<p>Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. We developed a novel multiplexed method for SNP genotyping based on four-color fluorophore tag-microarray minisequencing. This method allows simultaneous genotyping of 80 samples and up to 200 SNPs in any allele combination. In study I we set up the method for a panel of SNPs from genes in the type I interferon system, and applied it in study III. In study II we used the technique to genotype SNPs from the coding region of the mitochondrial genome. A panel of 150 SNPs was genotyped in 265 individuals representing nine different populations. We demonstrated that the multiplexed SNP genotyping method for mitochondrial DNA increases the power of forensic identification in combination with sequencing of the hypervariable region of mitochondrial DNA. </p><p>In study III we performed a genetic association study of SNPs in genes related to the type I Interferon system in Systemic Lupus Erythematosus (SLE). SLE is a chronic autoimmune inflammatory disease with a complex etiology. The SNPs were genotyped in DNA samples from Swedish, Finnish, and Icelandic patients with SLE, unaffected family members, and unrelated controls. The analysis identified SNPs in two genes, the tyrosine kinase 2 (TYK2) and interferon regulatory factor 5 (IRF5) genes that are highly associated with SLE with p-values <10^-7 for joint linkage and association. </p><p>Study IV describes the analysis of the TYK2 and IRF5 SNPs in a large Rheumatoid Arthritis (RA) sample cohort. We found that SNPs in the IRF5 gene were significantly associated with RA with a p-value = 0.00008. In contrast, we did not detect an association with SNPs in the TYK2 gene. These findings demonstrate that SLE and RA may have a common genetic background in the case of IRF5, while the TYK2 variants appear to be unique for SLE. </p>

<p>The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc.</p><p>Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies.</p><p>Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-<i>myc</i> amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. </p><p>In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.</p>

The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc. Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies. Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-myc amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. We developed a novel multiplexed method for SNP genotyping based on four-color fluorophore tag-microarray minisequencing. This method allows simultaneous genotyping of 80 samples and up to 200 SNPs in any allele combination. In study I we set up the method for a panel of SNPs from genes in the type I interferon system, and applied it in study III. In study II we used the technique to genotype SNPs from the coding region of the mitochondrial genome. A panel of 150 SNPs was genotyped in 265 individuals representing nine different populations. We demonstrated that the multiplexed SNP genotyping method for mitochondrial DNA increases the power of forensic identification in combination with sequencing of the hypervariable region of mitochondrial DNA. In study III we performed a genetic association study of SNPs in genes related to the type I Interferon system in Systemic Lupus Erythematosus (SLE). SLE is a chronic autoimmune inflammatory disease with a complex etiology. The SNPs were genotyped in DNA samples from Swedish, Finnish, and Icelandic patients with SLE, unaffected family members, and unrelated controls. The analysis identified SNPs in two genes, the tyrosine kinase 2 (TYK2) and interferon regulatory factor 5 (IRF5) genes that are highly associated with SLE with p-values &lt;10-7 for joint linkage and association. Study IV describes the analysis of the TYK2 and IRF5 SNPs in a large Rheumatoid Arthritis (RA) sample cohort. We found that SNPs in the IRF5 gene were significantly associated with RA with a p-value = 0.00008. In contrast, we did not detect an association with SNPs in the TYK2 gene. These findings demonstrate that SLE and RA may have a common genetic background in the case of IRF5, while the TYK2 variants appear to be unique for SLE.

Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used. Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting. Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades. Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.

Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used. Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting. Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades. Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.

<p>Vaccinia virus (VV) is the most thoroughly studied member of the Poxviridae family and the vaccine used to achieve the only successful eradication of a human disease. Over the years, it has proven itself as a useful tool for the study of antiviral immunity, vaccine development, and potentially cancer immunotherapy. VV is capable of eliciting a robust immune response; however the mechanisms by which VV accomplishes this task remain unknown. The overall goal of this thesis project is to determine how VV activates the innate immune system, and how this activation contributes to viral clearance in vivo. We determined that VV or VV-DNA activated the TLR8-MyD88 pathway in plasmacytoid dendritic cells (pDC), resulting in the production of type I interferons (IFN). We also demonstrated that TLR8-mediated production of type I IFN by pDC was crucial to efficient VV control and clearance in vivo. Moreover, we identified the polyA- and polyT-rich sequences in VV-DNA was the possible motif recognize by TLR8. Type I IFN, known for ability to establish the "antiviral state", are also critical mediators of NK cell activation. In the setting of VV infection, we demonstrated that direct action of type I IFN on NK cells, but not accessory cells such as DC, was necessary for NK cell activation in vivo. We further demonstrated that type I IFN-dependent activation of NK cells was required for optimal VV clearance in vivo. Given the importance of NK cells in anti-VV innate immunity, we next examined what role the TLR2-MyD88 pathway, critical for activation of cDC, played in the activation of NK cells. NK cells from TLR2-/- or MyD88-/- mice displayed a reduction in activation and cytolytic function, and this defect was independent of pro-inflammatory cytokine signaling. We were able to demonstrate that direct TLR2 signaling on NK cells was required for their optimal activation and function in response to VV infection. Moreover, we were able to demonstrate that TLR2-MyD88 signaling resulted in the activation of the PI3K-ERK pathway, which was necessary for NK cell cytotoxicity. In addition, we identified the NKG2D pathway as critical for efficient NK cell activation and function in response to VV infection, independent of the TLR2 pathway. Both the NKG2D and TLR2 pathways were crucial for optimal VV clearance and control in vivo. Collectively, this project illuminates the roles and mechanisms of the innate immune system in the control of VV in vivo.</p> / Dissertation

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology, Medical Microbiology and Immunology. Title from pdf file main screen (viewed on January 10, 2010). Includes bibliographical references.

Interferon regulatory factor-8 (Irf-8), a hematopoietic transcriptional regulator, controls myeloid-cell proliferation and coordinates innate and adaptive host immune responses. Mice from the BXH-2 recombinant inbred strain carry an endogenous R294C mutation in Irf-8. This loss-of-function mutation induces clonal infiltration of undifferentiated Mac-1+/Gr-1 + granulocytic precursors in BXH-2 mice, extramedullary hematopoiesis, and splenomegaly similar to those seen in human chronic myeloid leukemia. It also renders the host permissible to the otherwise avirulent Mycobacterium bovis (BCG), and negatively affects survival or recovery of these mice to other infectious pathogens. Here, we generated a polyc1onal anti-Irf-8 antibody to better characterize the effects of the R294C mutation on Irf-8 protein expression, stability, and inducibility in hematopoietic and non-hematopoietic tissues. We found that mutant Irf-8C294-expressing tissues consistently displayed reduced Irf-8 abundance compared to their wild-type counterparts in both primary splenocytes and following transfection into heterologous cells, presumably due to decreased stability or increased rate of degradation of the mutant isoform. Results also indicate that native Irf-8 is also expressed in the heart, and to a lesser extent, in the kidneys. Since neither of these organs is well-known to be associated with hematopoietic or immune functions, this finding strengthens the possibility that Irf-8 may exert additional regulatory functions in other cellular contexts. Taken together, our study provides a better understanding about the molecular features of the mutant Irf-8 C294 protein and contributes to a growing body of evidence in support of Irf-8 expression in non-hematopoietic tissues.

Abbreviations ALT – alanine aminotransferase AST – aspartate aminotransferase BMI – body mass index CHC – chronic hepatitis C EBR – early biochemical response EHR – early histological response EIA – enzyme immunoassay EVR – early virological response HAI – hepatitis activity index HCV – hepatitis C virus HCV RNA - hepatitis C virus ribonucleic acid Helicobacter spp. – Helicobacter species H. pylory – Helicobacter pylori IFN – interferon α-2b PCR – polymerase chain reaction PEG IFN – peginterferon RBV – ribavirin SVR – sustained virological response SBR – Sustained biochemical response INTRODUCTION Chronic hepatitis due to hepatitis C virus (HCV) infection is a worldwide disease representing a serious public health problem. Chronic hepatitis C (CHC) infection affects nearly 170-200 million people worldwide. The prevalence of anti-HCV at this time in the general adult population of Lithuania is nearly 50 thousand (0.9%). Without effective treatment strategies, hepatitis C - related morbidity and mortality is expected to increase nearly 3-fold by the year 2015. Current hepatitis C therapies are aimed at achieving eradication of HCV infection as a means of delaying progression to end-stage liver disease and preventing the development of hepatocellular carcinoma. The treatment options include interferon (IFN), ribavirin (RBV) and peg interferon’s a-2a and a-2b (PEG IFN). IFN-based regiments for the treatment of CHC have become increasingly effective and are to eradicate virus... [to full text]