Browsing by Subject "Comparative and Molecular Biosciences"
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Item Development of the larval zebrafish as a genetic model for the nicotine response.(2010-08) Petzold, Andrew MichaelTobacco use is predicted to result in over 1 billion deaths worldwide by the end of the 21st century. How genetic variation contributes to the observed differential predisposition in the human population to drug dependence is unknown. The zebrafish (Danio rerio) is an emerging vertebrate model system for understanding the genetics of behavior. We developed a nicotine behavioral assay in zebrafish and applied it in a forward genetic screen using gene-breaking transposon mutagenesis. We used this method to molecularly characterize bdav/ cct8 and hbog/gabbr1.2 as mutations with altered nicotine response. Each have a single human ortholog, identifying two points for potential scientific, diagnostic, and drug development for nicotine biology and cessation therapeutics. We show this insertional method generates mutant alleles that are reversible through Cre-mediated recombination, representing a conditional mutation system for the zebrafish. Additionally, we developed a conditioned place preference assay for use with larval zebrafish. This assay allows for the perturbation of the differences in genetic function between the physiological and learned response representing one of the first associative learning based assays in the larval zebrafish. The combination of this reporter-tagged insertional mutagen approach and zebrafish provides a powerful platform for a rich array of questions amenable to genetic-based scientific inquiry, including the basis of behavior, epigenetics, plasticity, stress, memory, and learning.Item Early localized SIV-specific CD8 T cells response to pathogenic SIV is correlated with successful SIV vaccine.(2010-01) Hong, Jung JooLive-attenuated lentivirus vaccines are the most effective in inducing protection against subsequent challenge with pathogenic lentiviruses. For example, infection with live-attenuated simian immunodeficiency viruses (SIVs) including nonpathogenic simian-human immunodeficiency virus (SHIV) 89.6, and SIV∆nef in macaques provides protection against subsequent challenge with highly pathogenic strains of SIV. The mechanism by which live-attenuated SIVs induce protection is not well understood. My central hypothesis is that an early SIV-specific CD8 T cell response in lymph nodes and at the site of infection is responsible for the protection induced from live-attenuated SIV vaccines. The rationale for this hypothesis stems from our previous studies showing that SIV disseminates throughout the body by one week post-infection but SIV-specific CD8 T cell responses are too late and not detected in situ until the second and third weeks post-infection. For the present study, rhesus macaques were immunized intravenously with live non-pathogenic SHIV89.6, or SIV∆nef and then challenged intravaginally with pathogenic SIVmac239, or SIVmac251. Using the experimental approach of in situ tetramer staining combined with immunohistochemistry, confocal microscopy and quantitative image analyses, I determined: 1) localization and quantification of virus-specific CD8 T cells; 2) phenotypic changes in lytic granule contents of virus-specific CD8+ T cells; and 3) virus-specific CD8 T cells interacting with CD83+cells in tissues from these animals. My main results show that an early but not necessarily robust SIV-specific CD8 T cell response localized to lymph nodes and genital tissues correlated with protection in these animals. In addition, the majority of SIV-specific CD8 T cells that I observed in vaccinated animals showed little to no perforin expression, compared to cells from non-vaccinated and pathogenic SIV-infected animals. Lastly, I also observed an increase in the interaction of vaccine-induced SIV-specific T cells with CD83+ dendritic cells after pathogenic SIV challenge in immunized animals. These results indicate that vaccine-induced protection correlates with early localized SIV-specific CD8 T cells that show little to no perforin expression at the portal of viral entry and lymph nodes. Additionally my studies indicate that vaccine-induced protection correlates with increased interactions of SIV-specific CD8 T cells with mature dendritic cells. These studies provide a better understanding of immune correlates involved in the protection afforded by live-attenuated SIV vaccines, and provide insights into what is needed to create a successful HIV vaccine.Item Expression profiling of the in vitro infection of cryptosporidium.(2011-10) Mauzy, Mary JeanCryptosporidiosis a global health concern in large part because the causative organism, Cryptosporidium , is a ubiquitous water contaminant throughout the planet. Transcriptional analysis of the parasite has been limited to the pre-attachment stage, the sporozoite. This research describes the transcriptome of the attached C. parvum over a 72 hr in vitro infection utilizing real time PCR. These data provide direct insight into the parasite's biochemical requirements over its developmental life cycle. The parasite gene transcription is dependent upon the developmental stage present and stage specific gene predictions were deduced. Furthermore, a comparison of the gene transcription between C. parvum and C. hominis in duplicated infection protocols indicates that although these two species are almost identical at the nucleotide level, phosphorylation related genes are not expressed at concurrent time points. Additional promoter analysis indicates that genes with differing expression between these two species are more likely to contain a gap in the upstream sequence.Item Functional Genomics of Round Heart (RH) Disease in the Turkey(2009-12) Mendoza, Kristelle MayItem Histological assessment of osteoarthritis lesions in mice.(2010-05) McNulty, Margaret AnnThe pathogenesis of osteoarthritis (OA) in both humans and animals is poorly understood. Histological assessment of tissue samples from joints is the most accurate way to diagnose OA severity. Unfortunately, current histological assessment grading schemes, such as the Mankin Histological-Histochemical Grading System (HHGS), are subject to observer variability and are unable to accurately distinguish varying levels of disease severity. The goals of this project were to: 1) develop a novel histological grading scheme to assess OA lesions in mice, 2) apply this scheme to several studies in which OA severity is expected to be influenced by treatment, and 3) compare the reliability and validity of this newly developed scheme to the widely used Mankin HHGS. Histological sections from the knee (stifle) joints from 5 studies (n=158 joints), including 2 studies using a surgically induced model and 3 studies of naturally occurring disease, were globally examined for changes associated with OA. Changes that were consistent were evaluated using histomorphometry or assigned a grade based on severity. The final scheme that was developed included 13 quantitative and 2 semi-quantitative parameters that evaluated changes in articular cartilage, chondrocytes, subchondral & periarticular bone, and meniscus. Principal Components Analysis combined these 15 parameters into 5 factors that globally evaluated changes in articular cartilage integrity, chondrocyte viability, subchondral bone, periarticular osteophytes, and menisci. This newly developed scheme was then applied to five studies of murine OA, and comparisons among intervention groups in the 15 parameters and 5 factors were evaluated. In addition, the original Mankin HHGS was applied to these same sections. The newly developed scheme identified numerous changes in several tissues in both surgically induced and naturally occurring disease and was able to characterize several changes associated with OA severity in the mouse. While the Mankin HHGS was able to identify significant differences in the surgically induced models, it overlooked changes associated with naturally occurring disease. Finally, a direct comparison between the two schemes revealed that the newly developed scheme was more reliable than the Mankin HHGS, and was also able to correctly distinguish the severities of OA as well as the Mankin HHGS.Item The major histocompatibility complex of the turkey.(2010-05) Chaves, Lee D.The ability to identify between self and foreign pathogens is a key function of the vertebrate immune system. To achieve this, vertebrates have developed complex genetic and epigenetic mechanisms to provide sufficient variability to respond to continuously evolving pathogens or transformed cells they face. One of these systems is the large and highly polymorphic major histocompatibility complex (MHC), responsible for the presentation of endogenous and exogenous peptide antigens to T cells. The degree of polymorphism, number of gene copies, and the co-dominant expression of genes of the mammalian MHC allows for a great number possible antigens to be presented. Compared to mammals, the chicken MHC (MHC-B) is greatly condensed, containing the major histocompatibility antigens in a region of just 50 kb with a reduction in gene copy numbers and a lack of co-dominant expression. In addition to the MHC-B, the chicken has a second MHC-like region (MHC-Y) located on the same microchromosome as B yet it is genetically unlinked. The work presented in this dissertation physically and genetically maps the homologous MHC regions in the turkey and identifies genes within these regions. Further work surveyed the polymorphism content within commercial and wild turkeys. A final study assayed the genome-wide diversity of the individual from which the MHC was sequenced to determine the appropriateness of this source DNA for whole genome sequencing. These data provide helpful background information to advance turkey whole genome sequencing and develops genomic resources for the study of the effect of MHC alleles on the outcome of pathogenic infections in the turkeyItem Regulation of airway epithelial cell migration by the cystic fibrosis transmembrane conductance regulator.(2010-05) Schiller, Katherine RebeccaThe airway epithelium is a critical barrier between the noxious particles in inspired air and the lung tissue. Mucociliary clearance is an essential function of the airway epithelium to protect against infection and airway damage. By this process, inhaled particles are trapped in the mucus lining the airway and propelled out of the airways by ciliary movement. Transport of Cl- and Na+ ions controls fluid secretion in the airways and the depth and viscosity of the periciliary liquid layer essential for cilia movement. The ion channel cystic fibrosis transmembrane conductance regulator (CFTR), transports Cl- and HCO3 - in the airways and other tissues, and controls the depth of the periciliary liquid layer. Cystic fibrosis is a fatal genetic disease in which CFTR is dysfunctional. As a result, mucociliary clearance is impaired and airways become chronically infected with microorganisms. Microorganism colonization results in recurrent inflammation and cycles of damage to the epithelial barrier followed by wound repair, eventually resulting in airway remodeling and ultimately respiratory failure. It has recently been demonstrated that the loss of CFTR function impairs epithelial restitution in the absence of infection. This suggests the CFTR plays a direct role in airway wound repair. To test this hypothesis, the role of CFTR in the initial step of epithelial wound repair-cell migration- was assessed. An impedance based assay was used to objectively measure cell migration rates of human bronchial epithelial cells. Inhibition of CFTR transport and silencing of CFTR protein expression were used to assess the effect of the loss of functional CFTR on cell migration rate. The migration rate of an airway epithelial cell line isolated from a cystic fibrosis patient was also compared to that of an airway epithelial cell line isolated from a healthy lung. A functional effect of CFTR on the process of lamellipodia protrusion during cell migration was also assessed morphometrically. Time lapse video images were also captured during wound closure of airway epithelial cells to assess how cell migration occurs. Additionally, the dependence of airway epithelial cell migration on Cl- and HCO3 - transport was assessed using ion substitution and selective inhibitors of CFTR and other transporters of these ions. Further, the effect of changes in extracellular pH on migration rate was assessed. The results of these studies revealed a role for CFTR transport of Cl- and HCO3 - to regulate airway epithelial cell migration. These studies contribute to the basic understanding of the repair process of the airways following damage and may potentially be important for the design of new treatments to limit airway damage in cystic fibrosis.Item Regulation of CD38 expression in human airway smooth muscle (HASM) cells.(2009-05) Jude, Joseph AntonyCD38 is a multifunctional enzyme-cum-receptor expressed in a variety of mammalian tissues including airway smooth muscles (ASM). The ADP-ribosyl cyclase activity of CD38 generates cyclic ADP-ribose (cADPR), a Ca2+ mobilizing agent in ASM cells. In vivo studies in mice showed that CD38 plays an important role in the development of airway hyperresponsiveness (AHR) following exposure to cytokines. In vitro studies demonstrated that a variety of cytokines, including the inflammatory cytokine TNF-alpha, induce CD38 expression in human ASM (HASM) cells. Studies also showed that the TNF-alpha-induced CD38 expression in HASM cells is mediated through both the transcriptional and post-transcriptional mechanisms and involves activation of mitogen-activated protein kinases (MAPKs) and transcription factors NF-kappaB and AP-1. The role of CD38 in the pathogenesis of AHR in human asthmatics is not known. The current studies demonstrate that HASM cells isolated from asthmatic patients show differentially elevated CD38 expression following TNF-alpha exposure compared to non-asthmatic HASM cells. Basal and TNF-alpha-induced activation of extracellular signal- regulated kinase (ERK) and p38 MAPK are elevated in asthmatic HASM cells, whereas the TNF-alpha-induced activation of c-jun N terminal kinase (JNK) is elevated in non-asthmatic HASM cells compared to asthmatic cells. The TNF-alpha-induced NF-kappaB activation is elevated in asthmatic HASM cells, indicating that the differentially elevated CD38 expression in asthmatic HASM cells is mediated through transcriptional mechanisms. The role of cross-talk between ERK and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathways in TNF-alpha-induced CD38 expression is investigated in the current studies. Two pharmacological inhibitors of PI3K, LY294002 and wortmannin, show differential effects on the TNF-alpha-induced CD38 expression in HASM cells. Transient expression of PI3K catalytic subunit (p110) elevates CD38 expression while transfection with phosphatase tensin homolog (PTEN) attenuates the TNF-alpha-induced CD38 expression, suggesting that PI3K/Akt pathway mediates CD38 expression in HASM cells in a non-ERK dependant manner. The role of adenylate-uridylate-rich elements (AREs) of the CD38 mRNA 3' untranslated region (3'UTR) in CD38 mRNA stability is currently being investigated. Preliminary findings show that, in HASM cells, the RNA-binding proteins HuR and TIA-1 selectively bind to an ARE of CD38 3'UTR in response to TNF-alpha-exposure, suggesting a potential role for the CD38 mRNA AREs in the post-transcriptional regulation of CD38 expression in HASM cells.Item Regulation of tumor cell expression of the carbohydrate epitope sialyl Lewis X in the tumor microenvironment: implications for metastasis(2010-01) Krieser, Katherine AnneThe association between inflammation and cancer has been recognized for almost 150 years. The processes of migration of cancer cells to metastatic sites and leukocyte recruitment to sites of inflammation have much in common. Both cell types display specialized carbohydrates modified with sialyl Lewis X (sLex) epitopes on their cell surfaces. These sLex epitopes are highly expressed on leukocytes and are ligands for selectin adhesion molecules present on activated vascular endothelium at sites of inflammation. The binding interactions between sLex ligands on leukocytes with endothelial selectins are well characterized and promote leukocyte trafficking to lymphoid tissues and sites of inflammation. Similarly, malignancy is associated with expression of sLex structures on cancer cells that aid tumor invasion and metastasis. High expression of sLex in many types of human carcinomas is associated with an advanced stage of disease and poor patient prognosis. Synthesis of sLex is dependent on activity of the glycosyltransferase enzymes α2,3-sialyltransferase and α1,3-fucosyltransferase-III (FucT-III). Exposure to the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) has been described to up-regulate FucT-III resulting in increased sLex in the airways of patients with respiratory disease, however, little is known about the molecular mechanisms involved in the regulation of sLex expression in the inflammatory lung tumor microenvironment. The overall objectives of this study are: 1) to investigate the role of cytokines and inflammatory cells in regulation of sLex expression on non small cell lung cancer cells, 2) to evaluate the role of FucT-III, sLex expression, and inflammatory cells in the acquisition of metastatic properties by lung cancer cells, and 3) to characterize the backbone protein and carbohydrate structures that display sLex on lung and colon cancer cells. Our major findings are presented in iv Chapters 2 and 3 and the conclusions drawn from this study are highlighted in Chapter 4. Our results suggest that FucT-III activity in non small cell lung cancers recruit neutrophils in the tumor microenvironment and enhance the metastatic potential of lung tumor cells through the promotion of sLex expression and acquisition of invasive and anchorage independent qualities. Thus, inhibition of FucT-III enzyme activity and sLex expression may represent promising therapeutic targets for the control of lung cancer metastasis.Item The role of HIV- and SIV-specific CD8+ T cells in the establishment of persistent HIV and SIV infections.(2009-12) Mattila, Teresa LeaCD8+ T cells are important in controlling viral infections. Although the appearance of HIV-specific CD8+T cells initially correlates with reduced viral load during HIV infection, for unknown reasons HIV is never fully cleared from the body. My central hypothesis is that B cell follicles are sites in which virus-producing cells are protected from virus-specific CD8+T cells. During the chronic stages of infection the majority of HIV-producing cells accumulate in B cell follicles. The localization and abundance of HIV-specific CD8+T cells relative to B cell follicles is not known. For these studies I determined the spatial localization of HIV and SIV-specific CD8+T cells relative to B cell follicles in lymph nodes from HIV-infected humans and SIV-infected rhesus macaques, using immunohistochemistry, in situ tetramer staining, confocal microscopy, and quantitative image analysis. My findings show that most HIV-specific CD8+T cells were concentrated in T cell zones and were largely excluded from areas within B cell follicles where HIV is concentrated. Because many similarities exist between HIV infection in humans and SIV infection in macaques, and SIV model systems are essential tools to understanding HIV/SIV infections and for the development of HIV vaccines, we set out to determine whether the exclusion of virus-specific CD8+T cells from B cell follicles also occurs in the SIV/rhesus macaque model of HIV infection. I found in a small cohort of animals that during the early and late stages of SIV infection, SIV-specific CD8+ T cells were concentrated in T cell zones of lymph nodes, and that within B cell follicles, concentrations of SIV-specific CD8+T cells were significantly lower than in T cell zones. Most B cell follicles showed an absolute exclusion of SIV-specific T cells from more than half of the B cell follicle area where SIV concentrates. These data support the hypothesis that B cell follicles are an immune privileged site in which HIV/SIV-producing cells are protected from HIV/SIV-specific CD8+T cells. These data have important implications for the development of a successful HIV vaccine and treatments to eradicate HIV.Item Strain dependent variations in iron metabolism of Mycobacterium avium subsp. paratuberculosis(2010-06) Janagama, Harish KumarJohne’s disease is a major animal health problem of ruminant species worldwide and imposes significant economic losses to the industry. Our ability to culture the causative agent--Mycobacterium avium subsp. paratuberculosis (MAP)--and therefore its rapid diagnosis and our understanding of its virulence is limited. MAP is difficult to culture because of its unusually strict iron requirements. For optimal growth in laboratory media, MAP requires a siderophore (mycobactin) supplementation that makes MAP fastidious, often requiring eight to sixteen weeks to produce colonies in culture – a major hurdle in timely diagnosis and therefore implementation of optimal control measures. Understanding iron regulatory networks in the pathogen in vitro is therefore of great importance. Several microbiological and genotyping studies and clinical observations suggest that Johne’s in certain hosts such as sheep, goats, deer, and bison is caused by a distinct set of strains that show a relatively high degree of host preference. At least two microbiologically distinct types of MAP have been recognized. A less readily cultivable type is the common, but not invariable, cause of paratuberculosis in sheep (type I), while another readily cultivable type is the most common cause of the disease in cattle (type II). In addition, since the MAP genome sequence was published in 2005, very little research has focused on iron physiology and its contribution to metabolic networks of this fastidious organism. Based on these observations, I hypothesize that iron dependent gene regulation is different between type I and type II MAP strains. Iron dependent Regulator (IdeR), a transcription factor, is an essential gene in MAP and differentially controls the expression of genes involved in iron physiology in the two strain types of MAP. We identified polymorphisms in the IdeR open reading frame (ORF) and the promoters of putative IdeR regulated genes between the type I and type II strains of MAP. Structure-function association studies revealed repression of an iron storage gene, bfrA in the presence of iron by type I MAP strain alone. In contrast, bfrA was upregulated in the presence of iron in type II MAP strain. This leads us to propose that type I MAP strains may experience iron toxicity when excess iron is provided in the medium. The rationale is that excess free iron is detrimental to the cells and must be stored in bacterioferritins, a feature that type I strains lack. Transcriptional and proteomic profiling of these MAP strains under iron-replete or –deplete conditions revealed that iron-sparing response to iron limitation was unique to the type II strain as evidenced by repression of non-essential iron utilization enzymes (aconitase and succinate dehydrogenase) and upregulation of proteins of essential function (iron transport, [Fe-S] cluster biogenesis and cell division). Under iron-replete conditions, type II MAP alone increased expression of BfrA (bacterioferritin) and MhuD (mycobacterial heme utilization, degrader) protein, which is intricately involved in iron recycling. These findings further supported the contention that type I MAP strains are metabolically inept under iron-replete conditions. The intracellular lifestyle of MAP in the intestines and lymph nodes of natural infection revealed that MAP deployed genes involved in maintaining iron homeostasis under iron stress in the tissues of infected animals. There was a clear dichotomy in in vitro infected macrophages and natural infection in the expression profiles of both iron acquisition genes and other virulence factors involved in MAP survival inside the host. In summary, our studies revealed that IdeR of type II strain regulates mycobactin synthesis and iron storage genes, similar to the function of IdeR in M. tuberculosis (MTB), while the type I strain is deficient in iron storage function. Given our inability to delete ideR, it appears that this is an essential gene (as in MTB) for MAP survival. MAP IdeR regulon studies led us to define a novel operon carrying genes encoding a potential secretory apparatus (ESX-3/type VII secretory system). Functional analysis of the iron-induced proteome also identified novel ESAT-6 (early secreted antigenic target) family of proteins belonging to ESX-5, which have been identified as major virulence factors in MTB. We also established that, type I MAP strains are more sensitive to fluctuations of environmental iron due to defective regulation of bfrA and may grow better under lower iron levels in the culture media. Taken together, our studies suggest that MAP employs a sophisticated repertoire of proteins that are inter-connected and function in response to environmental stress.Item Structural and functional characterization of porcine reproductive and respiratory syndrome virus N-glycans.(2011-12) Li, JuanPorcine reproductive and respiratory syndrome (PRRS) is one of the most severe infectious diseases facing the swine industry worldwide. The etiologic agent, PRRS virus (PRRSV), belongs to the order Nidovirales, family Arteriviridae, genus Arterivirus. It is a positive-sense ssRNA virus and has a smooth spherical envelope embedded with seven proteins, four of which are glycoproteins (GP). Since the major envelope proteins GP5 and matrix (M) have short ectodomains, the broadly distributed viral glycans likely cover the virion surface and stretch out as antennae, thus interacting with host cells and contributing to viral infection. Previous studies suggest potential roles of PRRSV envelope protein-linked glycans in virus assembly, virus attachment to target cells, virus neutralization and antigenicity. In particular, sialic acids on GP5 have been shown to bind sialoadhesin on porcine macrophages, mediating virus attachment and internalization. Nevertheless, the complete profile of GP5-linked glycan compositions and structures, and the role of specific glycan moieties in virus infection have yet to be determined. Herein, we purified the North American prototype PRRSV, VR-2332, and analyzed viral glycans in the aspects of composition, structures and functions in virus infection. Endoglycosidase digestion of virus showed that GP5 was the primary protein substrate among all the four envelope glycoproteins, and that the glycans were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers and terminal sialic acids, which was also confirmed by lectin co-precipitation. Based on the structural information, we further demonstrated that GlcNAc and LacNAc oligomer-specific lectins bound to PRRSV and blocked virus attachment, resulting in reduced infection. However, GlcNAc oligomers and LacNAc did not compete with virus to block infection, suggesting that GlcNAc and LacNAc oligomers are not directly involved in virus entry. Finally, removal or alteration of N-glycans from PRRSV envelope proteins did not affect infection, indicating that envelope protein-linked N-glycans are not required for PRRSV infection. In conclusion, GP5 contains most of the PRRSV glycans, which are primarily complex-type N-glycans. GlcNAc and LacNAc oligomers and sialic acids on the PRRSV envelope are accessible for specific recognition that may reduce infection by steric hindrance. Envelope protein-linked N-glycans are not required for PRRSV infection. Our findings provide a glycan database for molecular structural studies of PRRSV and facilitate a better understanding of molecular host-virus interactions.Item Survival strategies of Mycobacterium avium subsp. paratuberculosis in a variety of microenvironments.(2012-06) Lamont, Elise A.Mycobacteria, specifically Mycobacterium avium subsp. paratuberculosis (MAP), are extreme strategists and as a rule live by deception. Mycobacteria represent a group of closely related acid-fast bacilli that encompass a wide-range of host tropisms and diseases. Mycobacteria can be divided into two complexes: the Mycobacterium tuberculosis complex and the Mycobacterium avium complex (MAC). The MAC is comprised of M. avium subsp. avium (M. avium), MAP, M. intracellulare and M. avium subsp. hominissuis (M. hominissuis), all of which share an over 90 percent nucleotide similarity. Despite its genetic similarity, MAC elicits different diseases in both animals and humans including infections of the lung, lymph nodes, bones, skin and gastrointestinal tract. MAP is a unique member of MAC as it infects and establishes itself within the intestine of ruminants and other wildlife. Furthermore, MAP lives in a quiescent state in soil and aquatic environments. Since MAP encounters numerous environments, including those with unfavorable conditions, it has developed several strategies to survive. However, the mechanisms by which MAP survival is achieved remains incompletely understood. The goal of these studies was to determine how MAP may survive and disseminate under unfavorable conditions, which included nutrient starvation and host pressures. We have identified the development of a new MAP morphotype under prolonged nutrient starved conditions. This novel MAP morphotype resembles a spore-like structure and contains dipicolinic acid, which is used to protect DNA located within the core. These novel structures are heat resistant at 70oC and can be enriched for in multiple MAP strains. Furthermore, we describe an unrecognized mechanism by which MAP takes advantage of host responses at the epithelium interface to recruit macrophages to the site of initial infection. MAP is able to safely enter into macrophages and consequently ensures its establishment, survival and dissemination throughout the host. Lastly, we demonstrate the importance of host physiological relevant temperature on successful disease progression. Infection utilizing the temperature of MAP’s natural host, the cow, enhances the speed of infection as well as host and pathogen transcriptomic profiles. Taken together, data generated from these studies will provide the basis for understanding MAP persistence and survival in diverse conditions. The mechanisms by which MAP establishes, disseminates and/or survives difficult conditions may impact new programs to control JD as well as rational vaccine/therapeutic design and the way in which we view other mycobacterioses.Item Toll-like receptor interactions and their contribution to airway inflammation.(2010-06) Melkamu, TameneToll-like receptors (TLRs) are key components of the innate immune system involved in surveillance and early detection of infection, as well as coordination of the subsequent adaptive immune response. Pulmonary immune responses are initiated by TLRs expressed on airway epithelial cells. In this thesis, primary normal human bronchial epithelial (NHBE) cells, immortalized human bronchial epithelial (HBE) cells, a glandular lung adenocarcinoma cell line (Calu-3) and an alveolar epithelial adenocarcinoma cell line expressing NF-κB (A549/NF-κB-luc) were analyzed for expression of mRNAs for TLRs, coreceptors, adaptor proteins and non-TLR pathogen recognition receptors (PRRs). Quantitative RT-PCR analysis in Calu-3 and NHBE/HBE cells revealed mRNA expression of only TLRs 1-6, with TLR4/TLR6 having the lowest abundance and TLR3 exhibiting the highest level of expression. Conversely, in A549/NF-κB-luc cells mRNA expression of all TLRs 1-10 was detectable; with TLR5 mRNA expressed at the highest level. Poly i/c, a synthetic viral dsRNA analogue, elicited the greatest effect on expression of TLR subtypes. The effect was far more pronounced in NHBE/HBE cells as compared to Calu-3 and A549-NF-κB-luc cell lines. Poly i/c increased TLR2 mRNA expression by over sixty-fold and TLR3 by eleven-fold in NHBE cells, whereas TLR5 was significantly reduced. TLR2 protein was also enhanced by Poly i/c, but not by the TLR2 ligand, PAM3CSK4. Additionally, Poly i/c enhanced mRNA expression of adaptor molecules (MyD88, TIRAP, and TRIF) and co-receptors (Dectin-1, CD14) involved in TLR2-signaling. In contrast, mRNA and protein expression of co-receptor CD36 was significantly reduced by Poly i/c. Overall, Poly i/c activation of NHBE cells differentially regulated expression of multiple TLRs, non-TLR receptors, adaptor proteins and coreceptors. ELISA analysis of apical and basolateral solutions from Poly i/cstimulated NHBE monolayers revealed significantly higher levels of IL-6 and GMCSF. After priming with Poly i/c, an increase in IL-6 secretion was observed in cells stimulated with PAM3CSK4 and with Alternaria extract, a fungal allergen known to signal partly through TLR2. However, IL-6 secretion was not stimulated by other TLR2 ligands, zymosan or lipothechoic acid (LTA). Pretreatment with anti-TLR2 blocking antibody inhibited the PAM3CSK4-induced increase in IL-6 secretion after Poly i/c exposure. Up-regulation of TLR2 following exposure to dsRNA enhanced functional responses of the airway epithelium to certain (PAM3CSK4), but not all (zymosan, LTA) TLR2 ligands. In TLR3-deficient cells, induction of TLR2 mRNA expression by Poly i/c was reduced by thirteen-fold, indicating that Poly i/c-dependent TLR2 induction was TLR3-mediated. Furthermore, Poly i/c-dependent IL-6 secretion was abrogated by 83% in TLR3 knock-down cells. Following priming of NHBE cells with Poly i/c and subsequently with TLR5 ligand flagellin, no reduction in IL-6, GM-CSF, RANTES and IFN-β transcripts was observed. Similarly, IL-6 and GM-CSF protein secretion did not match Poly i/c-dependant down-regulation of TLR5. Using A549/NFκB-luc cells and wild-type (flagellated) and flagella-mutated Burkholderia (B.) cenocepacia, intact flagella were found to be critical for NF-κB activation and enhanced secretion of IL-8. To elucidate the in vivo actions of viral infection on the magnitude of TLR2 expression, and subsequent innate immune response to TLR2 activation, the effects of Poly i/c challenge on the inflammatory response of mouse airways to a selective TLR2 ligand (PAM3CSK4) and a common fungal allergen (Alternaria alternata) were investigated. Poly i/c enhanced mRNA and protein expression of TLR2 in the lung. It also elicited a marked induction of Th1 cytokines. However, priming with Poly i/c abrogated Alternaria-mediated Th2 cytokine production. Inflammatory cell recruitment into the airways, predominantly with neutrophils, was also observed. Taken together, both the in vitro and the in vivo studies show that activation of TLR3 by Poly i/c alters the inflammatory response of the airways to other microbial ligands, thereby modulating the severity of inflammation. This effect is partly due to changes in the expression of other TLR receptors and their associated co-receptors and adaptor proteins.