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Browsing by Author "Basaraba, Randall J., advisor"

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    Infectious disease, age, and environmental contaminants as neurotoxicants that modulate glia and contribute to neurodegenerative pathology
    (Colorado State University. Libraries, 2024) Latham, Amanda Shellee, author; Moreno, Julie A., advisor; Basaraba, Randall J., advisor; Tjalkens, Ronald B., committee member; Santangelo, Kelly S., committee member; Elf, Jessica, committee member
    Neurodegenerative disease cases are expected to double over the next twenty years. These diseases, which include Alzheimer's Disease (AD) and Parkinson's Disease (PD), are incurable with a largely unknown etiology. It is acknowledged within the field that age is the greatest risk factor for neurodegenerative disease, and that genetics and environmental factors, such as neurotoxicants and infectious agents, likely play a role. Despite this knowledge, it is not entirely understood why select individuals are pushed into a state of disease, while others progress into a state of normal brain aging. This is further complicated by the shared neuropathology between brain aging and neurodegenerative disease, which includes blood-brain barrier (BBB) modulation, gliosis, misfolded protein accumulation, and loss of function or degradation of neurons. To address these gaps in our understanding, the studies herein provide valuable insight as to how infectious disease, specifically through infection with Mycobacterium tuberculosis, contributes to the progression of neuropathology, evaluates an alternative model of brain aging that better recapitulates human disease, and provides mechanistic understanding of the neuroprotective and neurotoxic roles of glia in disease. Altogether, these data elucidate the etiology and mechanisms that drive neurodegenerative disease, as well as possible therapeutic avenues that may bring us one step closer to a cure.
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    Metformin: a tool to better understand T cell mediated protection against Mycobacterium tuberculosis
    (Colorado State University. Libraries, 2020) Haugen Frenkel, Jessica D., author; Basaraba, Randall J., advisor; Obregón-Henao, Andrés, committee member; Podell, Brendan K., committee member; Chicco, Adam, committee member; Avery, Anne, committee member
    To view the abstract, please see the full text of the document.
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    Pathogenesis of experimental tuberculosis in guinea pigs
    (Colorado State University. Libraries, 2010) Sivagiri Palanisamy, Gopinath, author; Basaraba, Randall J., advisor; Orme, Ian, advisor; Mason, Gary L., committee member; Bowen, Richard Arnold, committee member
    Tuberculosis is an important infectious disease of humans that can be modeled in a number of small laboratory animal species. In humans and guinea pigs, infection with the causative agent Mycobacterium tuberculosis, incites a chronic inflammatory response in the lung (pulmonary) and other (extra-pulmonary) tissues and organs of the body. Granulomatous inflammation can become organized into a distinct inflammatory mass referred to as a granuloma. Granuloma formation is thought to represent a favorable host response that functions to contain the infection, thus preventing spread within or between susceptible hosts. If infected cells within the granuloma die, intracellular bacteria are released and become entrapped in an extra-cellular microenvironment where they persist for long periods of time protected from drug therapy and the host immune response. The mechanism responsible for granuloma cell death (necrosis) is unknown but is important to understand as it represents a unique microenvironment for drug-tolerant bacilli to persist. One potential mediator of granuloma necrosis is the generation of cell and tissue damaging oxygen free radicals, also known as reactive oxygen species (ROS), a hypothesis tested in these studies. We used the guinea pig model of human tuberculosis to test what influence bacterial strain had on the development of pulmonary and extra-pulmonary granuloma necrosis. Our studies showed that the virulence of clinical isolates of M. tuberculosis was reflected in more severe and widely disseminated disease in experimentally infected guinea pigs and was a better predictor of virulence than the bacterial burden determined by culture. These data provide supporting evidence that the extent of lesion necrosis correlated with the severity of disease and is an important determinant in the clinical outcome of tuberculosis. We concluded that both host and pathogen factors contribute to the pathogenesis of lesion necrosis during M. tuberculosis infection. To determine the host factors that contribute to the pathogenesis of lesion necrosis, we focused on the role ROS generation has in the pathogenesis of lesion necrosis in experimental tuberculosis and explored whether this adverse response could be controlled therapeutically or through vaccination of guinea pigs with M. bovis BCG prior to virulent challenge. We found that depletion of host antioxidant defenses was a major determinant in the imbalance between the generation of ROS and host antioxidant capacity in this tuberculosis model. Moreover, we attributed the decreased expression of key antioxidant proteins to a defect in the function of a critical antioxidant transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2). We were able to partially restore Nrf2-mediated antioxidant defenses therapeutically in M. tuberculosis infected guinea pigs with the antioxidant drug N-acetylcysteine. We also established that low density lipoproteins were among the host macromolecules that are oxidized during the chronic inflammatory response typical of tuberculosis. Oxidized low density lipoproteins (OxLDL), known to be rich in cholesterol, accumulated in macrophages during infection and elevation of OxLDL levels was accompanied by increased expression of the OxLDL scavenger receptors CD36 and LOX-1. The significance of these data are that through the use of the guinea pig tuberculosis model, we have uncovered a previously unrecognized mechanism by which the host and pathogen interact to create a unique microenvironment that allows difficult to treat M. tuberculosis to persist. The characterization of these host-pathogen interactions may lead to the development of novel adjunct therapies aimed at preventing the adverse effect of M. tuberculosis infection in humans.
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    Potentiation of beta-lactam antibiotics against Mycobacterium tuberculosis by 2-aminoimidazoles: investigation into the mechanism of action and its relevance to mycobacterial bioenergetics
    (Colorado State University. Libraries, 2017) Jeon, Albert Byungyun, author; Basaraba, Randall J., advisor; Borlee, Brad, committee member; Gustafson, Daniel, committee member; Jackson, Mary, committee member; Melander, Christian, committee member; Obregón-Henao, Andrés, committee member
    Tuberculosis, caused by Mycobacterium (M.) tuberculosis, is a global health problem still causing morbidity and mortality due in part to the emergence of drug-resistance and the lack of new antimicrobial agents to treat the disease. While infection with drug-sensitive M. tuberculosis has cure rates between 90-95% with the conventional multidrug-regimen comprised of four different first-line anti-tuberculosis drugs, administered for a minimum of 6 months. In the event where premature termination of the treatment or poor patient compliance occurs, the disease may progress into latent tuberculosis, which holds the risk of reoccurring disease or even leads to development of drug-resistant strains that are refractory to first line anti-tuberculosis drugs. This persistence is a major hurdle in global tuberculosis control and warrants the development of a new class of anti-tuberculosis drugs or novel strategies to target persisting bacilli. However, the current anti-tuberculosis drug pipeline does not suggest an immediate solution required for the successful control of global tuberculosis epidemic. In sum, there is an urgent need for a new strategy to complement current tuberculosis chemotherapy. 2-aminoimidazoles and their derivatives have been shown to be effective inhibitors of bacterial biofilms. Not only does this class of small molecules inhibit the formation of or disperse biofilms, but they also exhibit a clinically relevant feature of potentially abrogating antibiotic resistance in important pathogenic bacteria. From the studies characterizing persistent M. tuberculosis bacilli after anti-tuberculosis therapy in animal models, it has been suggested that this subpopulation of bacilli share similarities with bacterial biofilms. Our group developed an in vitro culture system where M. tuberculosis can be cultured in biofilm-like surface-attached communities with host-derived macromolecules and showed they express extensive drug-tolerance to one of the first-line anti-tuberculosis drug, isoniazid. Based on the previous effects of 2-aminoimidazoles on biofilms and drug-resistant bacteria, we hypothesized that 2-aminoimidazoles could reverse phenotypic drug-tolerance expressed by M. tuberculosis in our model and demonstrated that, indeed, derivatives of 2-aminoimidazoles effectively resensitized drug-tolerant bacilli to isoniazid. Additionally, a fortuitous but critical observation was made in which one of the potent 2-aminoimidazole derivatives potentiated the effect of ß-lactam antibiotics against M. tuberculosis. As repurposing ß-lactams in tuberculosis treatment regimen has potential therapeutic value, which are described throughout this dissertation. In chapter 2, 2-aminoimidazole compounds are shown to be effective at potentiating multiple ß-lactam antibiotics. Minimum inhibitory concentrations, as well as bactericidal concentrations, of ß-lactams were dramatically reduced when combined with 2-aminoimidazoles. Through a transcriptional analysis of M. tuberculosis treated with 2B8, one of our lead 2-aminoimidazoles induced cell envelope related stress responses and suppressed mycolic acid biosynthesis. Thereafter, it was hypothesized that 2-aminoimidazoles disrupts one or more factors conferring M. tuberculosis ß-lactam resistance, which we shown in chapter 3 is in large part due to a reduction in secretion of the enzyme ß-lactamase and by increasing cell envelope permeability. 2B8 treated M. tuberculosis exhibited significantly lower ß-lactamase activity in culture supernatant, which was due to a general protein secretion defect, and not from direct inhibition of ß-lactamase enzyme activity by 2-aminoimidazole compounds. As expected from the transcriptional analysis, 2B8 induced alterations in cell envelope lipid composition highlighted by the accumulation of trehalose monomycolate, the reduction of trehalose dimycolate, as well as a decrease in mycolic acid biosynthesis. Additionally, increased sensitivity to the detergent SDS, increased permeability to multiple nucleic acid staining dyes, and increased bindings of peptidoglycan-targeting antibiotics were observed when with 2B8 treatment. Based on major findings from chapter 3, it was hypothesized that the underlying mechanisms of 2-aminoimidazoles are the disruption of proton motive force and the disturbance of mycobacterial bioenergetics. In chapter 4, the collapse of proton motive force with additional dose-dependent block of mycobacterial electron transport chain is highlighted. Through a series of assays, we determined that 2B8 blocks the M. tuberculosis electron transport chain downstream of complex I and II, but upstream of complex IV. Taken together, these results collectively extend our current understanding of the various effects 2-aminoimidazole treatment has on M. tuberculosis susceptibility to ß-lactam antibiotics through perturbation of mycobacterial bioenergetics which can provide a profound impact in improving current tuberculosis therapy. Furthermore, this study offers valuable information for the construction of the next generation of potent 2-aminoimidazoles to improve efficacy against M. tuberculosis as well as other compounds that may be developed as a new anti-TB drug targeting bioenergetics.
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    The pathogenesis of diabetes-tuberculosis comorbidity
    (Colorado State University. Libraries, 2014) Podell, Brendan K., author; Basaraba, Randall J., advisor; Dow, Steven, committee member; Rovnak, Joel, committee member; Jackson, Mary, committee member; Ishii, Douglas, committee member
    Exposure to the bacterium, Mycobacterium tuberculosis, only leads to the active form of tuberculosis disease (TB) in 5-10% of infected individuals. The development of active TB, at any stage of infection, is often the result of a known TB risk factor, either intrinsic to the individual or acquired as a communicable or non-communicable disease. An association between diabetes and TB has long been recognized, but only recently was diabetes confirmed to increase the risk of developing active TB disease. The convergence of a growing diabetes epidemic on regions with endemic TB has positioned diabetes as an emerging global threat to TB control. Of particular importance is the rapidly growing incidence of type 2 diabetes, which accounts for up to 95% of the global diabetic population. Since the potential impact of this growing comorbidity has only been recently emphasized, little is known regarding the mechanisms of dysregulated immune function and metabolism by which diabetes predisposes to active TB disease. The current understanding of this comorbidity is further limited by the lack of appropriate animal models that replicate the pathogenesis of both human type 2 diabetes and TB. The guinea pig is a well-established model of TB that replicates human pathology and disease progression. This species was emphasized in this series of studies with the goal of better understanding the impact of type 2 diabetes on TB progression and the mechanisms that may change the host response to M. tuberculosis infection. In Chapter 2, we investigated the impact of hyperglycemia alone, induced as post-prandial hyperglycemia through daily administration of sucrose, on TB disease progression in non-diabetic guinea pigs. Guinea pigs receiving daily sucrose developed both higher bacterial burdens in pulmonary and extrapulmonary tissue and also more severe pathology by day 60 of infection. This exacerbated disease manifestation was accompanied by the accumulation of advanced glycation end-products, which are inflammatory by-products of chronic hyperglycemia with known involvement in the development of diabetes-related complications. Interestingly, by monitoring glucose and lipid metabolism in these guinea pigs, we learned that TB alone leads to severe metabolic disturbances, manifesting as hyperglycemia and accumulation of circulating total free fatty acids. From this study, we were able to conclude that not only does mild post-prandial hyperglycemia worsen the course of TB disease in guinea pigs, but also, infection with M. tuberculosis alone induces metabolic disease resembling diabetes, similar to what has been previously reported in human TB. These conclusions rationalize the investigation of novel adjunctive therapies to restore metabolic homeostasis, which may improve the host response to infection, limit bacterial growth, and increase the efficacy of frontline antimicrobial drugs. In Chapter 3, we developed a novel model of type 2 diabetes in the guinea pig to be used in future investigations of type 2 diabetes-TB comorbidity. Previously, the guinea pig as a diabetic model has been described only in the context of β-cell cytotoxicity with the drug, streptozotocin (STZ), but with variable efficacy. In this study, we initially optimized the dose response and STZ preparation to achieve an induction of hyperglycemia that was uniform with limited mortality. This hyperglycemic response was transient but could be stabilized through continued β-cell stress, in the form of a high fat, high sugar diet. Feeding of this modified diet led to impaired glucose tolerance and a compensatory β-cell response that could be abrogated with the use of a single optimized dose of STZ. This novel model of type 2 diabetes develops both insulin resistance and β-cell failure, which replicate the typical progression of type 2 diabetes in humans, all within a reasonable experimental timeframe. From this study, two models emerged, a type 2 diabetic guinea pig as well as a model of impaired glucose tolerance, or prediabetes, that would be used to investigate the mechanisms of diabetes-TB comorbidity. In Chapter 4, the newly developed guinea pig models were used to investigate the overall impact of type 2 diabetes and impaired glucose tolerance on TB progression and the host immune response to M. tuberculosis infection. Although impaired glucose tolerance alone had limited impact on TB progression with exacerbation of disease only at chronic end points, M. tuberculosis infected type 2 diabetic guinea pigs closely resembled the reported manifestations of human diabetes-TB comorbidity including more severe TB disease, higher bacterial burdens, and a robust innate and cell-mediated immune response. Despite evidence of strong Th1 cell-mediated immunity, which is known to be critical for limiting bacterial growth and disease progression, diabetic guinea pigs were unable to control bacterial growth and developed damaging neutrophilic inflammation. To better understand the immune mechanisms leading to uncontrolled bacterial growth and severe disease, in Chapter 5, we investigated the innate and adaptive immune response over the course of early infection in type 2 diabetic guinea pigs. Diabetic guinea pigs were slow to develop early lesions with delayed bacterial transport to the lung draining lymph node, and a corresponding delay in antigen-specific Th1 immunity. Early alterations in cytokine expression were identified that may explain the delayed development of cell-mediated immunity and allow for substantial growth of M. tuberculosis in the lung of infected diabetic guinea pigs. These data indicate that not only does type 2 diabetes increase the severity of TB but also that the chronic inflammatory process associated with TB itself may worsen diabetes. This has important implications worthy of further investigation revolving around the diagnostic criteria for diabetes when associated with TB, the impact of active TB on medical management of diabetes, and the investigation of novel therapeutic targets, both metabolic and immunological, to enhance the host immune response to infection and limit TB disease severity in diabetics.