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Establishment and Improvement of Preclinical Murine Models of Severe Scrub Typhus to Define Immune Signatures and Pathogenic Mechanisms
(August 2023) Thiriot, Joseph David 1989-; Soong, Lynn; Sun, Keer; Walker, David; Comer, Jason; Riley, Sean
Scrub typhus is a leading source of febrile illness in endemic countries due to infection with Orientia tsutsugamushi (Ot), a seriously understudied intracellular bacterium. Scrub typhus causes 150,000 deaths per year, with an estimated 1 million cases, and a further 1 billion people at risk. No vaccine is available for this reemerging and severely neglected infection. Frustratingly, scrub typhus can result from infection due to a variety of Ot strains with limited cross protection. However, little is understood about strain-specific virulence factors or host immune response. While there are many established murine models of scrub typhus, differences in route of infection, host resistance, and clinically unrelated pathologic outcomes diminish the usefulness of model studies. Therefore, there is a great need for the development of small animal models that reproducibly mimic human diseases for immunologic investigation and future vaccine studies. Our recent studies have established a new inbred murine model of hematogenous spread. To expand these models, we have established an outbred murine model of hematogenous transmission that mimics tissue tropism and pathologic lesions of scrub typhus patients. Interestingly, we find this outbred model exhibits the same pro-inflammatory/Th1 skewed immune response as seen in our previously established inbred model. We further developed our outbred and inbred models by using two clinically prevalent O. tsutsugamushi strains, Karp and Gilliam, and reveal cellular immune responses in inflamed lungs and potential biomarkers of disease severity. We find that outbred CD-1 mice are highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice are susceptible to Karp, but resistant to Gilliam (with self-limiting infection). Our outbred preclinical model is particularly useful for future translational and vaccine studies for severe scrub typhus. This body of work provides the first evidence of differential tissue cellular responses in an inbred model against Karp vs. Gilliam infection, thus offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis.
HCV Core Protein affects Lipid Metabolism in a Genotype-Dependent Manner
(2020-08-01T05:00:00.000Z) Hull, Madeleine E
Chronic HCV infection is the leading cause of steatosis (fatty liver disease) and hepatocellular carcinoma (HCC). The virus establishes a chronic infection in 70% of patients and infects approximately 71 million people worldwide. Genotypes (gt) 1 and 3 are the most prevalent, with gt3 HCV being associated with more severe disease. It is known that core protein plays a role in the development of steatosis, but the precise mechanism is not yet understood. In this study, we investigate the role of genotypes in core-mediated differential regulation of lipid metabolism. Prior studies described the lipid induction by HCV infection or ectopic expression of core derived from gt1a and 3a HCV qualitatively. The goal of this thesis is to define the gt1a and 3a HCV core-mediated lipid regulation in a quantitative manner. Using FACS analysis, and immunofluorescence analysis by using confocal microscope, we found that gt3a core protein induces larger lipid droplet formation, although the quantity of lipids remains similar to that induced by gt1a core protein. We then attempted to determine the difference in fatty liver-associated gene expression levels induced by gt1a and gt3a core proteins. To do this, we utilized microarray to analyze the gene expression in cells transfected with different HCV core proteins. The analysis showed that both gt1a and gt3a core upregulated SOCS3; this upregulation has been shown to influence the liver response to previously used antiviral therapies, as well as the induction of insulin resistance. Additionally, genes that were downregulated by gt1a and gt3a core are largely associated with insulin resistance, which plays a role in the development of diabetes. Interestingly, more genes appear to be downregulated by gt3a than gt1a core protein, which could suggest that gt3a core protein induces insulin resistance and steatosis in a different manner, or to a more severe degree. Overall, these results present a potential mechanism for the relationship between insulin resistance and lipid metabolism deregulation induced by gt1a and gt3a core proteins.
Proviral Roles of Ebola virus VP35 Ubiquitination
(2022-05-01T05:00:00.000Z) Van Tol, Sarah Rose
Ebola virus’ structural protein VP35 is polyfunctional and plays vital roles in Ebola’s life cycle from antagonizing the host’s type I interferon pathway to acting as the polymerase co-factor. The mechanisms that regulate which function any given VP35 molecule engages remains unknown. Previously, we observed the host E3 ubiquitin ligase TRIM6 conjugates ubiquitin onto VP35 at lysine (K) 309. This post-translational modification was found to be proviral, but we did not know which VP35 role(s) ubiquitination regulated. We generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant’s suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This dysregulation of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. Blocking VP35/K309 ubiquitination enhanced interaction with the viral nucleoprotein and may trigger premature nucleocapsid packaging. Prior work also showed that at least one lysine residue other than K309 is ubiquitinated and that VP35 non-covalently interacts with ubiquitin. We observed that multiple residues in VP35’s N-terminus can receive covalent ubiquitin, including K119, 126, and 141. Substitution of K119-, 126-, and/or 141-to-R significantly attenuates VP35’s polymerase co-factor activity, but their mutation does not alter interactions with the viral polymerase or nucleoprotein. When looking into VP35’s non-covalent ubiquitin interaction, we found that VP35 specifically binds K63-linked ubiquitin chains via its C-terminus. The specific cleavage of unanchored ubiquitin chains, using the deubiquitinase isopeptidase T, impedes VP35’s non-covalent binding to ubiquitin and stunts EBOV’s polymerase activity. Finally, we also found that TRIM25, TRIM6’s relative, facilitates ubiquitin ligation onto VP35 and enhances VP35’s non-covalent interaction with ubiquitin. EBOV replication is attenuated 100-10,000 fold in cells lacking TRIM25. Overall, our data support that TRIM6- and TRIM25-mediated VP35 ubiquitination and VP35’s non-covalent interaction with unanchored ubiquitin is proviral.
Small Molecule Allosteric Modulation of G Protein-Coupled Receptors and Applications in the Pharmacological Targeting of 5-HT2 Receptors
(2021-05-01T05:00:00.000Z) Wold, Eric A.
The discovery of G protein-coupled receptors (GPCRs) and the extensive second messenger systems associated with their activation has ushered in an enormously productive period in drug discovery. The extent to which around one-third of FDA-approved medications target GPCRs. Predictably, small molecule allosteric modulation has emerged in recent years as a new means to control GPCR function with numerous examples nearing FDA approval. This body of work begins with a substantive review of drug discovery efforts in the development of Class A GPCR allosteric modulators as a means to compile successful strategies and take note of the distinct challenges in the field as our group approaches allosteric modulator discovery for the serotonin 5-HT2C receptor (5-HT2CR). From the time of its characterization, the 5-HT2CR has been marked by unique, untapped potential as a drug discovery target for numerous diseases and disorders of the central nervous system. Thus, the second chapter in this work provides a clear rationale for 5-HT2CR allosteric modulator discovery in context of the neurobiological framework wherein the 5-HT2CR plays an integral role in reward-related behaviors and cortical executive functions. With the rationale established, the following sections report our efforts in the discovery of novel 5-HT2CR positive allosteric modulators (PAMs) from their design and chemical synthesis to the in vitro and in vivo characterization of these molecules. Additionally, further work describing 5-HT2CR PAM pharmacokinetic properties as suitable for rodent behavioral assays and the structural determinants of 5-HT2CR PAM binding via molecular modeling are discussed. Having discovered allosteric modulators with functionality across the 5-HT2R subfamily and benefiting from the wealth of available structural data for these targets, the final chapter delves into a theoretical mechanism underpinning allosteric modulation of 5-HT2Rs. The enhanced activation state (EAS) is thus coined for the first time herein to describe 5-HT2R PAM functionality and is treated with a rigorous theoretical framing comprised of the observed pharmacological, structural, and computational studies that shape our understanding of GPCR dynamics, specifically the activation dynamics of 5-HT2Rs. The resultant body of work provides the reader a comprehensive understanding of allosteric modulation and its pharmacological utility in targeting 5-HT2Rs.
Opioid Prescribing and Opioid-Related Health Outcomes Among Cancer Survivors
(2023-05-01T05:00:00.000Z) Gibson, Derrick C
The US population of older long-term cancer survivors—Americans who are free of cancer 5-years post-cancer diagnosis and not receiving cancer treatment—is growing. The prevalence of pain among cancer survivors after curative treatment is approximately 40% and opioids are frequently prescribed to manage the pain. The purpose of this dissertation is to explore long-term opioid therapy and opioid-related harms in cancer survivors using Surveillance Epidemiology and End Results – Medicare linked datasets. First, we explored the temporal and geographical variation in long-term opioid therapy among cancer survivors in the United States. We found that long-term opioid therapy rates were highest in the south and lowest in the northeast and that long-term opioid therapy rates peaked in 2012 but declined until 2016. Second, we assessed if patient level pain conditions and provider specialties seen at outpatient visits by cancer were associated with long-term opioid therapy. We found that cancer survivors who had been diagnosed with chronic pain or noncancer pain conditions and who were treated by noncancer specialists were more likely to receive long-term opioid therapy. Third, we assessed if cancer survivors were more likely than noncancer controls—matched on age, gender, race, pain conditions, previous opioid use—to experience an opioid-related emergency department visit or hospitalization. We found that the incidence of opioid-related adverse events were five times higher among cancer survivors who used opioids previously than opioid naïve cancer survivors. We found cancer survivors were as likely as persons without cancer to experience an opioid-related emergency department visit or hospitalization. In conclusion, we found high prevalence rates of long-term opioid therapy that differed by time and US geographical region and the risk of an opioid-related emergency department visit and hospitalization is comparable between cancer survivors and persons without a history of cancer. Our findings support the idea that policies and guidelines should continue to promote and incentivize the use of nonpharmacological and nonopioid interventions for managing pain among older adults.