Hyperhomocysteinemia (hHcys), an important pathogenic factor contributing to the progression of end-stage renal disease (ESRD), has been shown to activate NOD-like receptor protein 3 (NLRP3) inflammasomes and cause podocyte dysfunction and glomerular sclerosis. hHcys induces aggregation of the three inflammasome components – NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1 – and its activation is indicated by increased caspase-1 activity and secretion of interleukin-1β (IL-1β). The aims of the present study sought to elucidate the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated redox signaling in hHcys-induced NLRP3 inflammasome activation, to dissect the contribution of common endogenous reactive oxygen species (ROS) including superoxide (O2•−), hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hydroxyl radical (•OH), and to explore the molecular mechanisms by which the NLRP3 inflammasome senses changes in oxidative stress through thioredoxin-interacting protein (TXNIP). Specific inhibition of the gp91phox subunit of NADPH oxidase markedly reduced Hcys-induced caspase-1 activity and IL-1β production in cultured podocytes. Concurrently, gp91phox−/− or administration of a gp91ds-tat peptide also exhibited diminished glomerular inflammasome formation and activation in mice fed a folate-free (FF) diet to induce hyperhomocysteinemia and displayed glomerular protection as shown by prevention of hHcys-induced proteinuria, albuminuria and glomerular sclerosis. Interestingly, dismutation of O2•− by 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl and administration of H2O2 decomposer catalase either in cultured podocytes or hyperhomocysteinemic mice inhibited hHcys-induced NLRP3 inflammasome aggregation and activation. Hyperhomocysteinemic mice also demonstrated a significant increase in glomerular TXNIP binding to NLRP3, confirmed by confocal microscopy, size-exclusion chromatography, and co-immunoprecipitation studies. Blockade of TXNIP by genetic interference or by the calcium channel blocker verapamil prevented this hHcys-induced TXNIP-NLRP3 binding, NLRP3 inflammasome formation and activation, as well as protected hyperhomocysteinemic mice from glomerular dysfunction and damaged morphology. In conclusion, hHcys-induced NADPH oxidase activation is importantly involved in the switching on of NLRP3 inflammasomes in podocytes, where NADPH oxidase-derived O2•− and H2O2 primarily contribute to NLRP3 inflammasome activation. TXNIP binding to NLRP3 is a key signaling mechanism allowing NLRP3 inflammasome to sense these changes in oxidative stress. These findings greatly enhance our understanding of the early pathogenesis of hHcys-induced glomerular sclerosis, which may identify new therapeutic targets for prevention or treatment of ESRD.

Aberrant metabolism has become an increasingly interesting area of cancer biology. In many cancers including lower grade glioma, glioblastomas and some leukemias, a mutation in the metabolic enzyme Isocitrate Dehydrogenase (IDH), has been found in more than 70% of cases and has been shown to lead to a distinct hypermethylator phenotype. IDH commonly converts isocitrate to alpha-ketoglutarate in normal cell metabolism. Three isoforms of this enzyme are found in humans: IDH1, IDH2 and IDH3. Studies on IDH1, the cytosolic isoform, have revealed that mutations in the enzyme’s binding site lead to a novel gain of function: the synthesis of an oncogenic metabolite, 2-hydroxyglutarate (2HG). 2HG competitively inhibits alpha-ketoglutarate dependent enzymes such as the TET 5-methylcytosine (5mC) oxygenases and histone demethylases. These oxygenases are responsible for the hydroxymethylation (5hmC) of cytosine residues, ultimately leading to demethylation and gene re-expression. Thus, mutant IDH may lead to an elevation in 5mC levels producing the hypermethylator phenotype described. A similar gain-of-function mutation in IDH2, the mitochondrial isoform of IDH1, has been associated with leukemias and gliomas lacking IDH1 mutations. Mutations in IDH1, IDH2 and TET2 are mutually exclusive, and each yields a similar hypermethylator phenotype. IDH2, along with IDH3, is primarily involved in the TCA cycle and energy production for the cell. Recently, the Taylor lab has uncovered evidence of 5mC and 5hmC residues in mitochondrial DNA, established and maintained by mtDNMT1 and TET2. Changing levels of mtDNMT1 appears to alter the patterns and levels of mtDNA transcription from the mitochondrial genome. We hypothesized that mutant IDH would produce a similar effect on the mitochondrial genome as that found in the nuclear genome and result in a decrease in the level of 5-hydroxymethylcytosine, as well as a subsequent increase in the level of 5-methylcytosine caused by the competitive inhibition of the TET enzymes by 2-hydroxyglutarate accumulation. Using molecular biology techniques such as Western blots and MeDIP (methylated DNA immunoprecipitation) I aim to uncover the role of IDH mutation on mitochondrial DNA methylation and its effect on energy production in mammalian cells.

A DIRECT AND INDIRECT MECHANISM FOR CCR5 IN OPIOID AND HIV-1 MEDIATED NEURODEGENERATION By Elizabeth M. Podhaizer, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2014 Major Director: Kurt F. Hauser, Ph.D., Professor of Pharmacology & Toxicology Human immunodeficiency virus (HIV)-1 infection currently affects over 34 million people worldwide, and despite the use of cART, the prevalence of HIV-1 associated neurocognitive impairments (HAND) has not declined. Additionally, other co-morbid factors such as the abuse of injection drugs (i.e. heroin, morphine) increase both the frequency and the speed by which patients progress to AIDS. To begin to understand the mechanisms, we chose to examine a pathway, through CCR5, which may act as a convergence point for opioids and HIV-1 proteins. C-C chemokine receptor 5 (CCR5) is an immune receptor involved in physiological processes in the brain in addition to mediating neuroinflammatory signaling events, and it is a co-receptor for HIV-1. CCR5 interacts directly with gp120 to facilitate HIV-1 infection, and may interact indirectly with HIV-1 Tat through convergent signaling mechanisms. Additionally, CCR5 is modified by opioid responses, and so may be central to opioid-HIV-1 interactions that are seen in our model. We hypothesized that CCR5 would mediate the opioid-HIV-1 interaction. We examined both HIV-1 gp120 and HIV-1 Tat, both for interactions with opioids and modification by the CCR5 antagonist, maraviroc. HIV-1 gp120ADA was neurotoxic on its own, but showed no interactions with morphine. However, further probing revealed that morphine can in fact modify the neurotoxic effects of gp120, but that the response is dependent on gp120 strain. We did, however, find that morphine did enhance the neurotoxicity of Tat, which we’ve shown previously, as well as that inhibition of CCR5 can prevent this interactive effect. Additionally, use of CCR5 knockout glia or neurons modified the response and suggests that neurons and glia play different roles in the integration of opioid and HIV-1 signals. Sublethal effects of morphine and Tat were also dampened by maraviroc pretreatment or use of knockout cells, as was the secretion of chemokine ligands. Manipulation of CCR5 showed utility in preventing neurodegenerative effects both to HIV-1 proteins alone as well as to the interactive opioid-HIV-1 signaling responses and suggests that maraviroc, a cART therapeutic used to prevent viral entry, may also aid in reducing the chronic inflammatory state of the CNS that leads to the persistent neurocognitive complications.

Cancer is the second most common cause of death in the US. The most frequently observed cancer type in women is breast cancer. A special type of breast cancer is triple negative (TNBC) cancer that is characterized by lacking three receptors: estrogen, progesterone and human epithelial growth factor (HER 2). The HS578t breast cell line is a model of TNBC that also has a mutation of the p53 protein. Ionizing radiation is used widely in the clinic to debulk tumors before surgery as well as post-surgery to eliminate residual tumor cells outside the surgical field. Previous studies from our laboratory showed that inhibition of autophagy does sensitize p53 wild type MCF-7 and ZR-75 breast tumor cells to radiation. However, this is not necessarily the response in all breast cell lines. The Hs578t cells did not appear to be sensitized to radiation after inhibition of autophagy using chloroquine as a pharmacological inhibitor. The present study was designed to build upon these previous findings and further confirm that the Hs578t breast cell line could not be sensitized to radiation through autophagy inhibition. Time course studies showed a reduction of viable cell number upon irradiation of Hs578t breast tumor cells and that both autophagy and senescence were induced. Acridine orange staining was used to examine the acidic vacuole formation while β-galactosidase staining indicated the promotion of senescence. Flow cytometry was used to quantify both autophagy and senescence. Inhibition of autophagy using pharmacological inhibitors such as ammonium chloride, or genetic silencing of autophagy by beclin1, which is a protein initiator of autophagy, did not sensitize Hs578t breast tumor cells to irradiation. It shows from these studies that autophagy is not necessarily cytoprotective in all breast cancer cell lines, which should be considered in current clinical trials designed to sensitize tumor cells to chemotherapy and radiation through inhibition of autophagy.

Vital pulp therapy is the group of procedures indicated to maintain pulp vitality allowing for continued root development and apical closure in the presence of pulp exposure due to caries or trauma. Complete maturation results in stronger tooth structure that is more able to withstand occlusal forces. Historically, calcium hydroxide (Ca(OH)2) was the material of choice for a vital pulpotomy. Recently mineral trioxide aggregate (MTA) has been used as a pulp sealing material because of its biocompatibility and its ability to induce hard tissue barrier formation. The purpose of this retrospective study was to evaluate the clinical and radiographic outcome of vital pulp therapy procedures performed with MTA. All patients receiving MTA pulpotomies and MTA pulp caps at Virginia Commonwealth University School of Dentistry Graduate Endodontic Practice between November 30, 2009 and August 15, 2013 were recalled and evaluated for presence or absence of clinical symptoms, pulp vitality, radiographic evaluation of continued root development, dentin bridge formation or pulp canal obliteration. Results were analyzed descriptively. At time of treatment caries was found to be the most common etiologic factor. Fifty-seven percent of cases were symptomatic at time of treatment (43% asymptomatic). Forty-seven percent of the teeth presented with immature apices (53% with radiographically closed apices). Overall recall rate was 50%. At recall all teeth were clinically asymptomatic. Forty seven percent of teeth tested normally to cold (33% were non-responsive to cold) at recall. Pulp canal obliteration was seen in 6 cases, 4 molars and 2 anterior teeth. In conclusion, MTA pulpotomy and MTA direct pulp caps are a predictable treatment modality for young vital permanent teeth affected by caries or trauma.

Objective: The purpose of this study was to determine the fluoride recharging capability of Opal Seal, a fluoride releasing orthodontic primer, as compared to Transbond XT, the control. Material and Methods: 1mm x 5mm disks of Opal Seal and Transbond were prepared according to the respective manufacturer’s instructions. Initially, the samples were stored in deionized water (DI) for 8 weeks. The samples were then randomly divided into one of two groups: Over-the-counter (OTC) fluoride mouthwash and prescription strength (PS) fluoride mouthwash. The OTC group samples were immersed in 5mL of 0.0219% sodium fluoride containing mouthwash for one minute every day for seven days. The PS group samples were immersed in 5mL of 0.2% sodium fluoride containing mouthwash for one minute. All of the samples were suspended in 5mL fresh DI water and fluoride release measurements were taken at baseline (the end of initial 8 weeks of storage), 24 hours, 3 days, 5 days, 7 days, and 14 days. Results: Opal Seal samples treated with the OTC fluoride mouthwash exhibited significant fluctuation in fluoride ion release across time (p=0.0058). However, there were no statistically significant differences in fluoride ion release between the individual timepoints and baseline. Similarly, Opal Seal samples treated with the PS fluoride mouthwash exhibited significant variation in the fluoride ion concentration across time (p< 0.001), and a statistically significant increase over baseline was seen at 24 hours only (p= 0.0006). The control group samples treated either with the OTC or PS mouthwash did not exhibit any significant difference in fluoride ion release between any individual timepoint and baseline. Conclusion: For Opal Seal and Transbond XT, there were no statistically significant differences of fluoride concentration at any timepoint compared to baseline measurements when using OTC mouthwash. When using PS mouthwash, there was a small, statistically significant increase of fluoride concentration of the Opal Seal samples after 24 hours but no differences were seen at any other timepoints. Opal Seal did not demonstrate a substantial amount of fluoride recharge when fluoride mouthwash is used as a fluoride delivery vehicle. Future well-designed randomized controlled trials are needed to evaluate the efficacy of Opal Seal primer when coupled with the use of fluoride mouthwashes.

HIV-1 enters the CNS soon after initial systemic infection. HIV-1 can induce a wide range of neurological deficits, collectively known as HIV-1-associated neurocognitive disorders (HAND). Mature neurons are not infected by HIV-1; instead, infected and/or activated glial cells release various viral and cellular factors that induce direct and/or indirect neuronal toxicity, leading to HAND. Injection drug abuse is a significant risk factor for HIV-infection, and opiate drug abusers show increased HIV-neuropathology, even with anti-retroviral treatments. Our previous work has largely modeled HIV-neuropathology using the individual viral proteins Tat or gp120, with murine striatal neurons as targets. To model disease processes more closely, the current study uses supernatant from HIV-1-infected cells. Supernatant from HIV-1SF162 (R5-tropic)-infected differentiated-U937 cells (HIV+sup) was collected and p24 level was measured by ELISA to assess the infection. We assessed HIV+sup effects on neuronal survival and neurite growth/pruning with or without concurrent exposure to morphine, an opiate that preferentially acts through µ-opioid receptors. Effects of HIV+sup ± morphine were assessed on neuronal populations, and also by time-lapse imaging of individual cells. HIV+sup caused dose-dependent toxicity over a range of p24 levels (10-500 pg/ml). Significant interactions occurred with morphine at lower p24 levels (10 and 25 pg/ml). In the presence of glia, selective neurotoxic measures were significantly enhanced and interactions with morphine were also augmented. Importantly, the arrest of neurite growth that occurred with exposure to HIV+sup was reversible unless neurons were continuously exposed to morphine. Thus, while reducing HIV-infection levels may be protective, ongoing exposure to opiates may limit recovery. During early stage of HIV-infection R5-tropic viruses are predominant, but during later stages of disease X4-tropic viruses are more predominant; co-receptor usage switch from CCR5 to CXCR4 is crucial in disease progression to AIDS. Some previous studies have shown that drugs of abuse interact with virus or viral proteins in strain/tropism-dependent manner. Therefore, we also assessed neurotoxic effects and interactions with opiates by supernatant from HIV-1LAI (X4-tropic)-infected H9 cells. Neurotoxic effects and the interactions with opiates of HIV-1LAI-supernatant are quantitatively similar to that of HIV-1SF162. Surprisingly, the cytokine/chemokine release profile of HIV-1LAI-infected H9 cells is similar to that of HIV-1SF162-infected U937 cells. Only in the presence of glia, HIV-1LAI virion induced neurotoxic effects, but no interactions with morphine were seen. Also our studies have shown that HIV-1LAI virions are slightly more neurotoxic than HIV-1SF162. Altogether, largely our results suggest that HIV+sup mediated neurotoxicity and the interactions with opiates are majorly attributed to cytotoxic factors released from infected and activated cells instead of viral strain specific factors. Although there is a correlation between opiate drug abuse and progression of HAND, the mechanisms that underlie interactions between HIV-1 and opiates remain obscure. Previous studies have shown that HIV-1 induces neurotoxic effects through abnormal activation of GSK3β. Interestingly, expression of GSK3β has shown to be elevated in the brains of young opiate abusers suggesting that GSK3β is also linked to neuropathology seen with opiate abusing patients. Thus, we hypothesized that GSK3β activation is a point of convergence for HIV- and opiate-mediated interactive neurotoxic effects. Cultures of striatal neurons were treated with HIV+sup (R5-tropic), in the presence or absence of morphine and GSK3β inhibitors. Our results show that multiple GSK3β inhibitors significantly reduce HIV-1-mediated neurotoxic outcomes, and also negate interactions with morphine that result in cell death. This suggests that GSK3β-activation is an important point of convergence and a potential therapeutic target for HIV- and opiate-mediated neurocognitive deficits.

Objective. The aim of this study was to determine the effects of interleukin-10 on mast cell development from bone marrow progenitors.Materials and Methods. Unseparated mouse bone marrow cells were cultured in IL-3+SCF, giving rise to mast cells and monocytes/macrophages. The addition of IL-10, and the use of Signal Transducer and Activator of Transcription (STAT)3-deficient bone marrow cells were employed to measure the effects of IL-10 and STAT3 expression on cell viability, proliferation, and differentiation. Bax-deficient and Bcl-2 transgenic bone marrow cells were used to determine the importance of the mitochondria in IL-10-mediated effects.Overview. Mast cells arise from hematopoietic stem cells and continue development in either connective tissue or mucosa. Th2 cytokines have been implicated in the regulation of mast cell development and subsequent function. Mast cells have also been shown to be essential players in many Th2 immune responses. In the following study we investigate the effects of the Th2 cytokine IL-10 on mast cell development from isolated bone marrow progenitors. The addition of IL-10 to whole murine bone marrow greatly reduced cell numbers and altered the phenotype of the developing progenitor cells. The reduction in cell numbers was due to apoptosis, as judged by DNA fragmentation and caspase activation. The apoptosis observed included alteration in mitochondrial membrane potential. Furthermore, apoptosis could be reduced by the overexpression of Bcl-2 or by ablating p53 expression. Utilizing a flox/cre system we found that IL-10 mediated apoptosis required expression of Stat-3, since Stat-3 deficient bone marrow cells did not undergo apoptosis in response to IL-10. In this study we also observed significant alterations in the mast cell growth factor receptors IL-3R and c-kit. The loss of these growth factor receptors may explain the apoptosis induced by IL-10. These data demonstrate the potent regulatory capabilities of Th2 cytokines on mast cells, a central effector in the Th2 response.

Pseudomonas aeruginosa secretes several proteases associated with pathogenesis, but the most abundant and active is elastase (M4 metalloendopeptidase). Elastase (lasB), is first synthesized as a preproenzyme, with a signal peptide, an 18-kDa N-terminal propeptide, and a 33-kDa mature domain. The propeptide functions as an intramolecular chaperone that is required for the folding and secretion of elastase, but ultimately is proteolytically removed and degraded. Previous research has identified the conserved residues in the propeptide of elastase as compared to other M4 protease precursors and showed some among them to be important for the production of active elastase. In this project, the ability of the propeptide alone to fold into a defined secondary structure was explored and a molecular model was created. Furthermore, the effects of substitutions on conserved residues in the propeptide of plasmid-encoded lasB pro alleles were assessed by expressing them in a lasB propeptide mutant. The kinetics of elastase activity in culture supernatants was quantitated using a fluorescent substrate, Abz-AGLA-p-Nitro-Benzyl-Amide, to provide an accurate assessment of the effects of mutant propeptides. In vitro refolding studies were also performed to determine the effects of specific substitutions on foldase activity of the propeptide. When wild-type propeptide and mature elastase were denatured as separate proteins in guanidine-HCl buffer and renatured together, restoration of activity of the refolded elastase was measured, which was propeptide-dependent. Several mutant propeptides have now been shown to have defects using this in vitro foldase assay. Additional mutants were near wild-type activity level suggesting their role in recognition by the secretion apparatus. Residue locations were determined on a molecular model of the complex and confirmed the role of the secretion mutants as residues on the exterior. Residues that had diminished ability to refold in the in vitro assay were found to be in the interior parts of the complex, confirming their ability to be critical residues at the interface of the proteins or important in the stability of the propeptide’s intrinsic structure. The goal was to perform a series of comprehensive analyses of the propeptide and its conserved residues in order to determine its role as an intramolecular chaperone.

Herpes simplex virus entry into cells is a multistep process that engages the host cell machinery. The proteasome is a large, ATP-dependent, multisubunit protease that plays a critical role in the maintenance of cell homeostasis. A battery of assays were used to demonstrate that proteasome inhibitors blocked an early step in herpes simplex virus entry that occurred after capsid penetration into the cytosol but prior to capsid arrival at the nuclear periphery. Proteasome-dependent viral entry was not reliant on host or viral protein synthesis. MG132, a peptide aldehyde that competitively inhibits the degradative activity of the proteasome, had a reversible inhibitory effect on herpes simplex virus capsid transport. Herpes simplex virus can use endocytic or nonendocytic pathways to enter cells. These distinct entry routes were both dependent on proteasome-mediated proteolysis. In addition, herpes simplex virus successfully entered cells in the absence of a functional host ubiquitin-activating enzyme, suggesting that viral entry is ubiquitin independent. Herpes simplex virus immediate-early protein ICP0 is a multifunctional regulator of herpes simplex virus infection. Late in infection ICP0 interacts dynamically with cellular proteasomes. ICP0 has a RING finger domain with E3 ubiquitin ligase activity that is necessary for its IE functions. The fundamental and functional properties of ICP0 that is present in the virion tegument layer have not been well characterized. For these reasons, I sought to characterize tegument ICP0 and determine the role of tegument ICP0 during proteasome-dependent entry of herpes simplex virus. Protein compositions of wild-type and ICP0 null virions were similar, suggesting that the absence of ICP0 does not grossly impair virion assembly. Virions with mutations in the RING finger domain contained greatly reduced levels of tegument ICP0, suggesting that the domain influences the incorporation of ICP0. Virion ICP0 was resistant to removal by detergent and salt and was associated with capsids, features common to inner tegument proteins. ICP0 mutations that resulted in the absence of ICP0 in the tegument layer, allow herpes simplex virus to enter cells independently of the proteasome activity. I propose that proteasomal degradation of virion and/or host proteins is regulated by ICP0 to allow for efficient delivery of incoming herpes simplex virus capsids to the nucleus.