Browsing by Subject "Pathology"
Results Per Page
Sort Options
Item Open Access Acute Pathogenesis of Recombinant Vesicular Stomatitis Virus Vaccine Vectors is Linked to Interleukin-1(2011) Athearn, Kathleen ConstanceRecombinant vesicular stomatitis virus (rVSV) is a promising candidate viral vaccine vector for use in humans. VSV is highly immunogenic, pre-existing immunity to VSV is rare, and VSV is able to grow to high titers in cell lines approved for vaccine use. Its potential reactogenicity is a barrier to its use in humans, with small laboratory animals developing fever and losing up to 20% of their pre-immunization body weight in the first four days after administration [1, 2], and the one person to date that has received an experimental rVSV vaccine developed headache, fever, and muscle pain within 12 hours and transient VSV viremia was detected [3]. The underlying cause of these reactions has not yet been studied. Here, we have found that IL-1β and/or IL-1α contributes to rVSV pathology after intramuscular immunization in mice and that IL-1 production is not required for control of rVSV replication in vivo, or for the generation of protective immune responses to VSV antigens. Suppression of IL-1 may be a safe strategy to reduce vector reactogenicity without affecting immunogenicity. Utilizing mice deficient in either ASC or caspase-1, we have also found that production of mature IL-1β in response to rVSV might be independent of inflammasome activation or caspase-1 cleavage. The exact mechanism is yet to be determined, but might depend upon which cell types secrete mature IL-1β after immunization.
Item Open Access Biochemical Characterization and Genetic Modeling of Glioma-Associated Mutations in Isocitrate Dehydrogenases.(2014) Lopez, Giselle YvetteGliomas are the most common tumors of the central nervous system. Our lab recently identified mutations in IDH1 and IDH2 as occurring frequently in progressive gliomas. We applied a series of biochemical and genetic approaches to explore the roles of the mutations in tumors and generate models for study.
IDH1/2 mutations have the potential to impact a number of metabolic pathways. IDH1/2 convert isocitrate to α-ketoglutarate while simultaneously converting NADP+ to NADPH. To assess changes in metabolism, we completed metabolic profiling and complementary studies in cell lines with and without mutant IDH1 or mutant IDH2. We identified a decrease in hypoxia signaling and a decrease in global 5-hydroxymethylcytosine in cell lines with mutant IDH1/2 .
Having observed mutations in IDH1/2 in a large fraction of progressive gliomas, we asked if the mutations were either 1) advantageous for growth in brain parenchyma, or 2) advantageous in a particular cell-of-origin. Sequencing of a series of metastases to the brain from non-central nervous system tumors identified no mutations in IDH1/2, lending less credence to the first hypothesis. To elucidate whether mutations in IDH1/2 can initiate glioma progression and explore the potential cell-of-origin for progressive gliomas, we generated mice in which we induced expression of mutant IDH2 in different populations of cells in the brain, either alone or in combination with TP53 deletion, another frequently altered gene in progressive gliomas. Mice with broad expression of mutant IDH2 developed hydrocephalus and encephalomalacia early in life, but did not develop tumors. Therefore, we restricted expression, and two brain tumors were identified in mice with both IDH2 mutation and TP53 deletion. While this suggests that both mutations might be required for the development of tumors, this is too small a number to draw significant conclusions. Further research with an expanded cohort of mice, utilization of additional drivers of expression, and further characterization of identified tumors will help in elucidating the role of mutant IDH2 and the cell-of-origin for progressive gliomas.
Item Open Access Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Regulates Dendritic Cells and Myeloid Derived Suppressor Cells Development in the Lymphoma Microenvironment(2016) Huang, WeiCalcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies.
We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.
In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.
Item Open Access Developing a Senomorphic Treatment Strategy in Osteoarthritis(2022) Chen, Yu-HsiuOsteoarthritis (OA) is the most common form of arthritis in the population worldwide, resulting in significant disabilities. Currently, no treatments can prevent or reverse the development of OA. Cellular senescence has been identified as a major contributing factor to OA. Therefore, a therapy targeting senescence could be an effective treatment for OA. Several strategies have been proposed to target senescence in OA, including neutralizing agents for the senescence-associated phenotypes (SASPs), senolytics for eliminating senescent cells, and senomorphics for modifying the senescence phenotype. In fact, a senolytic, UBX0101, showed a protective effect for post-traumatic OA (PTOA) development in mouse models by reducing both OA histological grading and OA related pain. However, it failed to meet the primary endpoint of relieving symptoms in a clinical phase 2 trial. The failure of the human trial may be related to a high placebo response rate from the control group or, more likely, the heterogeneous phenotypes involved in human OA disease differ from the PTOA in the mouse model. The lack of available senescent-specific biomarkers, which could be used to refine the phenotype of the subject enrollment or to monitor the occurrence of senescence presents a challenge to evaluate a trial successfully. Alternatively, senolytics may be detrimental in tissue with a higher proportion of senescent cells. After treatment, the remaining cells may not be able to maintain the integrity of the cartilage. Therefore, in the present study, we investigated the association of cellular senescence with OA disease severity, identified a biomarker dipeptidyl peptidase-4 (DPP4) for chondrocyte senescence and OA progression, and proposed a senomorphic treatment using chromobox 4 (CBX4) for modulating cell function of the replicative senescent model WI-38 cells and human osteoarthritic chondrocytes.First, we investigated the association of OA disease severity in human knee joints with the percentage of cells expressing senescence-associated β-galactosidase activity (SA-β-gal) and p16. We assessed three regions within the tibial plateau of the knee corresponding to a gradient of disease severities in tissue procured from human medial-dominant OA. We found that SA-β-gal and p16 were positively correlated with OA severity. Our result suggested cellular senescence could be involved in OA progression, and targeting senescence could be a promising treatment for OA. Subsequently, we identified Dipeptidyl-peptidase 4 (DPP4, also known as CD26) as a potential biomarker for OA senescence. We isolated chondrocytes from knee OA cartilage and determined the association of DPP4 expression with senescence markers, SA-β-gal and p16, by flow-cytometry. We also compared the expression of anabolic and catabolic genes, senescence-related genes, and senescence-associated secretory phenotypes (SASPs) in DPP4+ and DPP4- cells, isolated by two different methods: fluorescence-activated cell sorting and magnetic-activated cell sorting. Additionally, we quantified soluble DPP4 in synovial fluid (SF) and assessed its association with radiographic knee OA. DPP4 expression was associated with higher SA-β-gal, p16 expression, senescence-related gene and catabolic gene expression, SASPs secretion, and lower anabolic gene expression in chondrocytes. In addition, SF DPP4 was significantly associated with radiographic knee OA progression (β=4.657 p=0.015). Next, we identified the senomorphic effect of CBX4 in WI-38 cells. We first observed the decrease of CBX4 protein expression and increased senescence markers and gene expression during WI-38 serial culture. We next evaluated the presence of senescence markers and expression of senescence-related genes in the CBX4 activation and CBX4 knockdown compared to controls in pre-senescent WI-38. Compared to the control group, knockdown of CBX4 increased cellular senescence, whereas activation of CBX4 decreased senescence in the pre-senescent WI-38 cells. Based on these results, we identified that CBX4 regulates replicative senescence in WI-38 cells and functions as a senomorphic and potential anti-senescence target. Additionally, we explored the mechanisms of senescence regulation from CBX4 domains by using CBX4 mutated lentiviral particles and compared them with CBX4 wildtype in WI-38 cells. CDM, SIMs, and C-box domains are all involved in the regulation of senescence by CBX4; where CDM is mainly involved in cell cycle regulation, SIMs are involved in the cell proliferation, DNA damage repair, and SASPs secretion, and C-box is related to cell proliferation and SASPs secretion. Taken together, CBX4 is a multi-functional protein, and these mutants elucidated the different non-overlapping functions in senescence regulation. Finally, we identified the CBX4 senomorphic effect in osteoarthritic chondrocytes by comparing CBX4 wildtype and control transduced cells. Compared to control, CBX4 overexpressed chondrocytes demonstrated lower DPP4 expression and higher proliferation marker EdU. Overall, our study demonstrated that cellular senescence is positively correlated with OA disease severity, identified DPP4 as a potential biomarker for cellular senescence in OA, and explored CBX4 as a potential senomorphic treatment in human WI-38 fibroblasts and OA chondrocytes.
Item Open Access Driving Brain Tumorigenesis: Generation and Biological Characterization of a Mutant IDH1 Mouse Model(2014) Pirozzi, Christopher JamesDespite decades worth of research, glioblastoma remains one of the most lethal cancers. The identification of IDH1 as a major cancer gene in glioblastoma provides an exceptional opportunity for improving our understanding, diagnostics, and treatment of this disease. In addition to mutations in IDH1, recent studies from our laboratory have characterized the genetic landscape of gliomas and have shown the cooperation between IDH1 mutations and other oncogenic alterations such at TP53 mutations. Normally, IDH1 functions in the oxidative decarboxylation of isocitrate to α–ketoglutarate, however the mutant form confers neomorphic enzymatic activity by producing 2–hydroxyglutarate, an oncometabolite responsible for aberrant methylation in IDH1–mutated tumors, among other mutant IDH1–mediated phenotypes. To determine the role of mutant IDH1 in vivo, we generated a conditional knock–in mouse model. This genetically faithful system is both biologically and clinically relevant and will promote the understanding of mutant IDH1–mediated tumorigenesis while offering a route for therapeutic targeting.
We observed that broad expression of mutant IDH1 throughout the brain leads to hydrocephalus in 80% of animals. In assessing the earliest effects of mutant IDH1 on the brain, we determined mutant IDH1 confers a decrease in the proliferative cells of the subventricular zone of the lateral ventricle, the area which houses the neural stem cells in embryonic and adult animals. Additionally, a perturbation to the normal neural stem cell niche was observed in these animals. Combined, this data suggests that mutant IDH1 may be affecting the signaling pathways involved in differentiation in this population of cells. In vivo and in vitro studies will further elucidate mutant IDH1's effects on the differentiation patterns of neural stem cells expressing mutant IDH1.
To express mutant IDH1 in a more restricted manner and harness spatiotemporal control, we crossed mutant animals to a Nestin–CreERT2 strain of mouse that permits expression of floxed alleles upon treatment with tamoxifen. Animals were sacrificed at the onset of symptoms or at 1–year of age. We observed the development of both low– and high–grade gliomas in approximately 15–percent of E18.5 tamoxifen–treated animals. All tumors were found in a TP53–deleted background with mutant IDH1 being detected in only those tumors with the mutant allele. Lastly, to decrease the latency and increase the penetrance of tumor formation, an orthotopic intracranial injection model was generated to allow for visualization of tumor formation and development, as well as investigation of therapeutic modalities. The models generated and the knowledge gained from these studies will offer an understanding of the biological effects of the most common mutations found in the astrocytic subset of gliomas, bringing us strides closer to determining mechanisms and therapeutic targets for IDH1–mutated cancers.
Item Open Access Elucidating the Molecular Architecture of Cartilage by Proteomics(2015) Hsueh, MingFengArticular cartilage is a highly specialized avascular tissue and consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. The chondrocyte morphology and extracellular matrix properties vary with the depth of cartilage. Some past studies have defined the zonal distribution of a broad range of cartilage proteins in different layers. Based on the variations within each layer, the extracellular matrix can be further distinguished to pericellular, territorial and interterritorial regions. However, most of these studies used guanidine-HCl extraction that leaves an unextracted residual with a substantial amount of collagen. The high abundance of anionic polysaccharide molecules from cartilage adversely affects the chromatographic separation. Scatter oriented chondrocytes only account for the small proportion of the whole tissue protein extraction. However, the density of the cell varies with depth of cartilage as well. Moreover, the physiological status may also altered the extracellular matrix properties. Therefore, a comprehensive strategy to solve all these difficulties are necessary to elucidate the molecular structure of cartilage.
In this study, we used quantitative and qualitative proteomic analysis to investigate various cartilage tissue processing protocols. We established a method for removing chondrocytes from cartilage sections that minimized matrix protein loss. Quantitative and qualitative proteomic analyses were used to evaluate different cartilage extraction methodologies. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. The different extraction methods yielded different protein profiles. For instance, an overwhelming number of collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction.
Subsequently we applied these methods to extract cartilage sections from different cartilage layers (superficial, intermediate and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic). We also utilized lase capture microscopy (LCM) to harvest cartilage sample from individual subregions (territorial and interterritorial regions). The results suggested that there is more unique proteins existed in the superficial layer. By removing the chondrocytes, we were able to identify more extracellular matrix proteins. The phenotyping of cartilage subregions provided the chance to precisely localize the protein distribution, such as clusterin protein. We observed that the guanidine-HCl extractability (guanidine-HCl/ guanidine-HCl + in situ digestion extracts) of cartilage proteins. Proteoglycans showed high extractability while collagen and non-collagenous proteins had lower extractability. We also observed that the extractability might differ with depth of cartilage and also disease states might alter the characters as well.
Laser capture microscopy provides us the access to the cartilage subregions in which only few studies have investigated because of the difficulties to separate them. We established the proteomic analysis compatible-protocol to prepare the cartilage section for LCM application. The results showed that most of the proteoglycans and other proteins were enriched in the interterritorial regions. Type III and VI collagens, and fibrillin-1 were enriched in the territorial regions. We demonstrated that this distribution difference also varied with depth of cartilage. The difference of protein abundance between subregions might be altered because of disease states.
Last we were looking for the post-transliational modification existed in these subregions of cartilage. Deamidation is one of the modification without the enzyme involved. Previous studies have showed that deamidation may accumulated in the tissue with low turnover rate. Our proteomic analysis results suggests that abundance of deamidated peptides also varied in different layers and subregions of cartilage.
We have developed the monoclonal antibody based immunoassay to quantify the deamidated cartilage oligomeric matrix protein within cartilage tissue from different joints (hip and knee) and disease states (healthy, para-lesion, and remote lesion). The results suggests that the highest concentration of deamidated COMP was identified in arthritic hip cartilage.
The results of this study generated several reliable protocols to perform cartilage matrix proteomic analysis and provided data on the cartilage matrix proteome, without confounding by intracellular proteins and an overwhelming abundance of collagens. The discovery results elucidated the molecular architecture of cartilage tissue at different joint sites and disease states. The similarities among these cartilages suggested a constitutive role of some proteins such as collagen, prolargin, biglycan and decorin. Differences in abundance or distribution patterns, for other proteins such as for cartilage oligomaric matrix protein, aggrecan and hyaluronan and proteoglycan link protein, point to intriguing biological difference by joint site and disease state. Decellularization and a combination of extraction methodologies provides a holistic approach in characterizing the cartilage extracellular matrix. Guanidine-HCl extractability is an important marker to characterize the statue of cartilage; however it has not been fully understand. The protein distributions in matrix subregions may also serve as an index to characterize the metabolic status of cartilage in different disease states. A large sample cohort will be necessary to elucidate these characters.
Item Open Access Enhancing Cisplatin Delivery and Anti-tumor Efficacy Using Hyperthermia(2013) Landon, Chelsea DawnMild hyperthermia (39°C-43°C) has numerous therapeutic benefits as an adjuvant therapy in the treatment of a variety of tumor types. Hyperthermia increases tumor blood flow and vascular permeability, promoting drug delivery and tumor oxygenation. Hyperthermia enhances the uptake and efficacy of numerous chemotherapeutic agents, including cisplatin, resulting in increased cytotoxicity. In addition to these biological responses, hyperthermia can be used as a drug-release trigger for temperature-sensitive nanoparticles, resulting in an improved and more targeted drug delivery system. Cisplatin was chosen because 1) it shows broad spectrum activity against a wide range of heatable cancers (i.e., those in sites such as the pancreas, colon and rectum, cervix and bladder, and 2) the same hyperthermic temperatures that enable temperature-sensitive lipsome-drug release also enhance cisplatin-induced cytotoxicity.
The role of hyperthermia in enhancing cisplatin delivery and cytotoxicity was investigated at both the cellular and tissue levels. While hyperthermia treatment is applicable to a variety of tumor types, the focus of this work was on bladder cancer. The synergistic effects of hyperthermia and cisplatin were investigated, along with the role of copper transport protein 1 (Ctr1) in this process. In addition, cisplatin was encapsulated within temperature-sensitive liposomes, which were used in combination with hyperthermia for targeted drug delivery. These studies demonstrated that the combination of cisplatin and hyperthermia improved drug delivery, and potentially anti-tumor efficacy, and that targeted delivery was enhanced through incorporation of temperature-sensitive liposomes. As many current methods for administering bladder hyperthermia have drawbacks, such as invasiveness and regional heating, the final aim of this study was to develop and test a less-invasive and more focused preclinical bladder heating device in a rat model.
Hyperthermia sensitizes cells to the cytotoxic effects of the commonly used chemotherapeutic agent cisplatin by increasing drug accumulation and subsequent platinum-DNA adduct formation. However, the molecular mechanisms underlying this enhancement remain unclear. Understanding the fundamental mechanisms involved in the synergistic interaction is necessary to increase the therapeutic benefits of this combination in the clinic. The synergism between the anti-cancer benefits of cisplatin and the drug delivery benefits of hyperthermia may offer a novel and more effective treatment for many cancer patients. We hypothesized that hyperthermia increases cisplatin accumulation and efficacy in part by modulating the function of Ctr1, a major regulator of cellular cisplatin uptake. To test this hypothesis, we examined the significance of Ctr1 during combined hyperthermia and cisplatin therapies and assessed the importance of cisplatin- and hyperthermia-induced Ctr1 multimerization in enhancing cisplatin cytotoxicity. We observed increased Ctr1 multimerization following hyperthermia treatment (41°C) in vitro, compared to normothermic controls (37°C), suggesting that this may be a mechanism for increased cisplatin uptake in heat-treated cells. The impact of increased Ctr1 multimerization was evaluated by measuring platinum accumulation in wild-type (WT) and Ctr1-/- cells. WT cells contained greater levels of platinum compared to Ctr1-/- cells. A further increase in platinum was observed following hyperthermia treatment, but only in the WT cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in WT cells with a dose-modifying factor (DMF) of 1.8 compared to 1.4 in Ctr1-/- cells. Our data suggest that heat increases Ctr1 activity by increasing multimerization, resulting in enhanced drug accumulation. Although we recognize that the effect of heat on cells is multi-factorial, our results support the hypothesis that Ctr1 is, in part, involved in the synergistic interaction observed with cisplatin and hyperthermia treatment.
In addition to assessing cisplatin delivery at the cellular level, we evaluated cisplatin delivery at the tissue level, using novel cisplatin-loaded temperature-sensitive liposomes. We hypothesized that delivering cisplatin encapsulated in liposomes under hyperthermic conditions would improve the pharmacokinetic profiles of cisplatin, increase drug delivery to the tumor, decrease normal tissue toxicity, and enhance the anti-tumor activity of cisplatin. We successfully prepared temperature-sensitive liposomes loaded with cisplatin and demonstrated that heat (42°C) sensitizes cisplatin-resistant cells to the cytotoxic effects of cisplatin in vitro.
Decreased toxicity was observed in animals treated with the cisplatin liposome (± heat) compared to the free drug treatments. A pharmacokinetic study of cisplatin-loaded temperature-sensitive liposomes and free drug was performed in tumor-bearing mice under normothermic and hyperthermic conditions. Cisplatin half-life in plasma was increased following liposome treatment compared to free cisplatin, and cisplatin delivery to the tumors was greatest in mice that received liposomal cisplatin under hyperthermia. These initial in vivo data demonstrate the potential effectiveness of this cisplatin-loaded liposome formulation in the treatment of certain types of cancer. To assess the anti-cancer efficacy of the liposome treatment, a tumor growth delay study was conducted and demonstrated equivalent efficacy for the cisplatin-loaded temperature-sensitive liposome compared to free drug.
In addition to the liposome work, we developed and evaluated a novel heating device for the bladder. Despite the evidence that hyperthermia is an effective adjuvant treatment strategy, current clinical heating devices are inadequate, warranting the development of a new and improved system. We induced hyperthermia using ferromagnetic nanoparticles and an alternating magnetic field device developed by Actium Biosystems. Initial preclinical studies in a rat model demonstrated preferential bladder heating. However, our preliminary studies show severe toxicity with the direct instillation of the nanoparticles in the bladder, and further studies are needed to potentially modify the nanoparticle coating, the catheterization procedure, as well as to develop a different animal model.
Item Open Access Evaluation and Optimization of the Translational Potential of Array-Based Molecular Diagnostics(2012) Kernagis, DawnThe translational potential of diagnostic and prognostic platforms developed using expression microarray technology is evident. However, the majority of array-based diagnostics have yet to make their way into the clinical laboratory. Current approaches tend to focus on development of multi-gene classifiers of disease subtypes, but very few studies evaluate the translational potential of these assays. Likewise, only a handful of studies focus on development of approaches to optimize array-based tests for the ultimate goal of clinical utility. Prior to translation into the clinical setting, molecular diagnostic platforms should demonstrate a number of characteristics to ensure optimal and efficient testing and patient care. Assays should be accurate and precise, technically and biologically robust, and should take into account normal sources of biological variance that could ultimately affect test results. The overarching goal of the research presented in this dissertation is to develop methods for evaluating and optimizing the translational potential of molecular diagnostics developed using expression microarray technology.
The first research section of this dissertation is focused on our evaluation of the impact of intratumor heterogeneity on precision in microarray-based assays in breast cancer. We conducted genome-wide expression profiling on 50 needle core biopsies from 18 breast cancer patients. Global profiles of expression were characterized using unsupervised clustering methods and variance components models. Array-based measures of estrogen (ER) and progesterone receptor (PR) status were compared to immunohistochemistry. The precision of genomic predictors of ER pathway status, recurrence risk, and sensitivity to chemotherapeutics were evaluated by interclass correlation. Results demonstrated that intratumor variation was substantially less than the total variation observed across the patient population. Nevertheless, a fraction of genes exhibited significant intratumor heterogeneity in expression. A high degree of reproducibility was observed in single gene predictors of ER (intraclass correlation coefficient (ICC)=0.94) and PR expression (ICC=0.90), and in a multi-gene predictor of ER pathway activation (ICC=0.98) with high concordance with immunohistochemistry. Substantial agreement was also observed for multi-gene signatures of cancer recurrence (ICC=0.71), and chemotherapeutic sensitivity (ICC=0.72 and 0.64). Together, these results demonstrated that intratumor heterogeneity, although present at the level of individual gene expression, does not preclude precise micro-array based predictions of tumor behavior or clinical outcome in breast cancer patients.
Leading into the second research section, we observed that in some cancer types, certain genes behave as molecular switches and have either an "on" or "off" expression state. Specifically, we observed these molecular switch genes exist in breast cancer as robust diagnostic and prognostic markers, including ER, PR, and HER2, and define tumor subtypes associated with different treatment and patient survival. We hypothesized that clinically relevant molecular switch (bimodal) genes exist in epithelial ovarian cancer, a type of cancer with no established molecular subgroups. To test this hypothesis, we applied a bimodal discovery algorithm to a publically available ovarian cancer expression microarray dataset (GSE9891:285 tumors; 246 malignant serous (MS), 20 endometrioid (EM), 18 low malignant potential (LMP) ovarian carcinomas). Genes with robust bimodal expression were identified across all ovarian tumor types and within selected subtypes. Of these bimodal genes, 73 demonstrated differential expression between LMP vs. MS and EM, and 22 genes distinguished MS from EM. Fourteen bimodal genes had significant association with survival among MS tumors. When these genes were combined into a single survival score, the median survival for patients with a favorable versus unfavorable score was 65 versus 29 months (p<0.0001, HR=0.4221). Two independent datasets (n=53 high grade, advanced stage serous and n=119 advanced stage ovarian tumors) validated the survival score performance. Taken together, the results of this study revealed that genes with bimodal expression patterns not only define clinically relevant molecular subtypes of ovarian carcinoma, but also provide ideal targets for translation into the clinical laboratory.
Finally, the third research section of this dissertation focuses on development of robust blood-based molecular markers of decompression stress (DS). DS is defined as the pathophysiological response to inert gas coming out of solution in the blood and tissues when a body experiences a reduction in ambient pressure. To date, there are no established molecular markers of DS. We hypothesized that comparing gene expression before and after human decompression exposures by genome-wide expression profiling would identify candidate molecular markers of DS. Peripheral blood was collected 1hr before and 2hr after 93 hyperoxic, heliox experimental dives (n=54). Control arms included samples collected 1 hour before and 2 hours after high pressure oxygen breathing (n= 9) and surface exercise (n=9), and samples collected at 7am and 5pm for time of day (n=11). Pre and post-dive expression data collected from normoxic nitrox experimental dives were utilized for independent validation. Blood samples were collected into PaxGene RNA tubes. RNA was extracted and processed for globin reduction prior to cDNA synthesis and Affymetrix U133A GeneChip hybridization. 746 genes were differentially expressed following hyperoxic, heliox decompression exposures (permutation adjusted p-value cutoff 1.0E-4). After filtering control significant genes, 726 genes remained. Pathway analysis demonstrated a significant portion of genes were associated with innate immune response (p<0.0001). A 362 multi-gene signature of significant, covariant genes was then applied to the independent dataset and demonstrated differentiation between pre and post-dive samples (p=0.0058). There was no significant correlation between signature and venous bubble grade or bottom time in the validation study. Our results showed that expression profiling of peripheral blood following decompression exposures, while controlling for experimental and normal sources of biological variance, identifies a reproducible multi-gene signature of differentially expressed genes, primarily comprising genes associated with innate immune response and independent of venous bubble grade or dive profile.
Taken together, the research and results presented in this dissertation represent considerable advances in the development of approaches to guide microarray-based diagnostics towards the ultimate goal of clinical translation.
Item Open Access Evaluation of LMP-420: A Novel, Nontoxic Drug with Anti-Inflammatory Properties and Therapeutic Potential for CLL(2012) Mowery, Yvonne MarieB-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Although treatment of this disease has advanced considerably over the past decade, CLL remains incurable with current chemotherapeutics. In addition, available drug regimens for CLL are associated with frequent cytopenia-related complications, such as infection and fatigue. Thus, the major challenge in CLL treatment today is the need for alternative therapeutics with decreased toxicity and improved efficacy for disease refractory to currently available drugs.
CLL is characterized by slow accumulation of malignant cells, which are supported in the microenvironment by cell-cell interactions and soluble cytokines such as tumor necrosis factor (TNF). We evaluated the effect of the small molecule TNF inhibitor LMP-420 on primary CLL cells. LMP-420 exhibited cytotoxic activity against these cells in the MTS assay, with similar potency to the front-line CLL drug fludarabine. LMP-420 induced time- and dose-dependent apoptosis in CLL cells, as demonstrated by annexin V staining, caspase activation, and DNA fragmentation. These changes were associated with decreased expression of the anti-apoptotic proteins Mcl-1, Bcl-xL, Bcl-2, and XIAP. CLL cells from patients with poor prognostic indicators exhibited LMP-420 sensitivity equal to that for cells from patients with favorable characteristics. In addition, LMP-420 potentiated the cytotoxic effect of fludarabine and inhibited in vitro proliferation of CLL cells. In contrast to other CLL therapeutics, LMP-420 exhibited minimal effects on normal peripheral blood mononuclear cell viability, mitogen-stimulated B- and T-cell proliferation, and hematopoietic colony formation. Our data suggest that LMP-420 may be a useful treatment for CLL with negligible hematologic toxicities.
The effect profile of this compound in normal immune cells and the microarray studies in CLL cells indicate that the mechanism of action of LMP-420 likely involves modulation of the NF-kB pathway. Our initial studies demonstrate moderate but significant inhibitory activity against p65, a key member of the NF-kB transcription factor family. Research is ongoing to gain a better understanding of the specific cytotoxicity of LMP-420 for CLL cells and to elucidate other components of its mechanism of action. Regardless of the ultimate mechanistic findings with LMP-420, our studies support this molecule as a promising new CLL therapeutic that warrants further preclinical evaluation.
Item Open Access Evaluation of Vaccine‐induced Maternal Antibody Transfer and Impact on Infant Immune Responses in the Rabbit Model(2019) Jones, Dorothy IMaternal vaccination protects infants through transplacental transfer of vaccine-specific maternal IgG and milk transfer of IgG and IgA antibodies from mother to child. I performed experiments in the rabbit model, which models human maternal antibody transfer, to determine how maternal HIV vaccine formulations impact the passive transfer of maternal gp120-specific antibodies and to investigate potential side effects of vaccine-elicited maternal antibodies. Since the mammary gland is part of the mucosal immune system, mucosal administration of maternal vaccines may enhance milk transfer of maternal antibodies; however, the tendency of mucosal vaccines to induce lower serum IgG responses than injected vaccines could decrease transplacental transfer. Optimized intranasal boosting during pregnancy resulted in similar concentrations of gp120-specific IgG in infant serum, however milk gp120-specific IgA concentrations were not enhanced. Furthermore, intranasal boosting with chitosan-adjuvanted vaccines resulted in significantly higher transplacental transfer of maternal antibody than MPL-adjuvanted vaccines even though both formulations induced similar levels of gp120-specific IgG in maternal serum, indicating that maternal vaccine adjuvants may alter transplacental transfer of maternal antibodies.
Infant rabbits born to mothers that received the IM and IN maternal vaccine regimens were vaccinated with gp120 with or without adjuvant to investigate maternal antibodies interference with infant antibody responses to vaccination. Maternal gp120-specific IgG inhibited infant vaccination with unadjuvanted gp120, however inclusion of either alum or GLA-SE, a TLR4 agonist in an oil-in-water emulsion, was able to induce active antibody responses in infants. Furthermore, infant rabbits that received an alum-adjuvanted vaccine in the presence of maternal antibodies had enhanced serum gp120-specific and V1V2-specific IgG that infants vaccinated without maternal gp120-specific IgG present. GLA-SE did not enhance infant antibody responses to vaccination. Thus, maternal anti-gp120 IgG can enhance or inhibit infant antigen-specific responses to vaccination depending on the infant vaccine adjuvant.
While maternal antibodies protect the infant, there is evidence that some viruses, including HIV and Zika, use maternal antibodies to be transferred across the placenta, facilitating mother-to-child-transmission. As HIV infects and replicates poorly in rabbits, a rabbit model of Zika virus challenge was established and the impact of maternal vaccination or anti-flavivirus monoclonal antibody on pathogenesis was investigated. While Zika virus-specific antibodies altered maternal cytokine response to challenge, and there was an increased risk for fetal resorption in vaccinated rabbits compared to naïve rabbits, there was no significant impact on placental Zika virus RNA concentration. While further refinement is needed, Zika virus challenge of rabbits is a promising in vivo model for investigating the transplacental transfer of maternal antibody-pathogen complexes.
Item Open Access Ex Vivo MR Histology and Cytometric Feature Mapping Connect Three-dimensional in Vivo MR Images to Two-dimensional Histopathologic Images of Murine Sarcomas.(Radiology. Imaging cancer, 2021-05) Blocker, Stephanie J; Cook, James; Mowery, Yvonne M; Everitt, Jeffrey I; Qi, Yi; Hornburg, Kathryn J; Cofer, Gary P; Zapata, Fernando; Bassil, Alex M; Badea, Cristian T; Kirsch, David G; Johnson, G AllanPurpose To establish a platform for quantitative tissue-based interpretation of cytoarchitecture features from tumor MRI measurements. Materials and Methods In a pilot preclinical study, multicontrast in vivo MRI of murine soft-tissue sarcomas in 10 mice, followed by ex vivo MRI of fixed tissues (termed MR histology), was performed. Paraffin-embedded limb cross-sections were stained with hematoxylin-eosin, digitized, and registered with MRI. Registration was assessed by using binarized tumor maps and Dice similarity coefficients (DSCs). Quantitative cytometric feature maps from histologic slides were derived by using nuclear segmentation and compared with registered MRI, including apparent diffusion coefficients and transverse relaxation times as affected by magnetic field heterogeneity (T2* maps). Cytometric features were compared with each MR image individually by using simple linear regression analysis to identify the features of interest, and the goodness of fit was assessed on the basis of R2 values. Results Registration of MR images to histopathologic slide images resulted in mean DSCs of 0.912 for ex vivo MR histology and 0.881 for in vivo MRI. Triplicate repeats showed high registration repeatability (mean DSC, >0.9). Whole-slide nuclear segmentations were automated to detect nuclei on histopathologic slides (DSC = 0.8), and feature maps were generated for correlative analysis with MR images. Notable trends were observed between cell density and in vivo apparent diffusion coefficients (best line fit: R2 = 0.96, P < .001). Multiple cytoarchitectural features exhibited linear relationships with in vivo T2* maps, including nuclear circularity (best line fit: R2 = 0.99, P < .001) and variance in nuclear circularity (best line fit: R2 = 0.98, P < .001). Conclusion An infrastructure for registering and quantitatively comparing in vivo tumor MRI with traditional histologic analysis was successfully implemented in a preclinical pilot study of soft-tissue sarcomas. Keywords: MRI, Pathology, Animal Studies, Tissue Characterization Supplemental material is available for this article. © RSNA, 2021.Item Open Access Exploring the Role of Mitochondrial Bioenergetics and Metabolism in Heart Failure(2020) Davidson, Michael ThomasHeart failure is a worldwide public health problem with substantial clinical burden and economic costs. In the progression into failure, the heart undergoes dramatic alterations in mitochondrial fuel metabolism and bioenergetics. As such, there is considerable interest in the delineation of regulatory events involved in the metabolic dysfunction of heart failure. Previous collaborative work identified three metabolic signatures associated with early stage heart failure: 1) accumulation of acylcarnitine metabolites; 2) mitochondrial hyperacetylation; and 3) elevated ketone catabolism. The goal of this dissertation was to explore the role of these metabolic signatures in the pathogenesis of heart failure.
Tissue accumulation of acylcarnitine metabolites is characteristic of mitochondrial dysfunction and indicative of incomplete β-oxidation. This occurs when a large portion of the fatty acids (i.e., acyl groups) within the mitochondria are not fully catabolized and the resulting intermediates are transferred to carnitine esters, enabling the traversal of biological membranes and departure from the mitochondrial matrix.
Nϵ-acetylation in the mitochondrial matrix is a non-enzymatic, post-translational modification (PTM) that spontaneously arises from the relatively basic pH and abundance of acetyl-CoA. Accumulation of this PTM has been observed in other tissues and disease states with evidence suggesting it impairs mitochondrial metabolism and causes dysfunction. However, convincing studies are lacking to establish a direct causal connection between dysfunction and acetylation. To address this shortcoming, a novel assay platform for the comprehensive assessment of mitochondrial bioenergetic transduction was developed and validated. Next, we generated and validated a novel mouse model of cardiac mitochondrial hyperacetylation and utilized the bioenergetic assay platform to test the hypothesis that it causes metabolic perturbations. Surprisingly, these hyperacetylated mitochondria exhibited almost no deficits in mitochondrial oxidative metabolism. To determine if hyperacetylation causes mitochondrial dysfunction in vivo under pathologic stimuli, the mouse model and littermate controls were subjected to transaortic constriction, a surgical method to induce pressure-overload heart failure. The hyperacetylated animals did not exhibit enhanced sensitivity toward cardiac dysfunction relative controls. With these results, we concluded that mitochondrial hyperacetylation does not contribute to the pathogenesis of heart failure.
Elevated ketone catabolism was observed in early stage failing hearts. Through a series of murine and canine heart failure models, ketone catabolism was shown to be adaptive in response to pathological stress. Additionally, the mitochondrial bioenergetic assay platform was applied to cardiac mitochondria under substrate limited-conditions. These results indicate that ketone catabolism improves bioenergetic efficiency under constraints which mimic the failing heart. With these results, we conclude ketone catabolism is an important metabolic defense in response to the dysfunction of the failing heart.
Item Open Access Gastric Fluid Aspiration-Mediated Pulmonary Allograft Failure(2013) Chang, Jui-ChihSince its first success in 1983, lung transplantation has become the treatment of choice for selected patients with end stage lung disease. However, long-term graft survival is relatively low compared to other solid organs, such as heart, liver and kidney. The primary factor limiting long-term survival of pulmonary allografts is obliterative bronchiolitis (OB), a form of chronic pulmonary graft rejection characterized by submucosal fibroproliferation of the small airways, which leads to luminal compromise and respiratory failure. Due to their unique anatomic location, lungs are exposed to environmental factors in the air which include potentially toxic agents. This suggests a possible importance of non-alloimmune mediated factors in the pathogenesis of pulmonary rejection in addition to alloimmunity.
Clinical reports reveal that gastroesophageal reflux disease (GERD) is commonly observed following lung transplantation, and it has been postulated that GERD after lung transplantation could accelerate the development of OB and subsequent lung allograft failure. Although clinical studies point to the importance of GERD in the survival of the transplant, the potential effects of GERD on pulmonary allografts still need to be identified. In this dissertation, the effects of GERD on pulmonary allografts, including ischemia-reperfusion (I-R) injury and the pH of the gastric fluid aspirate, are evaluated. Further, this dissertation explores the mechanisms by which GERD affects chronic lung allograft rejection, particularly the role played by mast cells in rejection.
I-R injury of the pulmonary graft has been associated with a higher risk of developing bronchiolitis obliterans syndrome (BOS) in clinical practice, and prolonged ischemic time has been correlated with poorer long-term survival after lung transplantation. To explore the relationship between GERD, I-R injury, and OB formation after lung transplantation, the effect of long and short ischemic times on WKY-to-F344 rat orthotopic left lung transplants receiving 8 weekly aspirations of gastric fluid was assessed. In this study, described in Chapter 2, long ischemic times led to significantly (p < 0.05) greater development of OB compared to short ischemic times. However, the development of OB was dependent on aspiration, as controls receiving aspiration with normal saline showed little development of OB, regardless of ischemic time (p < 0.05). The data suggest that prolonged ischemic time, while insufficient by itself to lead to OB, worked synergistically with chronic aspiration of gastric fluid to exacerbate the development of OB.
Also, it remains unknown whether pharmaceutical-induced increases in gastric pH might effectively prevent pulmonary injury associated with chronic aspiration. The hypothesis that neutralization of gastric fluid would affect the development of aspiration-associated OB was tested. The results, described in Chapter 3, revealed that the pH of the aspirated gastric fluid did not significantly affect pulmonary graft rejection. This finding suggests that clinical management of lung transplant patients with GERD should probably include more than just pharmaceutical blockage of gastric protons and/or antacid to neutralize the pH of gastric fluid.
Mast cells, the first responders of the innate defense system, might play a role in pulmonary allograft rejection due to their ability to orchestrate innate and adaptive immune responses. Correspondingly, increased mast cell numbers associated with increased rejection grade as well as the presence of OB were reported in a retrospective clinical study. Identifying how mast cells are involved with gastric fluid aspiration-associated pulmonary allograft rejection could uncover a potential method for treatment. With this in mind, cromolyn, a mast cell membrane stabilizer which prevents mast cell degranulation, was utilized to investigate the role of mast cells in the pathogenesis of pulmonary allograft failure. The results, described in Chapter 4, reveal that pulmonary allografts in rats treated with cromolyn and aspirated with gastric fluid developed significantly fewer OB lesions than those treated with gastric fluid alone (p<0.001). Further, the number of mast cells per small bronchiole significantly increased in the animals aspirated with gastric fluid regardless of the treatment of cromolyn. Therefore, cromolyn ameliorates the development of OB in pulmonary allografts, perhaps by qualitative (preventing mast cells degranulation), and not quantitative (numbers of mast cells) changes in the mast cells in the bronchioles. These findings suggest that mast cells play a substantial role in gastric fluid aspiration-mediated pulmonary allograft failure.
Item Open Access Genetic Studies Identify Critical Biomarkers and Refine the Classification of Malignant Gliomas(2014) Killela, Patrick JGliomagenesis is driven by a complex network of genetic alterations and while the glioma genome has been a focus of investigation for many years; critical gaps in our knowledge of this disease remain. The identification of novel molecular biomarkers remains a focus of the greater cancer community as a method to improve the consistency and accuracy of pathological diagnosis. In addition, novel molecular biomarkers are drastically needed for the identification of targets that may ultimately result in novel therapeutics aimed at improving glioma treatment. Through the identification of new biomarkers, laboratories will focus future studies on the molecular mechanisms that underlie glioma development. Here, we report a series of genomic analyses identifying novel molecular biomarkers in multiple histopathological subtypes of glioma and refine the classification of malignant gliomas. We have completed a large scale analysis of the WHO grade II-III astrocytoma exome and report frequent mutations in the chromatin modifier, alpha thalassemia mental retardation x-linked (ATRX), isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), and mutations in tumor protein 53 (TP53) as the most frequent genetic mutations in low grade astrocytomas. Furthermore, by analyzing the status of recurrently mutated genes in 363 brain tumors, we establish that highly recurrent gene mutational signatures are an effective tool in stratifying homogeneous patient populations into distinct groups with varying outcomes, thereby capable of predicting prognosis. Next, we have established mutations in the promoter of telomerase reverse transcriptase (TERT) as a frequent genetic event in gliomas and in tissues with low rates of self renewal. We identify TERT promoter mutations as the most frequently mutated gene in primary glioblastoma. Additionally, we show that TERT promoter mutations in combination with IDH1 and IDH2 mutations are able to delineate distinct clinical tumor cohorts and are capable of predicting median overall survival more effectively than standard histopathological diagnosis alone. Taken together, these data advance our understanding of the genetic alterations that underlie the transformation of glial cells into neoplasms and we provide novel genetic biomarkers and multi – gene mutational signatures that can be utilized to refine the classification of malignant gliomas and provide opportunity for improved diagnosis.
Item Open Access Genomic approaches to guide the molecular classification of glioma(2019) Diplas, BillMalignant gliomas account for more than 80% of all primary brain malignancies and 14,000 deaths in the U.S. annually. Despite aggressive treatment, malignant gliomas are largely fatal, as their invasive nature renders them prone to rapid recurrence. Gliomas are classified according to histopathologic criteria which are critical to treatment planning, as certain subtypes show increased sensitivity to particular therapeutic agents. However, gliomas often exhibit extensive tumor heterogeneity and ambiguity among histologic features, leading to subjectivity in diagnosis and low concordance rates among neuropathologists.
Recently, a number of large-scale genomic studies identified mutations in the TERT promoter and IDH1/2 in ~80% of all gliomas. Based on the occurrence of these mutations, gliomas can be classified into objective molecular subtypes that stratify patients into clear prognostic subgroups more effectively than by histology alone. However, current sequencing-based methods to identify these alterations are limited by low sensitivity (40% tumor cellularity), a major constraint on their clinical utility in the context of diffusely infiltrative gliomas. Importantly, this work also revealed that 20% of glioblastomas lack these alterations, delineating a subset of tumors known as the TERT promoter wildtype-IDH wildtype (TERTpWT-IDHWT) glioblastomas.
Preliminary studies indicate that TERT promoter and IDH mutations can effectively stratify the majority (80%) of patients into clinically-relevant genetic subtypes, however current mutation detection methods lack sensitivity (Sanger sequencing) or are overly time-consuming (next-generation sequencing). Here, we report the development of a qPCR-based approach which can provide more sensitive and rapid detection of these mutations and practical utility in glioma diagnosis by detecting low-abundance mutations (e.g., poorly sampled tumors). Finally, we report the genetic landscape of TERTpWT-IDHWT glioblastomas using whole exome and whole genome sequencing, revealing that these tumors harbor a unique set of genetic alterations and exhibit distinct genetic mechanisms of telomere maintenance from other known subgroups of GBM, including recurrent SMARCAL1 mutations and rearrangements upstream of TERT. Using cell-based assays and markers of alternative lengthening of telomeres (ALT), we provide evidence showing that SMARCAL1 acts as a tumor and ALT suppressor and that loss of function cancer-associated mutations are involved in ALT mechanism of telomere maintenance.
These studies have identified the key underlying genetic alterations that characterize TERTpWT-IDHWT glioblastomas, and can serve as biomarkers for more accurate diagnosis and treatment of this glioma subgroup. By developing a sensitive diagnostic for the critical TERTp and IDH alterations, we facilitate accurate diagnosis and prognostication of glioma patients.
Item Open Access Glycans and Glycoconjugates as Biomarkers and Therapeutic Targets for Therapy-Resistant Prostate Cancer(2023) Butler, WilliamProstate Cancer (PCa) is the most common non-cutaneous malignancy and second leading cause of cancer-related mortality in men. Although most men are diagnosed with early-stage disease which is curable through prostatectomy or local radiation, a subset of men develop biochemically recurrent or metastatic disease requiring the use of hormonal therapy. Despite being initially successful, all men treated with hormonal therapy will eventually develop castration-resistant PCa (CRPC) in which the tumor cells are able to actively proliferate and metastasize despite continued androgen receptor (AR) inhibition. Furthermore, ~17-20% of CRPC tumors recur as small cell neuroendocrine carcinoma (SCNC), consisting entirely of AR-negative neuroendocrine (NE) cells which normally only represent 1% of the tumor cell population in the common, adenocarcinoma setting. Based on these observations, there remains an urgent need to discover important molecules in advanced form of PCa that can provide biological insight into CRPC and SCNC, serve as biomarkers for disease subsets, as well as have therapeutic potential. Although many authors have studied changes in the levels of many biomolecules, glycans and glycoconjugates have remained severely understudied due to the complexity of these structures as well as technological limitations. Therefore, the work in this dissertation has explored these structures specifically in the pursuit of developing novel biomarkers and therapeutic targets for advanced forms of PCa and hormone therapy-resistant tumor cells. Furthermore, the information from these studies provides novel information on how tumor glycosylation evolves throughout the evolution of PCa offering new biological insight. Chapter 1 provides a brief overview of PCa, including mechanisms of resistance to hormonal therapy as well as the concept of cellular heterogeneity and its role in disease progression and treatment resistance. Chapter 2 reviews classical and modern studies that have focused on glycans and glycoconjugates and their important role in the progression of PCa. Furthermore, Chapter 2 emphasizes the potential role these structures may serve as both diagnostic and prognostic biomarkers in the context of both tissue and liquid biopsy specimens. Chapter 3 demonstrates the expression of an oncofetal heparan-sulfate proteoglycan, Glypican-3 (GPC3), in NE cells of human PCa, including the highly lethal SCNC variant. Furthermore, Chapter 3 explores the function of GPC3 in NE tumor cells of human PCa as well as its potential molecular mechanism. Chapter 4 explores the utilization of N-glycan imaging mass spectrometry (IMS) to discover N-glycan markers of various stages of PCa progression, including the hormone-naïve, hormonally-treated, hormone-refractory, and NE subsets. Furthermore, the use of several of these discovered N-glycan structures as potential therapeutic targets is discussed as an important future direction to our work. Finally, Chapter 5 provides an outlook on the future exploitation of glycans and glycoconjugates as both biomarkers and therapeutic targets to improve the ability to diagnose clinically-relevant tumors as well as improve treatment options for patients with advanced disease.
Item Open Access Hyperinnervation and Chronic Mast Cell Activity in Bladder Pain Syndrome(2021) Hayes, ByronBladder Pain Syndrome (BPS) is a broad-spectrum pelvic pain disorder, characterized by pain and at least one underlying lower urinary tract symptom, that affects millions in the US. Despite its prevalence, the underlying etiology is unknown. Several clinical findings point to a potential role of peripheral nerves in the bladder, such as hyperinnervation and increased urinary nerve growth factor (NGF) in BPS patients. In addition, mast cell (MC) hyperplasia and a history of recurrent urinary tract infections (recUTIs) is also observed. Based on the similar findings of elevated urinary NGF in recUTI patients and the prominent role of MCs in bladder immunity to UTI, we examined whether mice that experience multiple UTIs develop BPS symptoms. We found that mice subjected to multiple bacterial bladder infections experienced both pelvic hypersensitivity and bladder frequency, assayed by mechanical stimulation of the pelvic region and cystometric analysis, respectively. These pathological outcomes were linked to extensive hyperinnervation, specifically in sensory nociceptive nerves in the bladder lamina propria, induced by recruited NGF-producing CCR2+ Ly6Chigh inflammatory monocytes and to the presence of activated MCs near sprouting nerves. Notably, both CCR2-/- and MC-deficient mice each were protected from developing BPS symptoms after multiple infections. Lastly, the MC product histamine was sufficient to trigger both hypersensitivity and frequency in a TRPV1 dependent manner. Taken together, these findings reveal chronic MC activity in a hyperinnervated bladder, triggered by past infections, could be an underlying basis for BPS.
Item Open Access IDH1 R132H Mutations Actively Contribute to the Epigenetic State of Glioma Cells(2019) Moure, Casey JosephPoint mutations in the active site of isocitrate dehydrogenases 1 and 2 (\textit{IDH}) occur in the majority of WHO grade II and III gliomas, resulting in a unique milieu of signaling and metabolism. IDH1/2 active site mutations confer a gain-of-function activity to the enzyme, which results in the production of the oncometabolite D-2-hydroxyglutarate (D-2HG). D-2HG accumulation in turn promotes tumor formation through competitive inhibition of $\alpha$-ketoglutarate dependent ($\alpha$-KG) enzymes. Inhibition of $\alpha$-KG-dependent enzymes, such as histone demethylases and DNA demethylases, is sufficient to induce tumor-promoting epigenetic changes, but can also impose situational constraints on cell proliferation. To develop better therapies for mutant IDH1-bearing gliomas, it is essential to determine whether the epigenetic changes induced by the mutant IDH proteins actively require the mutation after tumor formation. Furthermore, it is imperative to decode the molecular mechanisms that promote tumor cells’ fitness under IDH mutation-dependent constraints in representative models. Here, we describe and characterize CRISPR-Cas9 based isogenic cell line models using patient-derived IDH1$^{R132H/WT}$ glioma cell lines. We uncover that these models show persistent DNA hypermethylation in CpG loci of the glioma CpG island methylator phenotype even after D-2HG production has been abolished. We also report a genome wide pattern of DNA demethylation in CpG sites outside of CpG islands, which reflect the acquisition of a G-CIMP-low like state after loss of D-2HG production. Then, using these cell line tools, we performed an unbiased sub-genomic CRISPR-library screening to identify genes whose functions supported the growth of glioma cells bearing endogenous IDH1 mutations. This work thus provides new patient derived models for exploring novel therapeutic opportunities for IDH1 mutant tumors, and uncovers the extent to which IDH mutation linked hypermethylation profiles in glioma depend upon D-2HG production from the IDH mutation.
Item Open Access Immunotoxin Monotherapy and Combinatorial Therapy With Immune Checkpoint Inhibitors for Malignant Brain Tumors(2016) Bao, XuhuiGlioblastoma is the most common and aggressive malignant brain tumor among all primary brain and central nervous system (CNS) tumors. The median survival time for glioblastoma patients given the current standard of care treatment (surgery, radiation, and chemotherapy) is less than 15 months. Medulloblastoma is another major malignant brain tumor that most frequently occurs in children. Although recent advances in surgery, radiotherapy, and chemotherapy have led to an increase in 5-year survival rates of medulloblastoma patients, treatment-related toxicity often has a major impact on long-term quality of survival.
As a result, there is an urgent need to develop more efficient and novel therapeutic approaches that specifically target tumor cells while preserving the surrounding normal CNS to improve the poor survival and quality of life of patients with malignant brain tumors. To address this need, we have developed two novel targeted immunotoxins (ITs), D2C7-(scdsFv)-PE38KDEL (D2C7-IT) and NZ-1-(scdsFv)-PE38KDEL (NZ-1-IT). D2C7-IT was developed by fusing the single-chain variable fragment (scFv) of the D2C7 monoclonal antibody (mAb) with domains II and III of Pseudomonas exotoxin A (PE38KDEL), and NZ-1-IT was developed by fusing the scFv of the NZ-1 mAb with PE38KDEL. D2C7-IT reacts with both the wild-type epidermal growth factor receptor (EGFRwt) and the EGFR variant III (EGFRvIII), two overexpressed proteins in glioblastomas. NZ-1-IT reacts with podoplanin (PDPN), a protein that has a high expression in glioblastomas and medulloblastomas.
In vitro cytotoxicity data shows that both ITs effectively inhibited protein synthesis in a variety of epitope-expressing glioblastoma and medulloblastoma xenograft cells and human tumor cell lines. Furthermore, the direct anti-tumor efficacy of D2C7-IT was examined in orthotopic glioma models in immunocompromised mice, while the direct anti-tumor efficacy of NZ-1-IT was observed in medulloblastoma xenograft-bearing immunocompromised mice. Both immunotoxins showed a robust anti-tumor efficacy in the preclinical brain tumor models. D2C7-IT was first investigated in the subsequent studies to accelerate its translation to the clinic. The preclinical toxicity of intracerebral D2C7-IT infusion was subsequently determined in normal Sprague-Dawley (SD) rats. The maximum tolerated dose (MTD) of D2C7-IT was determined to be between a total dose of 0.10 and 0.35 μg, and the no-observed-adverse-effect level (NOAEL) of D2C7-IT was a total dose of 0.05 μg in SD rats. Both the MTD and NOAEL were utilized as references for the D2C7-IT clinical trial design.
In addition to direct tumor cell killing, immunotoxin monotherapy has been shown to induce a secondary anti-tumor immune response through the engagement of T cells. Therefore, the D2C7-IT-induced secondary anti-tumor immune response was investigated using syngeneic mouse glioma models in immunocompetent mice. Moreover, previous studies have demonstrated that immune checkpoint inhibitors have a robust anti-tumor efficacy by augmenting the T cell response to the tumor cells. Thus, immune checkpoint inhibitors were combined with D2C7-IT in order to enhance the immunotoxin-induced anti-tumor immune response to eliminate residual tumor cells and prevent tumor recurrence in the long term. Meanwhile, studies with NZ-1-IT remain preliminary; thus, this IT will not be as robustly discussed as D2C7-IT throughout this text.
Item Open Access Investigation of Chondroprotective Mechanisms of Selenium(2010) Cheng, Wai MingSelenium (Se) is an essential trace element and metalloid involved in several key metabolic activities: protection against oxidative damage, regulation of immune and thyroid function, and fertility. Several recent lines of evidence from epidemiology, genetic, and transgenic animal studies suggest that Se may play a protective role in Osteoarthritis (OA). However, the exact protective mechanism of Se is still unclear.
In this study, we hypothesized that Se exerts its chondroprotective benefit via an anti-oxidative and anti-inflammatory effect mediated by specific selenoproteins that neutralize cytokine-induced inflammatory responses in chondrocytes. We established an in vitro system for studying the effect of Se in the chondrosarcoma cell line SW-1353 and in human primary chondrocytes. Selenomethionine (SeMet) induced gene expression and enzyme activity of both antioxidative enzymes glutathione peroxidase (GPX) and thioredoxin reductase (TR) in SW-1353 cells. Our data suggest that Se may be protective against oxidative stress through regulation of the activity of these antioxidative enzymes.
As IL-1β is one of the primary pro-inflammatory cytokines contributing to the progression in OA, we next investigated the effect of Se on the gene expression induced by physiological doses of IL-1β. SeMet inhibited IL-1β induced catabolic gene expression of matrix metalloproteinase 1 (MMP1) and MMP13 as well as total MMP activity in chondrocytes. Similarly, SeMet inhibited chondrocyte gene expression of IL-1β induced nitric oxide synthase (iNOS) and cyclooxygenase (COX2) with corresponding reductions in nitric oxide (NO) and prostaglandin E2 (PGE2) production. In addition, SeMet pretreatment attenuated the IL-1β induced activation of p38 MAPK but not the ERK, JNK or NFkB pathways. Taken together, our results suggest that Se inhibits IL-1β induced expression of inflammatory and catabolic genes, partly through inhibition of IL-1β cell signaling.
Since Se may function through selenoproteins, we evaluated the role of three specific major selenoproteins, GPX1, TR1 and DIO2, in modifying the inflammatory response stimulated by IL-1β in chondrocytes by RNA interference. Based on RNA interference results, DIO2 and TR1 mediated the inhibitory effect of SeMet on IL-1β induced COX2 gene expression, while GPX1 did not show a significant inhibitory effect on Se. Depletion of DIO2 increased the IL-1β induced COX2 gene expression. This suggests that DIO2 may negatively modulate the IL-1β response. Our data also suggest that part of this inhibitory effect of DIO2 could be through regulation of IL-1β gene expression itself. These results highlight a potential new role of DIO2 in modulating the inflammatory response in chondrocytes
In summary, the result of this study suggests that Se may exert its chondroprotective effect through specific selenoproteins which neutralize oxidative stress and modify the inflammatory response in chondrocytes.