.

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

Influenza A virus (IAV) can evolve from low virulence in animal hosts to become highly virulent in humans. Pandemic Influenza A viruses such as the 1918 Spanish Influenza caused over 50 million deaths worldwide. However the genetic determinants of IAV host adaptation and virulence are largely uncharacterized. The IAV NS1 protein is a multifunctional interferon antagonist and a known virulence factor. We hypothesized that NS1 mutations selected upon IAV evolution to a novel host contribute to host adaptation by mechanisms involving increased gene expression and IFN antagonism. To this end, I phenotypically characterized the NS1 mutations selected upon adaptation of A/Hong Kong/1/1968 (H3N2) (HK-wt) to increased virulence in the mouse. Sequencing the NS genome segment of mouse-adapted variants revealed eleven mutations in the NS1 gene and four in the overlapping NEP gene. Using the HK-wt virus and reverse genetics to express recombinant HK NS1 mutant viruses, I demonstrated that all NS1 mutations were adaptive and enhanced virus replication (up to 100 fold) in mouse cells and/or lungs. All but one NS1 mutant was associated with increased virulence measured by survival and weight loss in the mouse. Ten of twelve NS1 mutants significantly enhanced IFN-β antagonism to reduce the level of IFN-β production relative to HK-wt in infected mouse lungs at 1 day post infection, where nine mutants induced viral yields in the lung that were ≥ HK-wt (up to 16 fold increase). Eight of 12 NS1 mutants had decreased binding affinity to the cleavage and polyadenylation specificity factor (CPSF30). The majority of mutant NS1 genes demonstrated increased viral polymerase activity and viral protein production in mouse cells. Viral protein production and viral growth were also assessed in human and canine cell lines; however these adaptive phenotypes were more robust in infected mouse cells. Adaptive NS1 mutations also increased cytoplasmic cellular localization of the NS1 protein in infected cells in a host cell-specific manner. Evaluation of phenotypic trends associated with the NS1 mutants demonstrated an inverse correlation between CPSF30 binding affinity and viral polymerase activity enhancement. This study demonstrates that NS1 is a multifunctional virulence factor subject to adaptive evolution.

Microglia activity has been detected in Parkinson’s disease (PD) post-mortem brains and experimental animal models; however the precise interplay between microglia and dopamine neurons of the SNpc is not well understood. In the blood plasma of PD patients, our laboratory found elevated levels of interferon-gamma (IFN-γ), a proinflammatory cytokine and potent activator of microglia. Given this, we sought to untangle the immune responses relevant to PD in mice, examining IFN-γ’s involvement and signaling mechanism using an inflammatory co-culture model of microglia and midbrain neurons treated with rotenone. By means of RT-PCR, we discovered IFN-γ mRNA transcripts are produced by microglia, and this expression increases upon exposure to rotenone. We delineated IFN-γ’s signaling mechanism in co-cultures using different IFN-γ receptor deficient cells, and showed it engages receptors in an autocrine (not paracrine) manner to further microgliosis and dopamine cell loss. After exploring the innate immune response in a model of PD, we subsequently shifted focus to an in vivo system to better investigate any involvement of the delayed humoral arm of the adaptive immune system. Needing a time appropriate death paradigm, we developed a protracted low dose regimen of MPTP, which elicits dopaminergic cell death after 2 weeks of treatment. Subjected to this paradigm, Rag 2 mutant mice (deficient in both T and B cells) exhibit resistance to dopamine cell loss, microglia activation and motor impairments. Further evidence in support of immune involvement came with the resensitization of Rag2 mice to MPTP after reconstitution with WT splenocytes. Additionally, mice deficient in Fcγ receptors exhibited neuroprotection in our protracted degeneration model. Taken together, these data indicate the innate and humoral arm can modulate the microglial response to dopaminergic degeneration and may participate in Parkinson's disease.

The data presented within this thesis shows the generation and characterisation of a complete-, macrophage/granulocyte- and T cell-specific IFNγR2 deficient mouse mutant. This mutant mouse is a valuable tool in dissecting the mechanism of action of the pleiotrophic cytokine IFNγ.The global mutant mouse was tested in three models in vivo - DSS induced colitis, Trichuris muris infection and EAE. The aim of the DSS-induced colitis model was to test the role of IFNγ in the innate immune system and, despite previous reports demonstrating IFNγ deficient mice are protected from DSS-colitis, our IFNγR2 deficient mice displayed equal or more severe colitis than control mice. We hypothesise that this discrepancy is due to differences in the gut microbiota.The Trichuris muris model was utilised as a method of examining the role of IFNγ in the adaptive immune system. The complete IFNγR2 mutant was resistant to a low dose T. muris infection; however, neither the T cell specific nor the macrophage/granulocyte specific mutant duplicated the resistant phenotype observed in the global knock-out mice. Analysis of a double conditional T cell and macrophage/granulocyte specific IFNγR2 mutant produced inconsistent results. Initial experiments suggested that, in combination, these deficiencies are sufficient to duplicate the resistant phenotype observed in the global mutant mice, but this was not reproducible.The final in vivo model that we used to analyse IFNγR2 mutant mice was EAE. This model was chosen as, for a long time, the mechanism of action and the involvement of IFNγ in EAE has been a matter of uncertainty. These results demonstrated that global IFNγR2 mutant mice demonstrate an atypical phenotype, with no signs of recovery. In contrast, control mice develop classical EAE symptoms with almost complete recovery prior to the termination of the experiment. The IFNγ receptor mutant mouse generated will be of great value to the scientific community as IFNγ has been demonstrated to play a role in multiple diseases and this tool allows the mechanism of action of this cytokine to be unravelled.

No description available.

<div> <div> <p>Purpose: Preclinical studies have demonstrated that cancer cells may produce innate immune signals such as type-I interferons following radiation damage, which derives from activation of the cGAS-STING pathway following detection of cytosolic dsDNA. Limited studies have explored how these mechanisms vary from the conditions of the radiation exposure. High- linear energy transfer (LET) radiation induces more DNA double-strand breaks (DSB) per dose than low-LET radiation, thus is expected to be more immunogenic. However, DNA damage in hypoxic cells is more probable to undergo chemical repair due to limitations in oxygen fixation, thus is expected to be more immunosuppressive. Our goal is to study and model the dose response characteristics of IFNβ and Trex1 in vitro following exposure of radiations with varying LET and to develop techniques for further study in vivo.<br></p><p><br></p> <p>Methods: Reference data from Vanpouille-Box (2017) on STING dose response was applied to develop empirical models of cytosolic dsDNA and Trex1 regulation as a function of dose and quantity of DNA DSB, the latter of which is dependent on particle LET and oxygenation and is calculated using Monte Carlo Damage Simulation (MCDS) software. These models were used as preliminary data to guide in vitro experiments using Merkel cell carcinoma cells. The dose response of pro-inflammatory IFNβ and exonuclease Trex1, an anti-inflammatory suppressor of cGAS-STING, was measured post-irradiation. MCDS was again used to model fast neutron relative biological effectiveness for DSB induction (RBEDSB) and compared to laboratory measurements of the RBE for IFNβ production (RBEIFNβ). RBEIFNβ models were applied to radiation transport simulations to quantify the potential secretion of IFNβ in representative clinical beams. To enable intra-tumor radiation targeting of tumor hypoxia, mice were seeded with syngeneic tumors and imaged longitudinally with PCT- spectroscopy to determine local variations hemoglobin concentration (Hb) and oxygen saturation (SaO2) over time. Hypoxia classification was based on SaO2 levels in voxels containing hemoglobin relative to a “hypoxia threshold” of SaO2 < 0.2.</p><p><br></p> <p>Results: Based on analysis of published data, our preliminary models of cytosolic DNA and Trex1 dose responses demonstrate dose enhancements from high-LET radiation, such as that at the distal edge of a Bragg peak, and suppression from cellular hypoxia. This manifests as an RBE-dependent ‘shift’ in STING response. Laboratory measurements in MCC13 cells show peak IFNβ production at 6.1 Gy following fast neutron irradiation and 14.5 Gy following x-rays (RBEIFNβ = 2.4). However, IFNβ signal amplitudes were not significantly different between these radiation types. Trex1 signal increased linearly with dose, with fourfold higher upregulation per dose for fast neutrons. Modeling of RBE in clinical beams suggests that ion sources may induce spatially localized IFNβ near their end of range, which is potentially advantageous for initiation of tumor-specific immune activity. Uncharged sources stimulate IFNβ more uniformly with depth. Longitudinal PCT-S scanning is able to localize and distinguish chronic and acute hypoxia in vivo. Changes in the hypoxic classification from tumor growth and following anti-angiogenic therapy are distinguishable.<br> </p><p> </p><div> <div> <div> <p>Conclusion: Radiation-induced immunogenicity can be induced differentially based on radiation quality and is expected to be affected by cellular oxygenation. High-LET radiation, such as fast neutrons, drives greater IFNβ innate immune response per dose than low-LET radiation, such as x-rays, which may enhance abscopal effects when used in combination with immune-stimulating agents. However, anti-inflammatory signaling is greater per dose for fast neutrons, and it remains unclear if high-LET radiations are therapeutically advantageous over low-LET radiation for pro-inflammatory tumor signaling. High resolution in vivo imaging of tumor hypoxia is feasible with photoacoustic techniques, which can potentially be leveraged to study selective immunogenicity enhancement of the hypoxic niche following radiation therapy. <br></p> </div> </div> </div> <p> </p> </div></div>