Now showing items 1-20 of 1934

    • University of Maryland School of Medicine State of the School 2020

      University of Maryland, Baltimore. School of Medicine, 2020
    • Informing the role of RIFINs in malaria pathogenesis, natural immunity, and design of a severe malaria vaccine

      Zhou, Albert; Travassos, Mark A.; Laufer, Miriam K. (2020)
      Plasmodium falciparum is a eukaryotic parasite that causes severe malaria and contributed to 405,000 deaths worldwide in 2018. Victims of severe malaria are predominantly sub-Saharan African children, who typically present with symptoms of severe anemia or unarousable coma. The pathogenesis of severe malaria is poorly understood but mediated by the expression of adhesive variant surface antigens (VSAs) on infected red blood cells. VSAs are involved in sequestration and rosetting, unique virulence processes that allow the parasite to evade host immune responses and prevent clearance in the spleen. A relatively unstudied family of VSAs, the repetitive interspersed family (RIFIN) proteins, have recently been found to be important in rosetting and host immune suppression. RIFINs also appear to be targets for protective immunity; humoral immune responses against RIFINs have been correlated with asymptomatic infections. In this dissertation, I applied a multi-faceted approach using protein and peptide microarrays, transcriptomics, and reverse vaccinology to identify appealing RIFIN candidates for inclusion in a future severe malaria vaccine. I show that serological responses against epitopes within the semi-conserved domain of RIFINs associated with severe malaria reflected age-related malaria exposure. Sequencing and identifying specific rif genes expressed in clinical infections have not been feasible. I have addressed these challenges by adapting a novel bioinformatic pipeline and developing an HMM-based tool to process, assemble, classify, and subtype RIFIN sequences from peripheral blood samples. This takes advantage of a targeted probe capture method that I determined yields more abundant, full-length RIFIN sequences than other library enrichment approaches. Finally, I performed a comprehensive genomic survey of RIFIN gene repertoires using publicly available whole genome data of sixteen P. falciparum isolates to identify highly conserved, strain-transcendent sequences. Together, these results provide insights and powerful tools that can advance our understanding of the role RIFINs play in severe malaria pathogenesis and the development of naturally-acquired immunity to severe malaria. This work will aid efforts to determine targets for vaccines to protect children from the deadliest consequences of malaria.
    • Inhibition of Traumatic Brain Injury (TBI)-Induced Neuroinflammation Using Pharmacological Modulators of Metabotropic Glutamate Receptors

      Vinueza, Gelareh; Faden, A. I.; 0000-0002-1036-6378 (2020)
      Chronic dysregulated microglial activation is a major hallmark of persistent inflammation and progressive neurodegeneration following traumatic brain injury (TBI). Thus, research has focused on strategies to inhibit chronically activated microglial responses following TBI. Metabotropic glutamate receptors (mGluRs) 4 and 5 are expressed on microglia and can modulate microglial activity; therefore, they may serve as potential therapeutic targets for inhibition of microglial-dependent neuroinflammation. In the first of these studies, based on its reported neuroprotective roles, we examined the effects of the mGluR5 positive allosteric modulator (PAM) VU0360172 in an established fluid percussion injury (FPI) rat model of TBI plus hypobaria (HB). Systemic administration of VU0360172 significantly reduced pro-inflammatory cytokines, chemokines and microRNAs (miRs) at 1- and 7- days following FP+HB. However, VU0360172 did not alter injury-induced behavioral deficits examined over the following 28 days. In order to assess potential mechanisms underlying the inflammatory changes, we used Nanostring analysis to identify miRs that modulate neuroinflammation and compared plasma changes for selected miRs with brain tissue changes. The pro-inflammatory miR-223 showed the strongest correlation between plasma and brain tissue expression levels at the 7d time-point in TBI+HB experimental rodent models. An additional series of studies addressed the purported anti-inflammatory effects of mGluR4 PAMs. We employed in vitro models of immortalized microglia cell lines and primary microglia to elucidate the molecular mechanisms responsible for the modulation of inflammation by ADX88178 and other mGluR4 PAMs. ADX88178 downregulated lipopolysaccharide (LPS)-induced expression of pro-inflammatory mediators in BV2 cells and primary microglia. However, ADX88178 anti-inflammatory effects appeared to be mGluR4-independent as mGluR4 expression in our in vitro models was very low and its actions were not altered by pharmacological or molecular inhibition of mGluR4. Moreover, we showed that putative mGluR4 PAMs attenuate pro-inflammatory pathways in BV2 microglia through mGluR4/Gi-independent mechanisms involving activation of cAMP-response element binding protein (CREB) and inhibition of NFkB. Overall, these studies show that mGluR4 and mGluR5 PAMs can significantly attenuate microglial activation. Therefore, further studies should examine their potential therapeutic effectiveness after TBI.
    • Transient mechanical stimuli elicit rapid mechano-chemical signal transduction in non-tumorigenic and malignant mammary epithelial cells

      Pratt, Stephen JP; Martin, Stuart S.; 0000-0003-3627-7258 (2020)
      Changes in are observed during breast tumor formation and progression, which promote malignant phenotypes in both normal mammary epithelial cells and breast cancer cells. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. This work applied time-lapse confocal microscopy and quantitative methods to define the rapid mechanically-stimulated calcium signaling mechanisms that occur in breast epithelial cells. While most tumor cell studies focus on long-term effects of mechanical stimulation (>24h), the current approach detected an immediate initiation of cytosolic calcium signals within 2 seconds after transient mechanical stimulation. Two novel methods were developed to describe this response and underlying mechanisms: a) the real-time scratch assay and b) scratch on low elastic dishes (SLED). The real-time scratch assay revealed an ATP/P2Y2/Ca2+ signaling axis in response to scratch with implications as a path toward EMT. The second method developed was SLED, a non-damaging application of mechanical stress to breast epithelial cells with physiologic implications as the elasticity of the cell substrate better matched that of the mammary gland in vivo than the typical glass or plastic experimental dishes. New data obtained using this novel approach revealed that normal breast epithelial cells are mechanically sensitive, responding to mechanical stimuli through a two-part calcium signaling mechanism. An immediate, robust rise in intracellular calcium (within seconds) was observed followed by a persistent extracellular calcium influx (up to 30 minutes). This persistent calcium was sustained via microtubule-dependent mechano-activation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on TRPM8 channels to prolong calcium signaling. Disruption of this conserved mechanobiology mechanism was possible through oncogenic activation such that, among common oncogenic mutations, constitutively-active KRas suppressed this signaling pathway. Therefore, certain oncogenes render cells mechanically unresponsive which could have interesting implications for the evolution of cancer cell mechanosensing in the tumor microenvironment as cells acquire new mutations. In addition, altered expression of the ROS generator (NOX2) and the ROS-responsive channel (TRPM8) are indicators of reduced overall survival in ER-negative breast cancer, suggesting that this mechano-pathway identified in breast epithelial cells may also be modified in patients in vivo.
    • FOXN2 Expression: from Pluripotent Stem Cells to Neural Progenitor Cells

      Saleh, Wissam; Ament, Seth A. (2020)
      Several transcription factors (TFs) have been demonstrated as risk genes for schizophrenia (SCZ) based on genome-wide association studies (GWAS), fine-mapping, and functional studies. In this project, we prioritized TFs that have multiple lines of evidence supporting their likelihood of being causal risk loci for SCZ. We integrated results from four published studies, which used GWAS, gene expression and chromatin conformation studies to identify genes and gene networks associated with SCZ. These analyses revealed that the TF FOXN2 is a strong candidate risk gene for SCZ. Next, we characterized the dynamic expression profile of FOXN2 in our stem-cell based cortical neurogenesis model by qPCR. This study helped in establishing FOXN2 as a risk gene for SCZ potentially involved in cortical neurogenesis and building a strong base for the future genetic editing experiments designed for functional characterization of FOXN2 in cortical neurogenesis, that might relate to the pathophysiology of SCZ.
    • Delineating the Role of NKT cell Activation in B cell Lymphoma

      Lee, Michael; Webb, Tonya J. (2020)
      Natural Killer T (NKT) cells play an important role in cancer surveillance and can reduce lymphoma burden in vivo; however, a hallmark of cancer is its ability to evade immune surveillance. Our goals were to elucidate novel mechanisms utilized by B cell lymphoma to evade NKT cell-mediated immune surveillance and determine the prognostic potential of assessing NKT cell function in lymphoma patients. We found that knockdown of sphingosine kinase 1 (SK1) in human lymphoma cells results in a significant increase in CD1d-mediated NKT cell activation. Lipidomic and co-culture studies identified cardiolipin as being upregulated in SK1 knockdown cells and implicated cardiolipin as an NKT cell-specific cancer neoantigen. We also sought to determine the efficacy of NKT cell-based therapy on survival and the induction of anti-tumor immune responses in a mouse model of B cell lymphoma. We found that activation of NKT cells via early administration of α-galactosylceramide (α-GalCer) only provided modest protection. Our data suggest that the lack of protection is due, at least in part, to the expansion of myeloid-derived suppressor cells in α-GalCer-treated tumor bearing mice. Lastly, we sought to identify novel immunological biomarkers in lymphoma patients. It was found that lymphoma patients have a reduction in NKT cell function compared to healthy donors. Furthermore, lymphoma patients have significantly higher levels of both pro- and anti-inflammatory cytokines in their sera compared to healthy donors. In addition, lymphoma patients who experience relapse have significantly reduced NKT cell function in the blood, compared to lymphoma patients who did not relapse. Collectively, our studies demonstrate the multifaceted role NKT cells play in immune responses to B cell lymphoma and will help inform the next generation of cancer immunotherapy.
    • Post-translational Regulation of Glucokinase in Hypothalamic Neurons

      McFarland, Jennifer; Rizzo, Megan A. (2020)
      Glucose-sensing tissues utilize glucokinase (GCK), the activity of which is rate-limiting for glucose metabolism, to sense and, consequently, counteract deviations from glucose homeostasis. Post-translational regulation of GCK is well defined in the liver and the pancreas, and is critical for the maintenance of glucose homeostasis; yet, post- translational regulation of GCK in hypothalamic neurons, which play a central role in maintaining glucose homeostasis, remains relatively unexplored. Here, we use a hypothalamically-derived, glucose-sensing GT1-7 neuronal cell line to provide evidence of a receptor-driven, ER Ca2+-mediated S-nitrosylation and activation of GCK. Strategic pharmacological manipulations were paired with the assessment of GCK activity, done by either measuring NAD(P)H autofluorescence while raising extracellular glucose, or through expression of a homotransfer FRET GCK biosensor. Further, a biotin-switch assay was used to confirm the presence of GCK S-nitrosylation. This work illustrates a central mechanism of post-translational GCK regulation, which may underlie metabolic signal integration in the hypothalamus and may contribute to the pathology of diabetes.
    • Novel PC2 regulation of ezrin in renal epithelia reveals insights into ADPKD cystogenesis

      Dixon, Eryn; Woodward, Owen Maxwell (2020)
      Autosomal dominant polycystic kidney disease is caused by the loss of function of either two transmembrane proteins, polycystin-1 or polycystin-2. In renal epithelia, the consequence of polycystin loss is the formation of progressive, focal, fluid-filled cysts. However, the function and associated downstream signaling pathways specific to the polycystins have not been defined. Therefore, a new in vitro tubuloid model was designed to investigate the proximate cellular changes in renal epithelial cells following inactivation of Pkd2, the gene that encodes for polycystin-2. This model system reinforced the relevance of proteins associated with cell junctions, adhesions, and matrix in the cyst mechanism. The impact of this model was further supported through morphometrical analysis of epithelial compartmentalization in human ADPKD tissue, demonstrating an altered apical compartment in emerging cysts compared to noncystic tubules. Seeking connection between the junctions and disrupted apical compartment led to investigation of ezrin, a master scaffold in the apical compartment in renal epithelial cells. Ezrin plays a critical role in regulation of polarity, cytoskeleton organization, and protein trafficking, and the downstream consequences of its disruption have not been elucidated. Investigation into the initiating events of cystogenesis in ADPKD revealed a dramatic change in ezrin, following loss of PC2 in our tubuloid model, cystic mouse model, and pathological human ADPKD tissue. Based on this novel regulatory relationship between PC2 and ezrin, as well as the antecedent loss of ezrin to cyst formation in mice, ezrin was overexpressed in the pkd2 morpholino zebrafish model. Increased expression of ezrin diminished the formation of pronephric cysts. This lead to the design of a cyst rescue mouse model, which has exhibited promising preliminary data for cyst area reduction with additional ezrin. The disruption of ezrin in Pkd2 inducible in vitro and in vivo model systems, changes in ADPKD patient tissue, and rescue of pronephric cysts in the pkd2 MO suggest there is a role of ezrin in renal cystogenesis. Understanding the relationship of ezrin, with PC2 in renal epithelial cells will help elucidate the mechanism of ADPKD cystogenesis and define important downstream pathways necessary for epithelial functions.
    • The Role of Type I and III Interferons in the Pathogenesis of Bordetella pertussis Infection and Disease.

      Ardanuy, Jeremy; Carbonetti, Nicholas H. (2020)
      Bordetella pertussis is a Gram-negative bacterial pathogen that infects human respiratory tracts and is the causative agent for the disease pertussis, otherwise known as whooping cough. In 2012, there were 48,277 reported cases in the United States, the most since 1955. Symptoms build up to severe paroxysmal coughing, often for 10+ weeks after onset. For infants, pertussis can be fatal due to complications including pulmonary hypertension, pneumothorax, high-level circulating lymphocytosis, and pneumonia. In adults, the disease is severe too, with long-lasting cough, lung damage, and serious symptoms resulting in hospitalization. A difficulty in treatment/prevention of pertussis is a suboptimal vaccine that confers waning immunity, and a lack of effective treatments available. Antibiotics are administered to patients to prevent transmission, but usually don’t change the clinical course of disease. Host directed therapeutics treating pertussis could benefit individuals with severe cough and save the lives of infected infants. Using RNA-sequencing transcriptomics we investigated lung gene expression responses to Bordetella pertussis infection in adult mice, revealing that type I and III interferon (IFN) responses and signaling pathways may play an important role in promoting inflammatory responses. In B. pertussis infected mice, lung type I/III IFN responses correlated with increased proinflammatory cytokine expression and lung inflammatory pathology. In mutant mouse models with increased type I IFN signaling, B. pertussis exacerbated lung inflammatory pathology, whereas knockout mice with deficiencies in type I/III IFN signaling had reduced lung inflammation compared to wild-type mice. In direct contrast, infant mice didn’t upregulate type I/III IFNs in response to B. pertussis infection and were protected from lethal infection by increased type I IFN signaling, indicating age dependent effects of type I/III IFN signaling during B. pertussis infection. The induction of type I/III IFNs was found to be MyD88 dependent, and TLR9 and STING were identified as DNA sensing pattern recognition receptors required for type I/III IFN responses, as well as for typical levels of lung inflammatory pathology. This observation, coupled with results showing DNase treatment of B. pertussis-infected mice causing reduced lung pathology, indicated a DNA dependent induction of type I/III IFNs, making these targets for therapeutic intervention.
    • A Bridge Between Integration Stations: Insula’s Connection with the ventral Bed Nucleus of the Stria Terminalis

      Girven, Kasey; Sparta, Dennis R. (2020)
      Individuals suffering from substance use disorder often experience relapse events that are attributed to drug craving. Insular cortex (IC) function is implicated in processing drug-predictive cues and is thought to be a critical substrate for drug craving. Here, we uncover the functional connectivity of a novel projection from the IC to the ventral bed nucleus of the stria terminalis (vBNST), a portion of the extended amygdala shown to modulate dopaminergic activity within the ventral tegmental area, and investigate the role of this pathway in establishing reward-predictive cues. We hypothesized that these cues activate IC projections that synapse onto projection neurons within the vBNST, which then activate the mesolimbic dopamine pathway, resulting in the acquisition of associations between exteroceptive stimuli and rewards. In addition, due to the BNST’s role in ethanol self-administration combined with IC’s role in processing interoceptive cues associated with addictive substances, we predicted exposure to ethanol would affect the characteristics of vBNST-projecting IC neurons. Here we utilized both ex vivo slice electrophysiology and in vivo optogenetics to examine the functional connectivity and bidirectionally control the IC-vBNST projection in various reward-related behavioral paradigms. We also examined the effect of alcohol consumption on the IC-vBNST projection in acute and repeated ethanol exposure as well as in withdrawal. This work provides a potential mechanism by which the IC processes exteroceptive triggers that are predictive of reward.
    • Whole-genome analysis of Plasmodium falciparum isolates to understand allele-specific immunity to malaria

      Shah, Zalak; Takala-Harrison, Shannon (2020)
      After repeated P. falciparum infections, individuals in high-transmission areas acquire clinical immunity to malaria. However, the genes important in determining allele-specific immunity are not entirely known. Previous genome-wide approaches explored signatures of selection in the parasite genome to identify targets of clinical immunity; however, these approaches did not account for individual level allele-specific immunity. Here we take a whole-genome approach to identify genes that may be involved in acquisition of allele-specific immunity to malaria by analyzing parasite genomes collected from infected individuals in Malawi. However, obtaining whole genome sequence data from clinical samples is one of the major hurdles in the field of malaria genomics. In order to obtain whole genome sequence data from non-leukocyte depleted, low parasitemia samples, we optimized a selective-whole genome amplification (sWGA) by filtering the DNA prior to sWGA, to generate high coverage, whole genome sequence data from P. falciparum clinical samples with low amounts of parasite DNA. Using this optimized approach, we successfully performed whole-genome sequencing on 202 parasite isolates. We compared parasite genomes from individuals with varying levels of clinical immunity, defined using an individual’s proportion of symptomatic infections during the course of the study, hypothesizing that individuals with higher immunity become symptomatically ill due to infection with parasites with less common alleles. Using FST, we identified 161 SNPs to be genetically differentiated between the two groups and the median allele frequency was significantly lower at these sites in individuals in higher immunity group compared to the lower immunity group. We also examined pairs of parasites collected at different time points from the same individuals and identified 225 loci in 174 genes that vary within same individuals more often than expected by chance. Using both of these approaches, we identified 25 genes that encode likely targets of immunity, including a known antigen, CLAG8. Further analysis of clag8 global diversity showed evidence of immune selection in the C-terminal region, supporting the use of this approach in identification of new vaccine targets. Identifying and further analyzing these genomic regions will provide insights into mechanisms involved in allele-specific acquired immunity.
    • Placebo Analgesia in Neuropathic Pain: A Translational Investigative Approach from Rodents to Humans

      Akintola, Titilola; Colloca, Luana (2020)
      Pain is a complex phenomenon which can be influenced by various factors. Placebo analgesia (PA) is the experience of pain relief after the administration of a physiologically inert intervention via the expectation of benefit. However, adequate animal models of PA in chronic neuropathic pain were unavailable to determine how PA occurs in neuropathic pain. Neuropathic pain (NP) is a chronic pain condition characterized by a dysfunction of the peripheral or central nervous system. There is still limited progress in translating the findings of preclinical studies to address the clinical burden of chronic pain. This is thought to partly reflect difficulties in reliably assessing pain in animals. Hence, we employ a translational approach in both rodents and humans to explore the occurrence of PA in chronic NP. First, I tested the hypothesis that the facial grimace scale is a useful metric of spontaneous pain in rodents. We performed a chronic constriction injury of the infraorbital nerve (CCI-ION) and tested for changes in mechanical hypersensitivity and grimace scores. Results showed rodents with CCI-ION had significantly higher grimace scores and lower mechanical withdrawal thresholds compared to controls. These changes were reversed by an opioid, indicating the grimace scale as a sensitive metric for assessing ongoing pain in CCI-ION. Secondly, I tested the hypothesis that pharmacological conditioning with fentanyl would produce PA in a rat model of CCI-ION. Rats were pharmacologically conditioned with or without contextual cues. We administered a placebo and found marginally significant PA effect via the grimace scale but not in mechanical sensitivity. These findings suggest that PA may be more challenging to induce in rodents. Finally, in humans, I investigated how NP-like symptoms in Temporomandibular Joint Disorder alter PA. The effect of NP on PA is yet to be fully understood. I tested the hypothesis that the presence of NP-like symptoms would decrease PA in TMD. NP assessment was carried out both in the orofacial region and across the whole body using validated screening tools. Our results showed that the presence of co-occurring NP-like symptoms increased PA in TMD. We also show that this effect is mediated by reinforced expectation.
    • Identifying pathogenic mechanisms and new therapeutic targets for Gaucher disease using induced pluripotent stem cells

      Srikanth, Manasa; Feldman, Ricardo A. (2020)
      Gaucher Disease (GD), the most common lysosomal storage disorder, is caused by mutations in the GBA1 gene, which codes for the lysosomal enzyme β-glucocerebrosidase (GCase). GCase breaks down sphingolipids but when it is mutated, it causes the accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). The common manifestations of GD include hepatosplenomegaly, anemia, thrombocytopenia, skeletal disease, and in case of severe mutations, there are also fatal neurological manifestations. The conventional treatment is not effective in managing the skeletal or neurological manifestations. Hence, a better understanding of the underlying mechanisms that cause GD pathology is required for development of effective therapeutic strategies. The goal of this thesis was to identify the molecular mechanisms responsible for phenotypic alterations in osteoblasts and neuronal cells from GD patients, thereby pinpoint molecular targets for therapeutic intervention. Our laboratory utilizes patient-specific induced pluripotent stem cells (iPSCs) harboring GBA1 mutations to model GD. We have previously differentiated these iPSCs to various cell types and have shown that we can recapitulate the pathologic hallmarks of GD. Thus, in this study, we generated GD-iPSC derived osteoblasts and neuronal cells and found that mutations in GBA1 disrupt the canonical Wnt signaling and lysosomal compartment in these cell types. The phenotypic consequence of this was observed in the form of defective osteoblast differentiation and maturation as well as loss of midbrain/hindbrain neuronal progenitors in the respective cell types. Due to the known lysosomal dysregulation in GD, we then explored the mTOR pathway which is upstream of lysosomal biogenesis. We found hyperactivation of mTOR in GD neuronal cells was mediated by the significant accumulation of GlcSph, a lysolipid of GlcCer. In addition, when we blocked the conversion of GlcCer to GlcSph using acid ceramidase inhibitors, we were able to reverse mTOR hyperactivation and restore lysosomal expression, suggesting that GlcSph is partly, if not fully, responsible for the lysosomal abnormalities observed in GD. In conclusion, our study reveals that activation of canonical Wnt pathway or suppression of mTOR pathway ameliorates the phenotypic abnormalities in GD and identifies b-catenin, mTOR and acid ceramidase as potential therapeutic targets for GD.
    • Expression patterns of the metastasis suppressor NME1 in metastatic melanoma

      Snyder, Devin; Kaetzel, David M. (2020)
      Localized melanoma is a curable form of skin cancer, with a 98% chance for survival. However, for individuals diagnosed with metastatic melanoma, the five-year survival rate is reduced down to 23%. To enhance patient survival, it remains imperative to fully differentiate melanoma metastasis mechanisms. Our motivation lies on depicting underlying patterns accompanying metastatic inhibition. Here, we examine inhibition of melanoma metastasis through analysis of the metastasis suppressor protein, NME1. NME1 is a known inhibitor of melanoma metastasis. Overall loss of NME1 protein in melanoma tumors is associated with reduced survival. Yet, not all melanoma tumors are the same. Melanomas are heterogenous and exhibit distinct differences across patients and across individual primary tumor cells. The prognostic implications of heterogenous NME1 expression within melanomas has not previously been defined. Our focus lies in characterizing how expression of NME1 across patients and across primary tumor cells may impact melanoma prognosis and overall metastatic incidence. We utilized a NME1-based approach to identify a mRNA expression signature associated with metastasis suppression in melanoma. Bioinformatic analysis of our metastasis suppression signature in a melanoma patient dataset exposed a subset of melanoma patients with altered survival. We were also interested in analyzing the heterogenous expression of NME1 protein within melanoma cells. Flow cytometry analysis revealed that melanoma cell lines contain a rare population of cells with low levels of NME1 protein. We utilized multiple methods to characterize the impact of NME1 loss on cell behavior. Experiments designed to mimic loss of NME1 through shNME1 produced results which suggest that NME1 is associated with melanoma spheroid formation. We further utilized an innovative approach, which relied on CRISPR technology, to study endogenous NME1 expression. Characterization of endogenous NME1 expression patterns lead to the identification of a previously unidentified group of cells, which have a neural crest-like phenotype and contain enhanced metastatic capacity. Overall, the expression pattern of NME1 within tumor cells and across patients provides an advantageous tool for identifying metastatic susceptibility.
    • Development and Application of Vertebrate Models to Investigate the Risk of Defense-Relevant Chemicals

      Narizzano, Allison; Pereira, Edna F. R.; Quinn, Michael J., Jr. (2020)
      Adverse effects from unintended exposure to chemicals have been widely described and have provided the basis for many chemical and environmental management regulations and policies that are intended to protect humans, animals, and/or the environment. The present work focuses on two classes of chemicals that are tested and/or used in training activities at military sites: insensitive munitions (IMs) and per- and polyfluoroalkyl substances (PFAS). As groundwater is a common source of drinking water, accurate risk assessments are critically needed to ensure protection of the environment and public health. IMs are more resistant to accidental, unintentional or incidental detonation than traditional explosives. Incomplete detonation may deposit munition components and by-products on impacted installations and, importantly, mobilize those constituents into groundwater. One component of several IM mixtures is 3-nitro-1,2,4-triazol-5-one (NTO). NTO is especially worrisome because it is highly water soluble and, thus, mobile in the environment. Additionally, laboratory tests with rodents have identified the testes and epididymides as targets of NTO. PFAS are also prevalent and problematic, nearly universal contaminants. PFAS are manufactured for use in paints, cleaning agents, non-stick cookware and food containers, water-impermeable products, and Aqueous Film Forming Foams (AFFFs). PFAS have attracted increased regulatory scrutiny because of their resistance to degradation, ability to bioaccumulate, and growing evidence of toxicity in animals. A considerable body of work has examined the effects of NTO in laboratory rodents. However, there is still uncertainty in the derivation of a safe workplace environmental exposure level (WEEL) for NTO. The present studies evaluate the effects of NTO in the Japanese quail (Coturnix japonica) and in Sprague Dawley rats. In concert, these toxicity data on NTO will improve risk estimation efforts, provide data to explore taxa read-across, and fill a regulatory needed data-gap. The toxicity of PFAS will be evaluated in the native mammalian species, Peromyscus leucopus. Given that the serum elimination half-lives within this class of chemicals can vary greatly from hours to years based on species, sex, and dose, extrapolating between species is difficult and inaccurate. As such, wild mammalian-specific data is novel and valuable for risk estimation in taxa that are directly site-specific.
    • Inducing DNA Repair Deficiencies in Triple Negative Breast Cancers Through Pharmacologic Stimulation of Innate Immune Signaling

      McLaughlin, Lena; Rassool, Feyruz V. (2020)
      Poly (ADP-ribose) polymerase inhibitors (PARPi) are FDA approved in a subset of patients with ovarian cancer or metastatic breast cancers who harbor BRCA gene mutations. These mutations generate homologous recombination deficiencies (HRD) and are the main predictor to PARPi sensitivity. Unfortunately, responses to therapy have not been durable and have failed for the majority of sporadic triple negative breast cancers (TNBC). We previously reported that DNA methyltransferase inhibitor (DNMTi) azacytidine (Aza) improves the efficacy of a new generation of PARPi, Talazoparib (Tal), through increased trapping of cytotoxic PARP-DNA complexes in both BRCA-mutant and -proficient TNBC. These trapped complexes lead to increased and persistent levels of lethal double strand breaks (DSBs), suggesting that DSB repair may also be impaired with this treatment. In the present study, we show that Aza/Tal treatment in BRCA-proficient TNBC cell lines significantly downregulates expression of HR and Fanconi Anemia (FA) genes, notably FANCD2, and decreases HR activity, thus generating HRD. DNMTi have also been established to induce a viral mimicry response which upregulate Type I interferon (IFN) signaling and production of inflammatory cytokines. We now link Aza/Tal facilitated HRD and induction of innate immune and inflammatory related genes, mediated in part through a STING dependent mechanism. Gene set enrichment analysis of RNA-Seq data derived from mono- and combination-treatments, reveal enrichment of innate immune and cytosolic DNA sensing pathways with significant increases of TNFα/NF-κB and IFNαβ gene sets. Overlap between HRD and immune related signaling was evaluated using the STRING database, which reveals a significant interaction specifically between FA pathway and TNFα/NF-κB and IFNαβ pathway genes. This inverse relationship was also validated in both METABRIC TNBC dataset and other TCGA data sets suggesting broad applicability of this observed transcriptional program independent of pharmacologic intervention. Additionally, Tal driven cytosolic DNA as well as an Aza augmentation in STING protein expression, emerges as the key node in Aza/Tal induced innate immune signaling to drive HRD. Induction of what we define as a pathogen mimicry response to drive HRD mechanism suggests that DNMTi-PARPi therapy strategies can expand the therapeutic scope of PARPi to encompass treatment of BRCA-proficient cancers.
    • Elucidating the Localization of Estrogen and Estrogen-related Receptors in the Inner Ear

      Lipford, Erika; Hertzano, Ronna P. (2020)
      Hearing loss is the most common sensory impairment, affecting hundreds of millions of people worldwide. Although both men and women are impacted by hearing loss, the incidence rate differs between the two sexes, with the prevalence of bilateral high-frequency hearing loss reported to be 2.7 times higher in males. Recent studies have implicated estrogen as having a protective effect against hearing loss in females. While the localization of the estrogen receptors within the cochlea is known, the roles of estrogen-related receptors in auditory function require further investigation. The aim of this study is to elucidate the localization of estrogen receptors 1 and 2 and estrogen-related receptors α, β, and γ within the inner ear of adult mice using RNAscope. Our results give insight into the molecular mechanism through which estrogen receptors and estrogen-related receptors may grant protection against hearing loss and contribute to the functionality of the inner ear.