Browsing by Subject "Microbiology, Immunology and Cancer Biology"

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Advancing novel cell-based therapies for the prevention of graft versus host disease (GVHD) and improvement of engraftment following bone marrow transplantation.
(2010-06) Highfill, Steven Lorenz
Bone marrow transplantation is a valuable treatment option for patients with underlying hematological disorders. Unfortunately, complications associated with this course of treatment, such as graft versus host disease and graft failure, results in significant morbidity and mortality. Current clinical methodology relies mainly on the use of corticosteroids and pan-T-cell depletion to restrain these complications, however, their global immunosuppressive attributes, toxicity and increased relapse rate of certain cancers has prompted the search for improved therapies. The research described herein investigates novel cell-based methods that may be used in place of current therapies. More specifically, we investigate the use of mutipotent adult progenitor cells (MAPCs) and myeloid-derived suppressor cells (MDSC) for ameliorating acute graft versus host disease (aGVHD) and the use of mesenchymal stem cells (MSCs) for enhancing bone marrow engraftment. For GVHD, we find that both MAPCs and MDSCs have the capacity to inhibit allo-T-cell reactions in vitro using two distinct mechanisms. However, we find that when these cells are applied to a murine model of aGVHD, only MDSC express the appropriate homing molecules that allows them to traffic to sites of allogeneic T-cell recognition and activation, and therefore, only MDSC decrease GVHD-related mortality when administered i.v. Another major concern following BMT is BM engraftment failure. Currently, MSCs are employed in the clinic as means to promote or enhance BM engraftment, however, there is no definitive mechanism of action identified to be responsible for this effect. We find that MSCs secrete copious amounts of a molecule known to have a positive influence on hematopoietic stem cell (HSC) survival and proliferation, prostaglandin E2 (PGE2). We show that the secretion of PGE2 by MSCs increases the number and differentiation capacity of HSCs and also show that inhibiting the production of PGE2 from MSCs by using COX inhibitors reverses these effects. These data are the first to define a mechanism of action that MSCs use to promote BM engraftment. In conclusion, the cutting edge preclinical research described here has advanced the field of cell therapies following BMT and will hopefully be used to design clinical regimes aimed at improving the lives of patients in need.
Analysis of the pre-immune T cell repertoire.
(2009-12) Chu, Hon Man Hamlet
Cell-mediated immune responses are initiated when a population of pre-immune (or naïve) T cells recognize their cognate ligands in the form of a specific peptide bound to a self major histocompatibility complex molecule (pMHC). This recognition is made possible by highly specific T-cell receptors (TCR) on individual T cell clones specific for a given pMHC complex. The pre-immune T cell repertoire is comprised of populations specific for at least 100,000 different pMHC, each containing multiple clones. It is important to understand the composition of this repertoire because it is the repository of all the host's potential for future cell-mediated immune responses to microbes and tumors, and in some cases its own tissues. . However, the study of pre-immune tumor antigen-specific, or any other pathogen-specific T cell populations within such a diverse T cell repertoire have been extremely difficult due to their low frequency in the body. A novel soluble pMHC magnetic enrichment technique was therefore developed to analyze naive T cell populations in mice and humans,. Using this procedure, different pMHCII-specific naive CD4+ T cell populations could be identified and enumerated. The size of these populations was found to vary depending on pMHC specificity. Additionally, these differences were directly proportional to the magnitude and TCR gene usage diversity of the primary CD4+ T cell response after immunization with relevant peptide. Thus, variation in naive T cell frequencies can explain why some peptides give rise to greater T cell responses than others. We explored this issue by enumerating pMHCII-specific CD4+ T cell populations that normally number 20 or 200 cells per mouse. Thymic positive or negative selection was required for optimizing the absolute size pf each population but did not alter the 10-fold ratio between the two populations. Large naïve population size was related to the presence of certain amino acids at T cell receptor contact sites within the peptide. These results suggest that certain MHCII-bound peptides are immunodominant because they contain amino acids with chemical properties that foster binding to many TCRs.
Antigen presenting cells In the retina
(2010-10) Lehmann, Ute
The presence and activity of dendritic cells (DC) in retina is controversial. This is in part due to the limited number of DC in the retina and the small number of reliable DC markers. In addition, functions ascribed to DC in other parts of the body are thought to be done by microglia in the central nervous system. CD11c is the cellular marker most associated with DC. In order to study the nature of retinal DC, transgenic mice that express green fluorescent protein (GFP) on the CD11c promoter were used to visualize, quantify, and characterize DC in both quiescent and injured retinas. In the quiescent retina it was found that compared to the number of neurons and microglial cells there were relatively few DC located in the nerve fiber layer and the outer plexiform layer. Upon retinal injury by either optic nerve crush or exposure to continuous bright light, the numbers of DC increased significantly and translocated to retinal layers associated with the injury. Surprisingly, the retinas of the eyes contralateral to the optic nerve crush also showed a significant increase in DC. The potential origin of the DC in retina was examined using a chimeric mouse paradigm. Most of the retinal DC were found to originate from circulating precursors, and a smaller number from a local progenitor cell population. This study suggests that retinal DC are a previously overlooked population, distinct from microglia, and may be important in the injury response and maintenance of homeostasis in the retina.
CD4 T cells in the protection and pathogenesis of persistent Salmonella infection
(2010-10) Johanns, Tanner Michael
CD4 T cells contribute a diverse and non-redundant role to host defense against infections by orchestrating the activation and quality of the innate and adaptive immune responses. The diversity of CD4 T cell function is accomplished by differentiating into a plethora of distinct effector and regulatory lineages that dictate the kinetics and extent of immune activation. However, due to the range and breadth of CD4 T cell function, the precise role and mechanism of these various effector and regulatory subsets in host immunity remains incompletely understood. As persistent infections represent a significant source of morbidity and mortality worldwide, and CD4 T cells play a critical role in protection against this class of pathogens, we sought to elucidate the relative contribution of effector and regulatory CD4 T cell subsets in the pathogenesis and protection of this class of pathogens. Using a murine model of persistent Salmonella infection, we demonstrate that CD4 T cells are required for protection during primary infection but dispensable for secondary immunity. Moreover, both host and pathogen factors limit the generation of a protective effector CD4 T cell response during primary disease including increased regulatory CD4 T cell suppressive function and Salmonella-associated virulence genes, respectively, that enables establishment and persistence of disease. Together, these findings provide novel insight into disease process of persistent Salmonella infection that will aid in the design of future therapeutic and prevention strategies.
Characterization of the ESCRT pathway in Candida albicans.
(2010-02) Wolf, Julie Marie
iv Abstract The human opportunistic pathogen Candida albicans has a signal transduction pathway unique to fungi, called the Rim101 pathway. The Rim101 pathway regulates the proteolytic activation of the transcription factor, Rim101, the activation of which is required for growth at neutral-alkaline pH. Many genes regulated by Rim101 play a role in C. albicans virulence, including genes involved in filamentation, cell wall structure, adhesion, and nutrient acquisition. The Rim101 pathway consists of two complexes: a signaling complex at the plasma membrane and a processing complex inside the cell, and both of these complexes are required for Rim101 activation. Rim101 activation also requires members of a second pathway, the endosomal sorting complex required for transport (ESCRT) pathway. The ESCRT pathway is required to generate multivesicular bodies prior to vesicle fusion with the vacuole. The ESCRT pathway consists of several polyprotein complexes recruited sequentially to the endosomal membrane to generate an intraluminal vesicle. The role of the ESCRT pathway has not been well characterized in C. albicans, and study of the ESCRT pathway is complicated by the secondary effect many ESCRT mutations have on Rim101 processing. These studies sought to separate iv Abstract The human opportunistic pathogen Candida albicans has a signal transduction pathway unique to fungi, called the Rim101 pathway. The Rim101 pathway regulates the proteolytic activation of the transcription factor, Rim101, the activation of which is required for growth at neutral-alkaline pH. Many genes regulated by Rim101 play a role in C. albicans virulence, including genes involved in filamentation, cell wall structure, adhesion, and nutrient acquisition. The Rim101 pathway consists of two complexes: a signaling complex at the plasma membrane and a processing complex inside the cell, and both of these complexes are required for Rim101 activation. Rim101 activation also requires members of a second pathway, the endosomal sorting complex required for transport (ESCRT) pathway. The ESCRT pathway is required to generate multivesicular bodies prior to vesicle fusion with the vacuole. The ESCRT pathway consists of several polyprotein complexes recruited sequentially to the endosomal membrane to generate an intraluminal vesicle. The role of the ESCRT pathway has not been well characterized in C. albicans, and study of the ESCRT pathway is complicated by the secondary effect many ESCRT mutations have on Rim101 processing. These studies sought to separate iv Abstract The human opportunistic pathogen Candida albicans has a signal transduction pathway unique to fungi, called the Rim101 pathway. The Rim101 pathway regulates the proteolytic activation of the transcription factor, Rim101, the activation of which is required for growth at neutral-alkaline pH. Many genes regulated by Rim101 play a role in C. albicans virulence, including genes involved in filamentation, cell wall structure, adhesion, and nutrient acquisition. The Rim101 pathway consists of two complexes: a signaling complex at the plasma membrane and a processing complex inside the cell, and both of these complexes are required for Rim101 activation. Rim101 activation also requires members of a second pathway, the endosomal sorting complex required for transport (ESCRT) pathway. The ESCRT pathway is required to generate multivesicular bodies prior to vesicle fusion with the vacuole. The ESCRT pathway consists of several polyprotein complexes recruited sequentially to the endosomal membrane to generate an intraluminal vesicle. The role of the ESCRT pathway has not been well characterized in C. albicans, and study of the ESCRT pathway is complicated by the secondary effect many ESCRT mutations have on Rim101 processing. These studies sought to separate ESCRT function from Rim101 function, and to investigate ESCRT pathway function in C. albicans virulence. In these studies, ESCRT and Rim101 pathway separation is demonstrated (1) at distinct domains on a single protein known to be part of both pathways by using alanine scanning mutagenesis and (2) at ESCRT pathway complexes by using deletion mutagenesis. The ESCRT pathway is demonstrated here to play a wholly Rim101-independent role in C. albicans virulence.
Characterization of the human cytomegalovirus tegument protein UL94 and its interaction with UL99.
(2012-06) Phillips, Stacia L.
Human cytomegalovirus (HCMV) is the largest and most complex of the human herpesviruses, both structurally and with respect to the coding capacity of the viral genome. Proteomic analysis of purified virions suggests that HCMV particles may be composed of as many as 71 virally encoded proteins. Despite intense investigation, the mechanisms by which HCMV particles are assembled in the cytoplasm of infected cells remain poorly understood. In an attempt to gain novel insight into the processes involved in virion assembly, we carried out a yeast two-hybrid screen designed to identify binary interactions among HCMV structural proteins. One interaction of particular interest that was identified in our screen was that between the herpesvirus-conserved tegument proteins UL94 and UL99. UL99 is essential for HCMV replication and functions at the stage of secondary envelopment in the cytoplasm. The function of UL94 is unknown. We hypothesize that the interaction between UL94 and UL99 is essential for HCMV replication. To investigate this hypothesis, we first sought to elucidate the function of UL94. In the absence of UL94, early events such as viral gene expression and DNA replication proceeded at wild type levels. However, we observed a dramatic defect in the localization of UL99 to the viral assembly complex in the cytoplasm of cells infected with the UL94-null mutant. In addition, ultrastructural analysis of cytoplasmic virus particles showed that in cells infected with the UL94-null mutant, there was a complete absence of enveloped virions, demonstrating that UL94, like UL99, is required for secondary envelopment. Finally, we mapped the domains of UL94 and UL99 that are required for their interaction. We then incorporated mutations that abolish the interaction into the viral genome. We showed that when the interaction between UL94 and UL99 was abolished during HCMV infection, both proteins exhibited aberrant localization and the production of infectious virus progeny was completely blocked. Taken together, our results suggest that UL94 functions at least in part to direct the proper localization of UL99 to the assembly complex through their interaction and that this event is essential for HCMV replication.
Characterization of the primitive neuroectodermal tumor genome using transposon-mediated insertional mutagenesis.
(2011-12) Larson, Jon David
Primitive neuroectodermal tumors (PNET) include medulloblastoma and supratentorial primitive neuroectodermal tumor subtypes that share histological features yet differ at the genomic level and in clinical outcome. Delineation of the genetic anomalies between PNET subtypes is a current challenge for establishing effective targeted therapeutic strategies against these aggressive tumors. Current efforts have demonstrated specific molecular pathways drive a minority of medulloblastoma and supratentorial PNET but the genetic basis of the majority of these tumors remains poorly understood and anecdotal. To better define the genetic causes of medulloblastoma and supratentorial PNET, we have developed a single Sleeping Beauty transposon insertional mutagenesis mouse model that recapitulates the morphological similarities and genetic heterogeneity of these tumor types capable of identifying genetic drivers important for PNETagenesis. This work has revealed biologically relevant candidate oncogenes and tumor suppressor genes for both medulloblastoma and supratentorial PNET in mice and humans. ARHGAP36 is a novel oncogene implicated in poorly understood MB molecular subgroups, and multiple RAS pathway effector genes were identified to be important for sPNET formation. Ultimately, these results present new understanding of the genetic basis for medulloblastoma and supratentorial PNET development and offer potential targets for therapeutic testing to improve patient clinical outcome.
Characterization of viral mutants for the functional analysis of ppUL69 during human cytomegalovirus replication.
(2009-11) Kronemann, Daniel Aaron
Human cytomegalovirus (HCMV) is a β-herpesvirus that infects over 80% of the human population. Although disease is rare in immunocompetent individuals, severe disease is common in immunocompromised patients, including neonates, transplant recipients and acquired immunodeficiency syndrome (AIDS) patients. HCMV UL69 is a viral protein packaged in the tegument of the virion and, as such, is present from the earliest moments of infection. Using transient transfection assays, UL69 has been shown to bind the cellular splicing and mRNA export factor U2AF65 associated protein 56 (UAP56), shuttle between the nucleus and the cytoplasm, and bind the cellular chromatin remodeling and transcriptional elongation factor Suppressor of Ty 6 (Spt6). In previously published work, these characteristics were shown to be required for transactivating the major immediate/early promoter (MIEP) and promoting the export of an intron-containing chloramphenicol acetyltransferase (CAT) reporter transcript. These results, in addition to the fact that most herpesviral transcripts are intronless, have led to a model for UL69 function during HCMV infection as a viral mRNA export factor. However, recently published work using a UL69 deletion viral mutant, termed TNsubUL69, has demonstrated that during viral infection, UL69 is not required for expression of immediate/early (IE) or early (E) genes. In contrast, deletion of UL69 results in a severe defect in late (L) gene expression. Additionally, the UL69 deletion mutant exhibits a defect in viral DNA replication and a MOI-dependent replication defect. These results demonstrate the importance of examining the functional role of UL69 in the context of a viral infection, in the presence of a full complement of viral factors at physiologically relevant levels. The goal of this thesis is to characterize viral mutants that contain mutations in the UL69 open reading frame (ORF) which have been previously described to abolish either binding to UAP56 (UL69mUAP), nucleocytoplasmic shuttling (UL69P603 or UL69E618), or binding to Spt6 (UL69C496). We demonstrate our own ΔUL69 viral mutant exhibits a similar replication phenotype, consistent with previously published results. We demonstrate that the UL69mUAP mutation abolishes UL69 binding of UAP56 but does not affect viral replication. The UL69P603 mutation, but not the UL69E618 mutation, abolishes UL69 shuttling and results in a defect in viral replication similar to the deletion mutant virus. However, the UL69P603 viral mutant is also defective for every other UL69 characteristic for which we have assayed, suggesting the UL69P603 mutation affects a core domain in the UL69 ORF and results in a global UL69 defect during viral infection. A similar result has been obtained for the UL69C496 viral mutant, which is defective for UL69 binding of Spt6, but also results in a defect in other UL69 functions. Using an shRNA strategy to further examine the role of Spt6 during HCMV replication, we demonstrate that partial knockdown of Spt6 negatively affects WT HCMV replication. Taken together, we conclude that UL69 binding of UAP56 is not required efficient HCMV replication, but are unable to make strong conclusions about UL69 shuttling or binding of Spt6 during viral replication. Since both UL69 shuttling and UL69 binding of UAP56 are required for export of an introncontaining transcript, and given that UL69 binding of UAP56 is not required for efficient viral replication, we predict that UL69 shuttling is also not important for efficient viral replication. Thus we propose three models for UL69 function, which center on UL69 binding of Spt6. Through its interaction with Spt6, UL69 functions as either a viral mRNA export factor, a chromatin remodeling factor, or a transcriptional elongation factor.
Co-stimulation and IL-2 in natural regulatory T cell development.
(2010-09) Dings, Kieng Bao
Treg development occurs via a two-step process. In step 1, signals through the T cell receptor and costimulation via CD28 initiate a Treg developmental program that is dependent on the NFkB signaling pathway. Notably, signals through CARMA1 and C-REL initiate a Treg progenitor in which cytokine signaling such as IL-2 (step 2) via STAT5 turns on Foxp3. Herein, we discuss in detail the molecular requirement for natural regulatory T cell development.
Derivation, maintenance, and functions of virtual memory cells.
(2011-09) Akue, Adovi Dodji
Memory phenotype CD8+ T cells are typically thought to have undergone an immune response to foreign antigen and to have differentiated from antigen-specific precursors in the naïve pool. However, using a peptide-MHC I tetramer enrichment technique, we identified foreign antigen-specific memory-phenotype CD8+ T cells in unimmunized mice. These cells (termed "virtual memory" T or VM cells) were observed in mice maintained in both specific-pathogen- and germ-free (SPF and GF respectively) housing. This thesis focuses on the relationship between VM cells and "conventional" memory cells: memory cells arising from homeostatic proliferation (HP), and innate-like memory CD8+ T cells such as IL-4 bystander memory CD8+ T cells. Our data indicate physiological HP and IL-4-driven bystander processes are the main mechanisms that drive the generation of VM cells and not the exposure to foreign antigens. VM cells arise in the periphery during the neonatal period and are maintained long term. We also show that VM cells respond in vitro to innate cytokines (similar to conventional memory CD8+ T cells) and they outcompete antigen-specific naive CD8+ T cells in in vivo responses. Overall our observations suggest that VM cells arise out of normal homeostatic and IL-4-driven bystander processes in unimmunized SPF and GF mice, and express at least some memory-like capabilities.
Developing novel strategies to enhance thymic recovery and T-cell reconstitution following bone marrow transplantation.
(2009-05) Kelly, Ryan Michael
Allogeneic HSCT is a valuable treatment option for many malignant and nonmalignant disorders. A significantly limiting factor for a favorable outcome following HSCT is the prolonged T-cell deficiency following transplant, which is primarily due to thymic injury caused by the intense chemotherapy/radiation-conditioning regimen given prior to transplant. The submitted work details the development of novel approaches to restore thymic function and enhance T-cell reconstitution following bone marrow transplantation (BMT). The preclinical research described in this dissertation investigates the therapeutic potential of combinatorial administration of keratinocyte growth factor, androgen regulators and general radioprotectants in restoring thymic function and T-cell reconstitution following BMT. The data suggest that pre-conditioning treatment of BMT recipients with combinations of these agents lead to rapid and durable restoration of thymic function and accelerated peripheral reconstitution of donor-derived, naïve CD4 and CD8 T-cells. Importantly, enhanced T-cell reconstitution correlates with superior antigen-specific CD4 and CD8 T-cell responses in vivo. This work also describes research aimed at characterizing the kinetics of depletion and recovery of thymic epithelial cells (TEC) following BMT and elucidating the role of thymocyte:TEC crosstalk in promoting TEC regeneration. A more thorough understanding of this process will allow for the identification of more focused targets for therapies aimed at promoting thymic and T-cell reconstitution following BMT. Taken together, this work has generated novel findings that will advance the field of immune reconstitution following bone marrow transplantation.
Enterococcus faecalis aggregation substance (Asc10) as liaison between bacterium and heart valve in endocarditis.
(2009-08) Chuang-Smith, Olivia Newton
Aggregation Substance proteins encoded by sex pheromone plasmids increase virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation Substance Asc10, encoded by the plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the vegetation was the best indicator of virulence. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative where glycine residues in two RGD motifs were changed to alanines showed the greatest reduction in virulence. Remarkably this strain, and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. In addition, mutants carrying Tn917 insertions in the prgB gene demonstrated that secreted N-terminal Asc10 fragments possess activity promoting endocarditis virulence. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis, and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo. Since Asc10 is important as a virulence factor in E. faecalis endocarditis pathogenesis, developing immunization approaches against this surface protein will be useful in combating endocarditis disease. Use of Fab fragment antibodies against Asc10 was found to decrease vegetation size and bacterial load in the rabbit endocarditis model. In addition, microarray and histological studies revealed two routes of infection in vegetation formation; one in the absence of Asc10, characterized by a robust inflammatory response, and the second in which the presence of Asc10 dampens this response, possibly impeding the influx of immune cells into the vegetation. We also employed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10+ E. faecalis and valve tissue, and to examine formation of biofilms. We found that the aggregation domains contribute most to Asc10-mediated E. faecalis valve adherence, whereas the RGD motifs have importance in later stages of valve colonization. Again, an N-terminal Asc10 fragment expressed from a prgB Tn917 insertion mutant mediated adherence of E. faecalis cells, emphasizing the importance of the aggregation domains in valve attachment. Most of the Asc10 mutants examined showed some defects in valve adherence at 4 h, corroborating results from our rabbit endocarditis model, and implying that Asc10 contributes mainly to persistence of E. faecalis during endocarditis infection. Extracellular matrix (ECM) protein studies to determine the eukaryotic Asc10 ligand in valve tissue found that fulllength Asc10 protein did not mediate E. faecalis binding to vitronectin, fibronectin, fibrinogen, von Willebrand factor, heparan sulfate, or chondroitin sulfate. In scanning electron microscopy analysis of the infected valve tissue, we found evidence of biofilm formation, including growing aggregates of bacteria, and the increasing presence of exopolymeric matrix over time. Additionally, E. faecalis cells preferentially bound to damaged tissue, though it was difficult to determine whether the bacteria caused the damage, or if it was due to deterioration of the tissue over time. This porcine heart valve tissue colonization model will serve as a useful tool in future studies of biofilm formation.
Epigenetic regulation of killer immunoglobulin-like receptor Gene expression in developing human natural killer cells.
(2010-05) Cichocki, Frank M.
The immune system is our primary defense against infection and disease. Immune cells need to recognize and efficiently destroy invasive pathogens while, at the same time, exercising tolerance towards normal cells and tissues within the body. Because pathogenic organisms are constantly evolving to evade detection, the immune system must employ multiple recognition strategies to keep pace. Natural killer (NK) cells have evolved a self versus non-self recognition strategy known as “missing self” that is based upon the recognition of self major histocompatibility complex (MHC) molecules by stochastically expressed inhibitory receptors on the surface of NK cells. When MHC expression is downregulated by a virus or cellular transformation event, the dampening signals that balance against NK cell activation are lost due to a lack of inhibitory receptor engagement. This lack of inhibitory signaling, along with the engagement of activating receptors, leads to the elimination of the distressed cell through targeted NK cell-mediated cytotoxicity. The work presented in this manuscript focuses on the transcriptional regulation of a critically important family of human NK cell inhibitory receptors known as killer immunoglobulin-like receptors (KIR). The KIR genes are present within the leukocyte receptor complex on chromosome 19 and are expressed in a variegated, clonally restricted pattern on fully differentiated NK cells. How this pattern of gene expression is regulated during NK cell development is not well understood despite the demonstrated clinical relevance of KIR during hematopoietic cell transplantation to treat patients with leukemia, the influence of the KIR repertoire on the progression of HIV to AIDS, and the importance of KIR during pregnancy. Progress in the elucidation of how KIR genes are regulated has been slow due to the complexity of the KIR locus and the lack of KIR genes in mice, which are much more amenable to genetic manipulation. We have shown that the 5’ upstream regulatory region of each KIR gene contains a previously uncharacterized distal promoter with a functional c-Myc binding site. Stimulation of primary peripheral blood NK cells with IL-15 induces c-Myc binding at the distal promoter, which acts to promote KIR transcription. We also found that the overexpression of c-Myc protein in the NK92 cell line, which lacks surface KIR due to dense methylation of CpG dinucleotides proximal to the transcriptional start site, causes de novo surface KIR expression. Taken together, these results suggest that IL-15 directly promotes KIR transcription by inducing the binding of c-Myc to the distal promoter. We hypothesize that the recruitment of c-Myc and the initiation of active transcription from the distal promoter may also be key steps in the removal of repressive epigenetic marks within KIR promoters during human NK cell development to allow for stable gene expression. In addition to identifying a novel distal promoter, our group has found that the conventional proximal KIR promoter exhibits bi-directional transcriptional activity, meaning that transcription can initiate in either the sense or antisense orientation. We observed a strong inverse correlation between the expression of KIR antisense transcripts and receptor expression on the cell surface, leading to the hypothesis that antisense transcripts directly participate in RNA-mediated transcriptional repression of individual KIR genes. We found that over-expressing full-length antisense transcripts during NK cell development led to an approximately 70% reduction in KIR expression compared to controls. Furthermore, we determined that full-length antisense transcripts are processed into a 28 base RNA with biochemical properties similar to those attributed to members of the PIWI family of small RNAs. We also demonstrate that the 28 base sequence is necessary for antisense transcript-mediated repression of KIR gene expression. This work establishes a direct association between KIR antisense transcription and the initiation of DNA methylation within the KIR promoter. Further elucidation of the mechanisms that regulate KIR expression during NK cell development may provide a basis for new strategies in the design of NK cell-based therapies
Examining immune responses in a mouse model of Salmonella infection
(2010-09) Griffin, Amanda Jill
Salmonella infections are responsible for significant morbidity and mortality throughout the world. Although extensive research has elucidated the mechanisms of protective immunity following vaccination with live vaccine strains (LVS) of Salmonella, very little work has been done to examine immune responses during and following antibiotic treatment of virulent Salmonella infections. We have developed a murine model of naturally acquired immunity to Salmonella, where susceptible mice are orally infected with virulent S. typhimurium and treated with antibiotics for an extended period of time. These mice demonstrate weak protective immunity to rechallenge with virulent Salmonella, which is due to Th1 and antibody responses and can be augmented by the administration of a TLR5 agonist. We have also used antibiotic treatment to examine the development of Th1 responses to LVS Salmonella, which are vital for mediating protective immunity to this pathogen. We show that Th1 cells develop after sustained exposure to Salmonella antigens. Eradication of the bacteria by antibiotic intervention within one week of primary infection has profound effects on the ability of mice to survive rechallenge with virulent Salmonella. We also establish that full effector/memory function of Th1 cells, as determined by robust production of Th1 cytokines, requires two weeks of exposure to Salmonella antigens. Finally, we use short-term antibiotic treatment to establish a model of relapsing virulent Salmonella infection where mice appear to have cleared the bacteria soon after they begin treatment but suffer recurrent and fatal Salmonella infection upon withdrawal. We demonstrate that Salmonella harbored in CD11b+Gr1- resident monocytes in mouse mesenteric lymph nodes (MLNs) are the source of relapsing infection. In addition, the MLNs appear to act as a filter prohibiting the dissemination of Salmonella to systemic tissues. By using antibiotic treatment to examine immune responses to Salmonella, this thesis work may contribute to future research in the development of efficacious therapeutic and/or preventative typhoid vaccines. Moreover, these studies may stimulate future studies using these same tools in other infectious disease models.
Gene therapy strategies for targeting the treatment refractory sites in Hurler syndrome.
(2009-03) Osborn, Mark John
The submitted work details the development of a novel gene therapy vector capable of expressing multiple genes from a single transcript. This vector allows for high level therapeutic gene expression that is coupled to a dual reporter system that allows for real time in vivo tracking of gene expression as well as cellular detection without need for antibody staining. Additionally, a fusion protein was designed to specifically target the α- L -iduronidase protein to the central nervous system by way of the transferrin receptor. This treatment resulted in a decrease in glycosaminoglycan storage material in the brain of mucopolysaccharidosis type I mice. Lastly, we implemented the use of an episomally maintained plasmid-based vector that mediates high levels of protein production in vivo over a long period of time. Cumulatively this work has generated novel findings that will contribute to the field of gene therapy as a whole.
Host and viral determinants of respiratory reovirus infection.
(2012-04) Nygaard, Rachel M.
Many viruses enter hosts by invading mucosal surfaces. Mammalian reoviruses gain access to the host by the gastrointestinal and respiratory tracts. Determinants of reovirus infection of the gastrointestinal tract are well- understood, however the host and viral determinants of respiratory reovirus infection are unknown. The work within this thesis characterizes the host and viral determinants of respiratory reovirus infection and systemic dissemination. To accomplish this, we developed a murine model of respiratory reovirus infection. Using this model, we showed that endogenous respiratory and inflammatory proteases can promote reovirus infection in vitro and that pre- existing inflammation augments in vivo infection in the murine respiratory tract. Through this work, we identified two laboratory isolates of T3D, T3DC and T3DF, that differ in their capacity to replicate in the respiratory tract and spread systemically, and we used these viruses to describe genetic polymorphisms that regulate reovirus replication and dissemination. The data presented in this thesis illustrate that reovirus infection of the respiratory tract and systemic dissemination are influenced by multiple factors including virion composition, resistance or sensitivity to protease-mediated inactivation and respiratory inflammation.
hTERT enables heterogeneity in a population of HMEC which sustains oncogene sensitive and resistant clones
(2012-12) Gomez-Garcia, Jose
Mammalian cells activate multiple mechanisms of defense against uncontrolled proliferation when oncogenic mutations occur. In this way, the process of transformation relies on breaching such defenses to promote cancer. Telomere maintenance, mediated by expression of telomerase in cancer cells, is associated with evasion of proliferative senescence, a known barrier against cancer. Whether telomerase contributes to deregulate activation of other mechanisms of defense is not clear in the field. We decided to study whether expression of telomerase deregulates the activation of known mechanisms of defense by comparing primary Human Mammary Epithelial cells (HMECS) and hTERT immortalized HMECS when they express oncogenic Ras. To our surprise we found that contrary to primary cells, hTERT immortalized cells respond differently against oncogenic Ras by undergoing post-replication arrest and destructive autophagy. Although these barriers were available, the population of cells did not respond uniformly against oncogenic Ras. Instead, we observed variability in the execution of these responses. Our results suggest that the process of immortalization fosters the opportunity for evolution of the population. In these conditions many cells can harness mechanisms of defense while a minority of cells becomes resistant to them, an important characteristic for further cancer progression.
Hydrocarbon biosynthesis by bacteria : genes and hydrocarbon products.
(2010-12) Sukovich, David John
Various publications have reported that microorganisms have the ability to produce hydrocarbons. One of these organisms, Vibrio furnissii M1, was reported to produce n-alkanes. Genomic analysis and biochemical studies revealed that the findings reported by Park et al. were not reproducible in our laboratory. Other heterotrophic bacteria were shown to produce hydrocarbons though. One of these organisms, Shewanella oneidensis MR-1, was found to produce 3,6,9,12,15,19,22,25,28-hentriacontanonaene. Hydrocarbon production in S. oneidensis was dependent upon the polyunsaturated fatty acid synthesis pathway and a relationship between temperature and hydrocarbon production was identified. Genomic analysis and mutation studies found that hydrocarbon production was dependent upon a gene cluster, designated oleA, oleB, oleC, and oleD. The OleA protein condenses two fatty acyl CoA chains in a head-to-head manner to produce a compound that, if the OleBCD proteins are not present, is spontaneously decarboxylated to a ketone. Homologs to the oleABCD genes were found in all heterotrophic bacteria reported to produce hydrocarbons. Searches of genomic databases found that 1.9% of all sequenced genomes have oleABCD gene homologs. These bacteria include members from the γ- and δ-Proteobacteria, Actinobacteria, Verrucomicrobia, Planctomycetes, and Chloroflexi Phyla. Bacteria containing the oleABCD homologs not previously characterized for hydrocarbons were obtained and tested for polyolefin production. It was found that if the genes were present, bacteria produced alkenes. A correlation between OleA amino acid sequence and product formation was also discovered. When different OleA proteins were expressed heterologously in non-native bacterial backgrounds, the bacteria hosts were able to produce ketones. Ketone production could be increased using alternative plasmid promoters and regulation sequences. Preliminary experiments investigated strategies for cloning and expressing an oleA gene in cyanobacteria heterologously. Also, exploratory experiments were conducted to determine if ketone production by Shewanella might enhance cell growth when antibiotics, detergents, or other potentially inhibitory chemicals were added to the growth media.
Identification and analysis of candidate MLL-AF9 cooperating genes in acute myeloid leukemia.
(2010-07) Bergerson, Rachel Joy
Human patients with a t(9;11) translocation (MLL-AF9) develop acute myeloid leukemia (AML) but evidence suggests that the product of the translocation requires additional cooperating genetic events for full-blown disease to develop. A retroviral insertional mutagenesis screen was performed in mice transgenic for the Mll-AF9 fusion oncogene, which also developed myeloid leukemia with a reduced latency compared to controls. We identified 88 candidate cancer genes near common sites of proviral insertion, including Fosb and Mn1. We found elevated expression of some candidate genes in leukemic tissues that were also upregulated in human AML harboring MLL gene translocations. A functional validation of several candidate genes was performed using RNAi lentiviral vectors in vitro and BMTT assays in vivo. We found the Open Biosystems libraries were not optimized for a hematopoietic system and shRNAs were not effective in all cells lines of causing gene knockdown or phenotype change. However, we still observed a requirement of FOSB for the maintenance the human U937 myeloid leukemia cell line. We also showed MN1 cooperated with Mll-AF9 in leukemogenesis in an in vivo bone marrow viral transduction and transplantation assay. To further investigate these leukemias, we transplanted bone marrow from the infected Mll-AF9 leukemic mice into recipient animals, which also succumb to myeloid leukemia. We established four AML cell lines from the recipient bone marrow with different signaling profiles and used them to test inhibitors against molecules in the receptor tyrosine kinase (RTK) and related pathways. The inhibitors were mostly ineffective in low doses but when cells were treated with combinations of drugs, dramatic changes in cell cycle and strong inhibitory effects on intracellular signaling were observed with variability for each cell line. The best combinations in all cell lines affected more biochemical targets and caused a prolonged apoptotic induction and inhibition of cell proliferation after three days of treatment. Our model of Mll-AF9 myeloid leukemia, induced with cooperating mutations provided by MuLV, helps define the genetic alterations in genes and pathway that are important in progression of leukemia with an MLL fusion. Furthermore, cell lines created from these leukemias are a valuable preclinical tool for assessment of cellular and biological response to inhibitors and therapeutic agents in AML cells with the Mll-AF9 translocation.
Identification and characterization of novel genetic determinants of biofilm formation in Enteroccous faecals.
(2010-05) Ballering, Katie Sunrise
The nosocomial pathogen Enterococcus faecalis is a normal resident of the intestinal tracts of many vertebrates and invertebrates, including humans, and is readily isolated from many other environments. It has been well documented that E. faecalis can form biofilms on biotic and abiotic surfaces, and enterococcal biofilms likely play a role in virulence, persistence, and horizontal gene transfer of this organism. In my thesis research I used a large genetic screen to identify over 68 potential determinants of enterococcal biofilm formation. This, in conjunction with another genetic screen from our lab, constitute the first comprehensive examination of the core genome of E. faecalis for genetic determinants of biofilm formation. I characterized the role in biofilm formation of a novel transcription factor, Enterococcal Biofilm Regulator (EbrA), identified in a Recombinase Based In Vivo Expression Techonology (RIVET) screen. This transcription factor was differentially expressed between biofilm and planktonic cells at 24 hours, and a mutant strain in which the open reading frame was deleted was defective in its ability to form biofilms as compared to wild-type. To determine the regulon of EbrA, I utilized methods that examined the proteome and the transcriptome and determined that EbrA is responsible for modulating the metabolic rate to promote survival of the biofilm population when it undergoes nutrient stress. As a function of this survival role, my experiments suggest that EbrA is also a negative regulator of the cell lysis mechanism, that is thought to mediate eDNA release for biofilm matrix production. The current model of eDNA release did not include a negative regulator. Biofilm development is a dynamic process in which the population undergoes changes in both gene expression and metabolic activity. Previous characterizations have focused mostly on biofilms grown for at least 24 h. There is little information about the cellular processes associated with this transition from planktonic to biofilm growth in non-motile species, such as E. faecalis. The low amounts of biomass present in these initial stages of biofilm development preclude the use of standard expression profiling analyses of mRNA and protein. My thesis research also describes comparative analysis of the increase in biomass and adherent bacterial populations in the early stages of biofilm formation by the laboratory strain in relation to several clinical isolates. I then combined this analysis with high-resolution Field Emission Scanning Electron Microscopy (FESEM) analysis of the cell surface and the extracellular matrix of the developing biofilms. These studies revealed a dramatic temporal change in the appearance and biochemical composition of the extracellular matrix that has not been previously reported. The data also suggest that the biochemical composition of the biofilm matrix changes over time. One of those phenotypes has not been previously described. This work highlights the importance of careful kinetics studies, including very early time points, to identify novel phenotypes in complex biofilm growth systems.

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