Browsing by Author "Morrison, Holly A."

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    Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporation
    Hendricks-Wenger, Alissa; Aycock, Kenneth N.; Nagai-Singer, Margaret A.; Coutermarsh-Ott, Sheryl; Lorenzo, Melvin F.; Gannon, Jessica; Uh, Kyungjun; Farrell, Kayla; Beitel-White, Natalie; Brock, Rebecca M.; Simon, Alexander; Morrison, Holly A.; Tuohy, Joanne L.; Clark-Deener, Sherrie; Vlaisavljevich, Eli; Davalos, Rafael V.; Lee, Kiho; Allen, Irving C. (Nature Research, 2021-04-07)
    New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients’ anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.
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    Improved Therapeutic Delivery Targeting Clinically Relevant Orthotopic Human Pancreatic Tumors Engrafted in Immunocompromised Pigs Using Ultrasound-Induced Cavitation: A Pilot Study
    Imran, Khan Mohammad; Tintera, Benjamin; Morrison, Holly A.; Tupik, Juselyn D.; Nagai-Singer, Margaret A.; Ivester, Hannah; Council-Troche, McAlister; Edwards, Michael; Coutermarsh-Ott, Sheryl; Byron, Christopher; Clark-Deener, Sherrie; Uh, Kyungjun; Lee, Kiho; Boulos, Paul; Rowe, Cliff; Coviello, Christian; Allen, Irving C. (MDPI, 2023-05-24)
    Pancreatic tumors can be resistant to drug penetration due to high interstitial fluid pressure, dense stroma, and disarrayed vasculature. Ultrasound-induced cavitation is an emerging technology that may overcome many of these limitations. Low-intensity ultrasound, coupled with co-administered cavitation nuclei consisting of gas-stabilizing sub-micron scale SonoTran Particles, is effective at increasing therapeutic antibody delivery to xenograft flank tumors in mouse models. Here, we sought to evaluate the effectiveness of this approach in situ using a large animal model that mimics human pancreatic cancer patients. Immunocompromised pigs were surgically engrafted with human Panc-1 pancreatic ductal adenocarcinoma (PDAC) tumors in targeted regions of the pancreas. These tumors were found to recapitulate many features of human PDAC tumors. Animals were intravenously injected with the common cancer therapeutics Cetuximab, gemcitabine, and paclitaxel, followed by infusion with SonoTran Particles. Select tumors in each animal were targeted with focused ultrasound to induce cavitation. Cavitation increased the intra-tumor concentrations of Cetuximab, gemcitabine, and paclitaxel by 477%, 148%, and 193%, respectively, compared to tumors that were not targeted with ultrasound in the same animals. Together, these data show that ultrasound-mediated cavitation, when delivered in combination with gas-entrapping particles, improves therapeutic delivery in pancreatic tumors under clinically relevant conditions.
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    NF-κB Inducing Kinase Attenuates Colorectal Cancer by Regulating Noncanonical NF-κB Mediated Colonic Epithelial Cell Q1 Regeneration
    Morrison, Holly A.; Eden, Kristin; Trusiano, Brie; Rothschild, Daniel E.; Qin, Yufeng; Wade, Paul A.; Rowe, Audrey J.; Mounzer, Christina; Stephens, Morgan C.; Hanson, Katherine M.; Brown, Stephan L.; Holl, Eda K.; Allen, Irving C. (Elsevier, 2024-06)
    BACKGROUND & AIMS: Dysregulated colonic epithelial cell (CEC) proliferation is a critical feature in the development of colorectal cancer. We show that NF-𝜅B-inducing kinase (NIK) attenuates colorectal cancer through coordinating CEC regeneration/ differentiation via noncanonical NF-𝜅B signaling that is unique from canonical NF-𝜅B signaling. METHODS: Initial studies evaluated crypt morphology/functionality, organoid generation, transcriptome profiles, and the microbiome. Inflammation and inflammation-induced tumorigenesis were initiated in whole-body NIK knockout mice (Nik⁻/⁻) and conditional-knockout mice following administration of azoxymethane and dextran sulfate sodium. RESULTS: Human transcriptomic data revealed dysregulated noncanonical NF-𝜅B signaling. In vitro studies evaluating Nik⁻/⁻ crypts and organoids derived from mature, nondividing CECs, and colonic stem cells exhibited increased accumulation and stunted growth, respectively. Transcriptomic analysis of Nik⁻/⁻ cells revealed gene expression signatures associated with altered differentiation-regeneration. When assessed in vivo, Nik⁻/⁻ mice exhibited more severe colitis with dextran sulfate sodium administration and an altered microbiome characterized by increased colitogenic microbiota. In the inflammationinduced tumorigenesis model, we observed both increased tumor burdens and inflammation in mice where NIK is knocked out in CECs (NikΔCEC). Interestingly, this was not recapitulated when NIK was conditionally knocked out in myeloid cells (NikΔMYE). Surprisingly, conditional knockout of the canonical pathway in myeloid cells (RelAΔMYE) revealed decreased tumor burden and inflammation and no significant changes when conditionally knocked out in CECs (RelAΔCEC) CONCLUSIONS: Dysregulated noncanonical NF-𝜅B signaling is associated with the development of colorectal cancer in a tissue-dependent manner and defines a critical role for NIK in regulating gastrointestinal inflammation and regeneration associated with colorectal cancer.
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    NLRX1 Deficiency Alters the Gut Microbiome and Is Further Exacerbated by Adherence to a Gluten-Free Diet
    Morrison, Holly A.; Liu, Yang; Eden, Kristin; Nagai-Singer, Margaret A.; Wade, Paul A.; Allen, Irving C. (Frontiers, 2022-04-28)
    Patients with gluten sensitivities present with dysbiosis of the gut microbiome that is further exacerbated by a strict adherence to a gluten-free diet (GFD). A subtype of patients genetically susceptible to gluten sensitivities are Celiac Disease (CeD) patients, who are carriers of the HLA DR3/DQ2 or HLA DR4/DQ8 haplotypes. Although 85-95% of all CeD patients carry HLA DQ2, up to 25-50% of the world population carry this haplotype with only a minority developing CeD. This suggests that CeD and other gluten sensitivities are mediated by factors beyond genetics. The contribution of innate immune system signaling has been generally understudied in the context of gluten sensitivities. Thus, here we examined the role of NOD-like receptors (NLRs), a subtype of pattern recognition receptors, in maintaining the composition of the gut microbiome in animals maintained on a GFD. Human transcriptomics data revealed significant increases in the gene expression of multiple NLR family members, across functional groups, in patients with active CeD compared to control specimens. However, NLRX1 was uniquely down-regulated during active disease. NLRX1 is a negative regulatory NLR that functions to suppress inflammatory signaling and has been postulate to prevent inflammation-induced dysbiosis. Using Nlrx1(-/-) mice maintained on either a normal or gluten-free diet, we show that loss of NLRX1 alters the microbiome composition, and a distinctive shift further ensues following adherence to a GFD, including a reciprocal loss of beneficial microbes and increase in opportunistic bacterial populations. Finally, we evaluated the functional impact of an altered gut microbiome by assessing short- and medium-chain fatty acid production. These studies revealed significant differences in a selection of metabolic markers that when paired with 16S rRNA sequencing data could reflect an overall imbalance and loss of immune system homeostasis in the gastrointestinal system.
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    NLRX1 Is a Multifaceted and Enigmatic Regulator of Immune System Function
    Nagai-Singer, Margaret A.; Morrison, Holly A.; Allen, Irving C. (2019-10-11)
    Over the last decade, significant progress has been achieved in defining mechanisms underlying NLR regulation of immune system function. However, several NLR family members continue to defy our best attempts at characterization and routinely exhibit confounding data. This is particularly true for NLR family members that regulate signaling associated with the activation of other pattern recognition receptors. NLRX1 is a member of this NLR sub-group and acts as an enigmatic regulator of immune system function. NLRX1 has been shown to negatively regulate type-I interferon, attenuate pro-inflammatory NF-kappa B signaling, promote reactive oxygen species production, and modulate autophagy, cell death, and proliferation. However, the mechanism/s associated with NLRX1 modulation of these pathways is not fully understood and there are inconsistencies within the field. Likewise, it is highly likely that the full repertoire of biological functions impacted by NLRX1 are yet to be defined. Recent mouse studies have shown that NLRX1 significantly impacts a multitude of diseases, including cancer, virus infection, osteoarthritis, traumatic brain injury, and inflammatory bowel disease. Thus, it is essential that the underlying mechanism associated with NLRX1 function in each of these diseases be robustly defined. Here, we summarize the current progress in understanding mechanisms associated with NLRX1 function. We also offer insight into both unique and overlapping mechanisms regulated by NLRX1 that likely contribute to disease pathobiology. Ultimately, we believe that an improved understanding of NLRX1 will result in better defined mechanisms associated with immune system attenuation and the resolution of inflammation in a myriad of diseases.
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    Noncanonical NF-kappa B Signaling Upregulation in Inflammatory Bowel Disease Patients is Associated With Loss of Response to Anti-TNF Agents
    Nguyen, Vu Q.; Eden, Kristin; Morrison, Holly A.; Sammons, Megan B.; Knight, Kristin K.; Sorrentino, Siena; Brock, Rebecca M.; Grider, Douglas J.; Allen, Irving C.; Sorrentino, Dario (2021-06-10)
    Objectives: Targeting tumor necrosis factor (TNF) with biologic agents, such as infliximab and adalimumab, is a widely used and effective therapeutic strategy in inflammatory bowel disease (IBD). Unfortunately, a significant number of patients fail to respond or lose response over time to these agents. Previous studies have defined multiple complex roles for canonical NF-kappa B signaling in the pathogenesis of IBD. However, preliminary evidence suggests that the lesser defined noncanonical NF-kappa B signaling pathway also contributes to disease pathogenesis and response to anti-TNF agents. The objective of this study was to evaluate this hypothesis in Crohn's disease (CD) and ulcerative colitis (UC) patients. Design: A total of 27 subjects with IBD (19 with CD and 8 with UC) and 15 control subjects were tested. Clinical criteria, patient history, and endoscopic disease activity were factors used to categorize patients and define therapeutic response. Biopsy specimens were collected during colonoscopy and expression was determined for 88 target genes known to be associated with noncanonical NF-kappa B signaling and IBD. Results: Noncanonical NF-kappa B signaling was significantly upregulated in IBD patients and was associated with increased gastrointestinal inflammation, epithelial cell death, lymphocyte migration, and Nod-like receptor signaling. Furthermore, noncanonical NF-kappa B signaling was further upregulated in patients unresponsive to anti-TNF agents and was suppressed in responsive patients. MAP3K14, NFKB2, CCL19, CXCL12, and CXCL13 were significantly dysregulated, as were genes that encode pathway regulators, such as CYLD, NLRP12, and BIRC2/3. Conclusion: Our study identifies a previously uncharacterized role for the understudied noncanonical NF-kappa B signaling pathway in the pathogenesis of IBD and anti-TNF therapy responsiveness. The genes and pathways identified may ultimately prove useful in IBD management and could potentially be used as biomarkers of drug response.
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    The Pro-Inflammatory Chemokines CXCL9, CXCL10 and CXCL11 Are Upregulated Following SARS-CoV-2 Infection in an AKT-Dependent Manner
    Callahan, Victoria; Hawks, Seth A.; Crawford, Matthew A.; Lehman, Caitlin W.; Morrison, Holly A.; Ivester, Hannah M.; Akhrymuk, Ivan V.; Boghdeh, Niloufar; Flor, Rafaela; Finkielstein, Carla V.; Allen, Irving C.; Weger-Lucarelli, James; Duggal, Nisha K.; Hughes, Molly A.; Kehn-Hall, Kylene (MDPI, 2021-06-03)
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a ‘cytokine storm.’ In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.
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    Pulmonary Exposure to Magnéli Phase Titanium Suboxides Results in Significant Macrophage Abnormalities and Decreased Lung Function
    McDaniel, Dylan K.; Ringel-Scaia, Veronica M.; Morrison, Holly A.; Coutermarsh-Ott, Sheryl; Council-Troche, McAlister; Angle, Jonathan W.; Perry, Justin B.; Davis, Grace; Leng, Weinan; Minarchick, Valerie; Yang, Yi; Chen, Bo; Reece, Sky W.; Brown, David A.; Cecere, Thomas E.; Brown, Jared M.; Gowdy, Kymberly M.; Hochella, Michael F. Jr.; Allen, Irving C. (Frontiers, 2019-11-28)
    Coal is one of the most abundant and economic sources for global energy production. However, the burning of coal is widely recognized as a significant contributor to atmospheric particulate matter linked to deleterious respiratory impacts. Recently, we have discovered that burning coal generates large quantities of otherwise rare Magnéli phase titanium suboxides from TiO2 minerals naturally present in coal. These nanoscale Magnéli phases are biologically active without photostimulation and toxic to airway epithelial cells in vitro and to zebrafish in vivo. Here, we sought to determine the clinical and physiological impact of pulmonary exposure to Magnéli phases using mice as mammalian model organisms. Mice were exposed to the most frequently found Magnéli phases, Ti6O11, at 100 parts per million (ppm) via intratracheal administration. Local and systemic titanium concentrations, lung pathology, and changes in airway mechanics were assessed. Additional mechanistic studies were conducted with primary bone marrow derived macrophages. Our results indicate that macrophages are the cell type most impacted by exposure to these nanoscale particles. Following phagocytosis, macrophages fail to properly eliminate Magnéli phases, resulting in increased oxidative stress, mitochondrial dysfunction, and ultimately apoptosis. In the lungs, these nanoparticles become concentrated in macrophages, resulting in a feedback loop of reactive oxygen species production, cell death, and the initiation of gene expression profiles consistent with lung injury within 6 weeks of exposure. Chronic exposure and accumulation of Magnéli phases ultimately results in significantly reduced lung function impacting airway resistance, compliance, and elastance. Together, these studies demonstrate that Magnéli phases are toxic in the mammalian airway and are likely a significant nanoscale environmental pollutant, especially in geographic regions where coal combustion is a major contributor to atmospheric particulate matter.