Browsing by Author "Allen, Irving C."

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    Ablative and Immunostimulatory Effects of Histotripsy Ablation in a Murine Osteosarcoma Model
    Hay, Alayna N.; Imran, Khan Mohammad; Hendricks-Wenger, Alissa; Gannon, Jessica M.; Sereno, Jacqueline; Simon, Alex; Lopez, Victor A.; Coutermarsh-Ott, Sheryl; Vlaisavljevich, Eli; Allen, Irving C.; Tuohy, Joanne L. (MDPI, 2023-10-09)
    Background: Osteosarcoma (OS) is the most frequently occurring malignant bone tumor in humans, primarily affecting children and adolescents. Significant advancements in treatment options for OS have not occurred in the last several decades, and the prognosis remains grim with only a 70% rate of 5-year survival. The objective of this study was to investigate the focused ultrasound technique of histotripsy as a novel, noninvasive treatment option for OS. Methods: We utilized a heterotopic OS murine model to establish the feasibility of ablating OS tumors with histotripsy in a preclinical setting. We investigated the local immune response within the tumor microenvironment (TME) via immune cell phenotyping and gene expression analysis. Findings: We established the feasibility of ablating heterotopic OS tumors with ablation characterized microscopically by loss of cellular architecture in targeted regions of tumors. We observed greater populations of macrophages and dendritic cells within treated tumors and the upregulation of immune activating genes 72 h after histotripsy ablation. Interpretation: This study was the first to investigate histotripsy ablation for OS in a preclinical murine model, with results suggesting local immunomodulation within the TME. Our results support the continued investigation of histotripsy as a novel noninvasive treatment option for OS patients to improve clinical outcomes and patient prognosis.
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    Analysis of the Murine Immune Response to Pulmonary Delivery of Precisely Fabricated Nano- and Microscale Particles
    Roberts, Reid A.; Shen, Tammy; Allen, Irving C.; Hasan, Warefta; DeSimone, Joseph M.; Ting, Jenny P.-Y. (PLOS, 2013-04-12)
    Nanomedicine has the potential to transform clinical care in the 21st century. However, a precise understanding of how nanomaterial design parameters such as size, shape and composition affect the mammalian immune system is a prerequisite for the realization of nanomedicine's translational promise. Herein, we make use of the recently developed Particle Replication in Non-wetting Template (PRINT) fabrication process to precisely fabricate particles across and the nano- and micro-scale with defined shapes and compositions to address the role of particle design parameters on the murine innate immune response in both in vitro and in vivo settings. We find that particles composed of either the biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) or the biocompatible polymer polyethylene glycol (PEG) do not cause release of pro-inflammatory cytokines nor inflammasome activation in bone marrow-derived macrophages. When instilled into the lungs of mice, particle composition and size can augment the number and type of innate immune cells recruited to the lungs without triggering inflammatory responses as assayed by cytokine release and histopathology. Smaller particles (80×320 nm) are more readily taken up in vivo by monocytes and macrophages than larger particles (6 µm diameter), yet particles of all tested sizes remained in the lungs for up to 7 days without clearance or triggering of host immunity. These results suggest rational design of nanoparticle physical parameters can be used for sustained and localized delivery of therapeutics to the lungs.
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    Antibiotics ameliorate lupus-like symptoms in mice
    Mu, Qinghui; Tavella, Vincent J.; Kirby, Jay L.; Cecere, Thomas E.; Chung, Matthias; Lee, Jiyoung; Li, Song; Ahmed, Sattar Ansar; Eden, Kristin; Allen, Irving C. (Nature, 2017-10-20)
    Gut microbiota and the immune system interact to maintain tissue homeostasis, but whether this interaction is involved in the pathogenesis of systemic lupus erythematosus (SLE) is unclear. Here we report that oral antibiotics given during active disease removed harmful bacteria from the gut microbiota and attenuated SLE-like disease in lupus-prone mice. Using MRL/lpr mice, we showed that antibiotics given after disease onset ameliorated systemic autoimmunity and kidney histopathology. They decreased IL-17-producing cells and increased the level of circulating IL-10. In addition, antibiotics removed Lachnospiraceae and increased the relative abundance of Lactobacillus spp., two groups of bacteria previously shown to be associated with deteriorated or improved symptoms in MRL/lpr mice, respectively. Moreover, we showed that the attenuated disease phenotype could be recapitulated with a single antibiotic vancomycin, which reshaped the gut microbiota and changed microbial functional pathways in a time-dependent manner. Furthermore, vancomycin treatment increased the barrier function of the intestinal epithelium, thus preventing the translocation of lipopolysaccharide, a cell wall component of Gram-negative Proteobacteria and known inducer of lupus in mice, into the circulation. These results suggest that mixed antibiotics or a single antibiotic vancomycin ameliorate SLE-like disease in MRL/lpr mice by changing the composition of gut microbiota.
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    ASC-Mediated Inflammation and Pyroptosis Attenuates Brucella abortus Pathogenesis Following the Recognition of gDNA
    Tupik, Juselyn D.; Coutermarsh-Ott, Sheryl; Benton, Angela H.; King, Kellie A.; Kiryluk, Hanna D.; Caswell, Clayton C.; Allen, Irving C. (MDPI, 2020-11-30)
    Brucella abortus is a zoonotic pathogen that causes brucellosis. Because of Brucella’s unique LPS layer and intracellular localization predominately within macrophages, it can often evade immune detection. However, pattern recognition receptors are capable of sensing Brucella pathogen-associated molecular patterns (PAMPS). For example, NOD-like receptors (NLRs) can form a multi-protein inflammasome complex to attenuate Brucella pathogenesis. The inflammasome activates IL-1β and IL-18 to drive immune cell recruitment. Alternatively, inflammasome activation also initiates inflammatory cell death, termed pyroptosis, which augments bacteria clearance. In this report, we assess canonical and non-canonical inflammasome activation following B. abortus infection. We conducted in vivo studies using Asc−/− mice and observed decreased mouse survival, immune cell recruitment, and increased bacteria load. We also conducted studies with Caspase-11−/− mice and did not observe any significant impact on B. abortus pathogenesis. Through mechanistic studies using Asc−/− macrophages, our data suggests that the protective role of ASC may result from the induction of pyroptosis through a gasdermin D-dependent mechanism in macrophages. Additionally, we show that the recognition of Brucella is facilitated by sensing the PAMP gDNA rather than the less immunogenic LPS. Together, these results refine our understanding of the role that inflammasome activation and pyroptosis plays during brucellosis.
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    The Biological and Immunological Effects of Irreversible Electroporation and Combination Therapy Options for the Improving the Treatment of Pancreatic Cancer
    Brock, Rebecca Michaela (Virginia Tech, 2020-06-05)
    While cancer treatments have advanced for multiple cancers, pancreatic cancer remains a lethal cancer with few therapy options available. This is due to the inaccessibility of the tumor by surgical and thermal ablative means, high potential for chemoresistance and metastasis, and strongly immunosuppressive tumor microenvironment that makes new treatment measures ineffective in clinic. Irreversible electroporation (IRE) utilizes short, high voltage electrical pulses to form microlesions in cell membranes and induce cell death. While IRE has had significant impact in pancreatic cancer treatment in clinical trials, little is known on the biological and immunological effects of IRE on pancreatic cancer. By studying the effects of IRE on pancreatic tumor biology and the host immune system, I hypothesize I can identify potential combination therapy targets for IRE. I utilized in vitro, ex vivo, and in vivo animal models of both human and murine cancer to study the effects of IRE on pancreatic cancer progression and its potential for immunomodulation. My findings have shown that IRE can significantly delay cancer progression by inducing necroptosis in the tumor and altering the tumor microenvironment by increasing inflammatory signaling. IRE can also produce viable antigens for presentation to induce local and systemic immunosurveillance. However, these effects are limited by countering expression of programmed-cell death ligand 1 (PD-L1), a checkpoint protein that inhibits cytotoxic lymphocyte activity and allows the tumors to recur. The effects of IRE can therefore be expanded by multiple combination therapy approaches, such as chemotherapeutic application (potentially with nanoparticle packaging), PD-1/PD-L1 antibody immunotherapies, and small molecule inhibitors directed at tumor growth signaling that previously showed limited efficacy in clinic.
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    Caspase-11 Modulates Inflammation and Attenuates Toxoplasma gondii Pathogenesis
    Coutermarsh-Ott, Sheryl; Doran, John T.; Campbell, Caroline; Williams, Tere M.; Lindsay, David S.; Allen, Irving C. (Hindawi, 2016-06-09)
    Toxoplasma gondii is an obligate intracellular parasite that is the etiologic agent responsible for toxoplasmosis. Infection with T. gondii results in activation of nucleotide binding domain and leucine rich repeat containing receptors (NLRs). NLR activation leads to inflammasome formation, the activation of caspase-1, and the subsequent cleavage of IL-1β and IL-18. Recently, a noncanonical inflammasome has been characterized which functions through caspase-11 and appears to augment many biological functions previously considered to be dependent upon the canonical inflammasome. To better elucidate the function of this noncanonical inflammasome in toxoplasmosis, we utilized Asc−/− and Casp11−/− mice and infected these animals with T. gondii. Our data indicates that caspase-11 modulates the innate immune response to T. gondii through a mechanism which is distinct from that currently described for the canonical inflammasome. Asc−/− mice demonstrated increased disease pathogenesis during the acute phase of T. gondii infection, whereas Casp11−/− mice demonstrated significantly attenuated disease pathogenesis and reduced inflammation. This attenuated host response was associated with reduced local and systemic cytokine production, including diminished IL-1β. During the chronic phase of infection, caspase-11 deficiency resulted in increased neuroinflammation and tissue cyst burden in the brain. Together, our data suggest that caspase-11 functions to protect the host by enhancing inflammation during the early phase of infection in an effort to minimize disease pathogenesis during later stages of toxoplasmosis.
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    Characterization of Biomedical and Incidental Nanoparticles in the Lungs and Their Effects on Health
    McDaniel, Dylan K. (Virginia Tech, 2018-11-20)
    Nanomaterials are defined as any material with at least one external dimension less than 100 nm. Recently, nanomaterials have become more common in medicine, technology, and engineering. One reason for their increased interest is due to nanomaterials having unique properties that allow them to interact effectively with biological systems. In terms of drug delivery, the lungs are a highly desirable site to administer therapeutic nanoparticles. Indeed, inflammatory diseases such as asthma and emphysema could potentially benefit from nanoparticle-mediated delivery. However, the lungs are also in constant contact with airborne particulate matter. Thus, harmful nanoparticles can enter the lungs and cause or even exacerbate inflammatory diseases. Our work focused on characterization of both therapeutic and potentially harmful nanoparticles in the lungs. We found that fluorescently-labeled nanoparticles were phagocytosed by macrophages and did not induce apoptosis or inflammation in the lungs, making them potentially useful as a therapeutic for inflammatory diseases. We also characterized a rare form of titanium-based particles called Magnéli phases, which have been shown to be produced via coal burning. We found that while these particles are non-inflammatory in the lungs of mice, they lead to apoptosis of macrophages as well as a change in gene expression associated with increased fibrosis. Ultimately, this was shown to lead to a decrease in lung function parameters and airway hyperresponsiveness, indicating increased lung stiffness after long-term nanoparticle exposure. Our data adds significant contributions to the field by assessing two nanoparticles with vastly different compositions in the lungs. Overall, we found that the unique properties of both particle types allows for interactions with cells and tissues. These interactions can have important outcomes on health, both in terms of disease treatment and exacerbation.
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    Characterization of NLRP12 during the Development of Allergic Airway Disease in Mice
    Allen, Irving C.; Lich, John D.; Arthur, Janelle C.; Jania, Corey M.; Roberts, Reid A.; Callaway, Justin B.; Tilley, Stephen L.; Ting, Jenny P.-Y. (PLOS, 2012-01-23)
    Among the 22 members of the nucleotide binding-domain, leucine rich repeat-containing (NLR) family, less than half have been functionally characterized. Of those that have been well studied, most form caspase-1 activating inflammasomes. NLRP12 is a unique NLR that has been shown to attenuate inflammatory pathways in biochemical assays and mediate the lymph node homing of activated skin dendritic cells in contact hypersensitivity responses. Since the mechanism between these two important observations remains elusive, we further evaluated the contribution of NLRP12 to organ specific adaptive immune responses by focusing on the lung, which, like skin, is exposed to both exogenous and endogenous inflammatory agents. In models of allergic airway inflammation induced by either acute ovalbumin (OVA) exposure or chronic house dust mite (HDM) antigen exposure, Nlrp122/2 mice displayed subtle differences in eosinophil and monocyte infiltration into the airways. However, the overall development of allergic airway disease and airway function was not significantly altered by NLRP12 deficiency. Together, the combined data suggest that NLRP12 does not play a vital role in regulating Th2 driven airway inflammation using common model systems that are physiologically relevant to human disease. Thus, the allergic airway inflammation models described here should be appropriate for subsequent studies that seek to decipher the contribution of NLRP12 in mediating the host response to agents associated with asthma exacerbation.
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    Characterization of NLRP12 during the In Vivo Host Immune Response to Klebsiella pneumoniae and Mycobacterium tuberculosis
    Allen, Irving C.; TeKippe, Erin McElvania; Wilson, Justin E.; Lich, John D.; Arthur, Janelle C.; Sullivan, Jonathan T.; Braunstein, Miriam; Ting, Jenny P.-Y. (PLOS, 2013-04-05)
    The majority of nucleotide binding domain leucine rich repeats-containing (NLR) family members has yet to be functionally characterized. Of the described NLRs, most are considered to be proinflammatory and facilitate IL-1β production. However, a newly defined sub-group of NLRs that function as negative regulators of inflammation have been identified based on their abilities to attenuate NF-κB signaling. NLRP12 (Monarch-1) is a prototypical member of this sub-group that negatively regulates both canonical and noncanonical NF-κB signaling in biochemical assays and in colitis and colon cancer models. The role of NLRP12 in infectious diseases has not been extensively studied. Here, we characterized the innate immune response of Nlrp12−/− mice following airway exposure to LPS, Klebsiella pneumoniae and Mycobacterium tuberculosis. In response to E. coli LPS, Nlrp12−/− mice showed a slight decrease in IL-1β and increase in IL-6 production, but these levels were not statistically significant. During K. pneumoniae infection, we observed subtle differences in cytokine levels and significantly reduced numbers of monocytes and lymphocytes in Nlrp12−/− mice. However, the physiological relevance of these findings is unclear as no overt differences in the development of lung disease were observed in the Nlrp12−/− mice. Likewise, Nlrp12−/− mice demonstrated pathologies similar to those observed in the wild type mice following M. tuberculosis infection. Together, these data suggest that NLRP12 does not significantly contribute to the in vivo host innate immune response to LPS stimulation, Klebsiella pneumonia infection or Mycobacterium tuberculosis.
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    Characterizing the roles of gut microbiota, probiotic Lactobacilli and CX3CR1 in the development of autoimmunity in MRL/lpr mice
    Cabana-Puig, Xavier (Virginia Tech, 2022-08-18)
    Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with no known cure. The crosstalk between the gut microbiota and the immune system plays an important role in the tolerance induction to self-antigens both in the intestinal mucosa and at the systemic level. The MRL/lpr mouse model exhibits lupus-like symptoms early in life due to multiple SLE susceptible loci of the MRL background, plus the Faslpr mutation that offers an accelerated model. Recently, we experienced a loss of disease phenotype in our in-house colony compared to the previous published phenotype of MRL/lpr mice. We thus compared mice newly obtained from The Jackson Laboratory (JAX) with our in-house MRL/lpr mice and found that the phenotypic drift, most significantly the attenuation of glomerulonephritis, was present in both colonies. In addition, while JAX mice and mice in our colony are genetically identical, there were minor differences in disease that might be due to differences in splenic microRNAs and the gut microbiota. Once confirming that our MRL/lpr mouse model was as good as that from JAX, we continued our investigation of the role of Lactobacilli in the pathogenesis of lupus-like disease in MRL/lpr mice. We previously published that the mixture of Lactobacillus reuteri (L. reuteri), L. oris, L. johnsonii, L. gasseri, and L. rhamnosus significantly attenuated disease in MRL/lpr mice by restoring the imbalance between regulatory T cells and T helper-17 cells. To further understand the role of Lactobacillus spp., we treated MRL/lpr mice with the combined culture supernatant of the 5 strains containing secreted metabolites, given that the metabolites may induce an immunosuppressive response. The results showed significant attenuation of the inflammation of the spleen and renal lymph nodes similar to the effect of the bacteria themselves. There was also a trending decrease of double-stranded DNA autoantibodies with the combined supernatant. We thus tested the strains individually but none was able to recapitulate the effect of the bacterial mixture. This suggests cell-to-cell contact among different strains of lactobacilli may be required in ameliorating the disease. With these results, we now have a better understanding of the role of probiotic Lactobacillus spp. against SLE. Future investigations will focus on the potential therapeutic effect of Lactobacillus spp. as a combination. Additionally, our group generated a Cx3cr1-deficient MRL/lpr mouse which exhibits a distinct phenotype of exacerbated glomerulonephritis with concurrent change of the gut microbiota composition compared to Cx3cr1+/+ MRL/lpr littermates. Interestingly, upon correction of the gut microbiota with Lactobacillus administration, the phenotype of exacerbated glomerulonephritis was reversed, suggesting that CX3CR1 controls glomerulonephritis in MRL/lpr mice through a gut microbiota-dependent mechanism. In addition, a collaborative project revealed that Cx3cr1 deficiency-mediated pathogenic mechanisms also contributed to SLE-associated cardiovascular disease in MRL/lpr mice. The results of these studies will lead to the identification of new therapeutic targets for the treatment of two severe manifestations, glomerulonephritis and cardiovascular disease, that together account for most of the morbidity and mortality in SLE.
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    Cold sensor, hot topic: TRPM8 plays a role in monocyte function and differentiation
    Trusiano, Brie; Tupik, Juselyn D.; Allen, Irving C. (Wiley, 2022-05)
    Understanding the innate immune system and how aberrant activation or impaired inhibition leads to the development of hyperinflammatory conditions, including inflammatory bowel disease, is crucial for patient management and treatment. An emerging area of interest surrounding dysregulated inflammation focuses on membrane bound transient receptor potential (TRP) ion channels. These channels are permeable to calcium and other cations involved in the balance of leukocyte membrane potential and function, as well as afferent neuron signaling within the myenteric plexus of the GI tract, bladder, and skin. A particular channel, TRPM8, is an important cell surface marker for prostate cancer and participates in the function of cold sensing neurons. Specifically, this ion-gated receptor is shown to be activated by agonists such as menthol and eucalyptus, which aid in the soothing, cooling effects of these agents. Furthermore, the TRPM8 channel is also identified on the surface of resident tissue M phi s and is also linked to the protective role and release of calcitonin gene-related peptide (CGRP) by sensory neurons.
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    Computational and Experimental Investigation of the Critical Behavior Observed in Cell Signaling Related to Electrically Perturbed Lipid Systems
    Goswami, Ishan (Virginia Tech, 2018-10-16)
    Problem Statement: The use of pulsed electric fields (PEFs) as a tumor treatment modality is receiving increased traction. A typical clinical procedure involves insertion of a pair of electrodes into the tumor and administration of PEFs (amplitude: ~1 kV/cm; pulse-width: 100 μs). This leaves a zone of complete cell death and a sub-lethal zone where a fraction of the cells survive. There is substantial evidence of an anti-tumor systemic immune profile in animal patients treated with PEFs. However, the mechanism behind such immune profile alterations remains unknown, and the effect of PEFs on cell signaling within sub-lethal zones remains largely unexplored. Moreover, different values of a PEF pulse parameter, for e.g. the pulse-widths of 100 μs and 100 ns, may have different effects on cell signaling. Thus, the challenge of answering the mechanistic questions is compounded by the large PEF parameter space consisting of different combinations of pulse-widths, amplitudes, and exposure times. Intellectual merit: This Ph.D. research provides proof that sub-lethal PEFs can enhance anti-tumor signaling in triple negative breast cancer cells by abrogating thymic stromal lymphopoietin signaling and enhancing stimulatory proteins such as the tumor necrosis factor. Furthermore, experimental evidence produced during this Ph.D. research demonstrates that PEFs may not directly impact the intracellular mitochondrial membrane at clinically relevant field amplitudes. As demonstrated in this work, PEFs may influence the mitochondria via an indirect route such as disruption of the actin cytoskeleton and/or alteration of ionic environment in the cytoplasm due to cell membrane permeabilization. Thus, a reductionist approach to understanding the influence of PEFs on cell signaling is proposed by limiting the study to membrane dynamics. To overcome the problem of investigating the entire PEF parameter space, this Ph.D. research proposes a first-principle thermodynamic approach of scaling the PEF parameter space such that an understanding developed in one regime of PEF pulse parameter values can be used to understand other regimes of the parameter space. Demonstration of the validity of this scaling model is provided by coupling Monte-Carlo methods for density-of-states with the steepest-entropy-ascent quantum thermodynamic framework for the non-equilibrium prediction of the lipid membrane dynamics.
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    The Design of Biodegradable Polyester Nanocarriers for Image-guided Therapeutic Delivery
    Jo, Ami (Virginia Tech, 2018-09-12)
    Multiple hurdles, such as drug solubility, stability, and physical barriers in the body, hinder bioavailability of many promising therapeutics. Polymeric nanocarriers can encapsulate the therapeutics to protect non-target areas from side effects but also protect the drug from premature degradation for increased circulation and bioavailability. To capitalize on these advantages, the polymer nanoparticle must be properly engineered for increased control in size distribution, therapeutic encapsulation, colloidal stability, and release kinetics. However, each application requires a specific set of characteristics and properties. Being able to tailor these by manipulation of different design parameters is essential to optimize nanoparticles for the application of interest. This study of nanoparticle fabrication and characterization takes us a step closer to building effective delivery systems tailored for specific treatments. Poly(ethylene oxide)-b-poly(D,L-lactic acid) (PEO-b-PDLLA) based nanoparticles were produced to range from 100-200 nm in size. They were fluorescently labeled with a hydrophobic dye 6-13 bis(triisopropylsilylethynyl) pentacene (TIPS pentacene) at an optimal loading of 0.5 wt% with respect to the core. Surfaces were successfully coated with streptavidin to be readily functionalized with various biotinylated compounds such as PD-L1 antibodies or A488 fluorophore. Using the same PEO-b-PDLLA, iron oxide and a conjugated polymer poly(2- methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene) (MEH-PPV) were co-encapsulated to form fluorescently labeled magnetic particles. Using poly(lactic-co-glycolic acid), CRISPR-Cas9 plasmids were encapsulated at 1.6 wt% and most of the payload released within the first 24 hours. The incorporated plasmids were intact enough to have mammalian macrophages successfully express the bacterial protein Cas9. Using similar PLGA based particles, the surface was functionalized with streptavidin and bound to the surface of bacteria as an active carrier for increased penetration of solid tumors averaging ~23 particles per bacterium. PEO-b-PLGA based particles were used in conjunction with a hydrophobic salt former to encapsulate a peptide designed to reduce platelet binding to cancer cells and mitigate extravasation. The peptide encapsulated was increased from < 2 wt% without salt former to 8.5 wt% with the used of hexadecyl phosphonic acid. Although the applications across these projects can be broad, the fundamentals and important design parameters considered contribute to the overarching field of effective carriers for drug delivery.
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    Detecting DNA: An Overview of DNA Recognition by Inflammasomes and Protection against Bacterial Respiratory Infections
    Tupik, Juselyn D.; Markov Madanick, Justin W.; Ivester, Hannah M.; Allen, Irving C. (MDPI, 2022-05-19)
    The innate immune system plays a key role in modulating host immune defense during bacterial disease. Upon sensing pathogen-associated molecular patterns (PAMPs), the multi-protein complex known as the inflammasome serves a protective role against bacteria burden through facilitating pathogen clearance and bacteria lysis. This can occur through two mechanisms: (1) the cleavage of pro-inflammatory cytokines IL-1β/IL-18 and (2) the initiation of inflammatory cell death termed pyroptosis. In recent literature, AIM2-like Receptor (ALR) and Nod-like Receptor (NLR) inflammasome activation has been implicated in host protection following recognition of bacterial DNA. Here, we review current literature synthesizing mechanisms of DNA recognition by inflammasomes during bacterial respiratory disease. This process can occur through direct sensing of DNA or indirectly by sensing pathogen-associated intracellular changes. Additionally, DNA recognition may be assisted through inflammasome–inflammasome interactions, specifically non-canonical inflammasome activation of NLRP3, and crosstalk with the interferon-inducible DNA sensors Stimulator of Interferon Genes (STING) and Z-DNA Binding Protein-1 (ZBP1). Ultimately, bacterial DNA sensing by inflammasomes is highly protective during respiratory disease, emphasizing the importance of inflammasome involvement in the respiratory tract.
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    Determining the Oncological and Immunological Effects of Histotripsy for Tumor Ablation
    Hendricks, Alissa Danielle (Virginia Tech, 2021-05-28)
    Histotripsy is an emerging non-invasive, non-thermal, image-guided cancer ablation modality that has recently been approved for its first clinical trial in the United States (NCT04573881). Histotripsy utilizes focused ultrasound to generate acoustic cavitation within a tumor to mechanically fractionate targeted tissues. While pre-clinical work has demonstrated the feasibility of applying histotripsy to solid tumors including primary liver and renal tumors, there is still a need to investigate the potential of histotripsy to treat additional malignancies. In investigating the potential for treating other malignancies there are two avenues that need to be considered: 1) the feasibility for treating tissues with more complex stromal structures and 2) the ability of histotripsy to modulate the tumor microenvironment. To determine the safety and feasibility of additional applications of histotripsy, we conducted dose studies ex vivo on human tumors and human liver to establish dosimetry metrics for applying histotripsy to more fibrotic tumors such as cholangiocarcinoma while sparing nearby critical structures, such as bile ducts and blood vessels. Learning the safety dose-margins from the excised tissues, we performed an in vivo study using mice bearing patient-derived xenograft cholangiocarcinoma tumors. With this model, we were able to demonstrate our ability ablate the stiff cholangiocarcinoma tumors without causing any debilitating off- target damage. To gain a more robust understanding of the effects of histotripsy ablation on potentially difficult to treat tumors, we developed a porcine xenograft tumor model and utilized veterinary cancer patients. These studies have helped established protocols for utilizing histotripsy with ultrasound guidance to treat tumors that are more difficult to treat and can withstand mechanical ablation, including pancreatic adenocarcinoma, osteosarcomas, and soft tissue sarcomas. Pigs share many similarities with human anatomy and physiology, making them an ideal model organism for testing new medical devices and regimes for treating new targets. Using pigs, we were able to establish a procedure to utilize histotripsy to target the pancreas in vivo without causing any lasting or major side effects, such as off-target damage or pancreatitis. One limitation to the porcine model and veterinary patients, is the limitation of gaining rapid insight into the immunological effects of histotripsy. Established cancer mouse models offer the opportunity to rapidly test many organisms with an intact immune system. We used these mice to study pancreatic adenocarcinoma to determine the immune response after histotripsy ablation. For these tumors the general response was an increase in immune cell infiltration post-treatment and a shift in the tumor microenvironment to a more anti-tumor environment. The results of this dissertation provide insight into establishing protocols for treating new types of tumors with histotripsy and immunological effects that lay groundwork for improving future co-therapeutic treatment planning. Future work will aim to translate histotripsy into clinical applications and determining co-therapies that can help control metastasis.
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    Discovery and Characterization of a Novel Regulatory Small RNA, VcrS, Required for Virulence in Brucella abortus
    King, Kellie Alexandra (Virginia Tech, 2022-02-01)
    Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses, such as acidic conditions and reactive oxygen species, as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation through the use of small regulatory RNAs (sRNAs). sRNAs bind to messenger RNA (mRNA) targets via complementary base pairing. sRNAs are a class of regulatory molecules in bacteria that elicit rapid post-transcriptional regulation. sRNA-mRNA binding can positively or negatively influence gene expression. Positive regulation can occur through sRNA binding to protect the mRNA from RNases. sRNA binding can also alleviate the secondary structure and reveal the ribosomal binding site. Alternatively, sRNA-mRNA interactions can have negative consequences on gene expression through degradation via RNases or sRNA binding can occlude the ribosomal binding site. Although some sRNAs have been discovered in B. abortus, few have been characterized in regards to virulence. In this study, B. abortus was stressed in conditions relevant to the macrophage, including, including low pH, oxidative stress, and nutrient limitation. Transcriptomic analysis revealed high levels of transcripts in intergenic regions, a hallmark of sRNAs, which led to the discovery of VcrS for virulence and cell wall regulating sRNA. A ΔvcrS was engineered and this mutant was used to infect both naïve murine macrophages, as well as BALB/c mice. Both virulence studies demonstrated significantly decreased bacterial recovery of ΔvcrS compared to the wildtype strain. Quantitative proteomics revealed that one protein, BAB1_1454, is 30-fold over-produced in ΔvcrS compared to wildtype. This essential protein encodes MurF, which catalyzes the final cytoplasmic step of generating the mura-pentapeptide precursor for peptidoglycan synthesis. VcrS is hypothesized to interact with murF mRNA and interfere with translation initiation. Sequence data indicates a putative 6 nucleotide motif in VcrS that has complementarity to the ribosomal binding site of murF. Identification of the binding site and further characterization of VcrS will showcase the importance of sRNA regulation in the virulence of B. abortus.
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    Divergent age-dependent peripheral immune transcriptomic profile following traumatic brain injury
    Hazy, Amanda; Bochicchio, Lauren; Oliver, Andrea; Xie, Eric; Geng, Shuo; Brickler, Thomas; Xie, Hehuang David; Li, Liwu; Allen, Irving C.; Theus, Michelle H. (Springer Nature, 2019-06-12)
    The peripheral immune system is a major regulator of the pathophysiology associated with traumatic brain injury (TBI). While age-at-injury influences recovery from TBI, the differential effects on the peripheral immune response remain unknown. Here, we investigated the effects of TBI on gene expression changes in murine whole blood using RNAseq analysis, gene ontology and network topology-based key driver analysis. Genome-wide comparison of CCI-injured peripheral whole blood showed a significant increase in genes involved in proteolysis and oxidative-reduction processes in juvenile compared to adult. Conversely, a greater number of genes, involved in migration, cytokine-mediated signaling and adhesion, were found reduced in CCI-injured juvenile compared to CCI-injured adult immune cells. Key driver analysis also identified G-protein coupled and novel pattern recognition receptor (PRR), P2RY10, as a central regulator of these genes. Lastly, we found Dectin-1, a c-type lectin PRR to be reduced at the protein level in both naive neutrophils and on infiltrating immune cells in the CCI-injured juvenile cortex. These findings demonstrate a distinct peripheral inflammatory profile in juvenile mice, which may impact the injury and repair response to brain trauma.
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    Dysregulation of Noncanonical NF-κB Signaling in Gastrointestinal Diseases
    Morrison, Holly Ann (Virginia Tech, 2023-09-01)
    Regulation of host health is intricately coordinated by a diverse interplay of immune cells detecting assaults from pathogens via recognition of pathogen associated molecular patterns (PAMPs) to mount an immune response, as well as detecting damage associated molecular patterns (DAMP) to indicate an area of damage and signal tissue repair. The gastrointestinal tract is a major signaling hub for such immune responses, as intestinal epithelial cells (IECs) compose the epithelial barrier, immune cells surveillance breached barriers to regulate the gut microbiome, and intestinal stem cells (ISCs) proliferate to replenish the IEC pool. One such method for regulating these cellular functions downstream of PAMPs/DAMPs within the gastrointestinal tract is via NF-κB signaling. This cellular signaling pathway is activated by one of two pathways: the well- defined canonical NF-κB pathway and the understudied noncanonical NF-κB pathway. The noncanonical NF-κB pathway is unique as it requires NIK, the NF-κB-inducing Kinase, to further elicit signal transduction of this pathway. Noncanonical NF-κB activation is critical to maintaining gut health, as signaling is regulated at a precise level to ensure a balance of pro-/anti-inflammatory signals to elicit a proper damage response. Any perturbations to NIK-activated signaling significantly predisposes the gastrointestinal niche towards chronic inflammatory conditions of the gastrointestinal tract. In this work, we explore the potential involvement of dysregulated noncanonical NF-κB signaling in inducing chronic inflammatory diseases of the gut, including Eosinophilic Esophagitis (upper GI tract), Celiac Disease/Non-Celiac Gluten Sensitivities (small intestine), Inflammatory Bowel Disease (entire intestine/large intestine), and an inflammatory subtype of colorectal cancer being Colitis-Associated Colorectal Cancer (large intestine). We study this pathway via the use of murine models bearing genetic deletions, cellular models, and the generation of miniature organs (i.e. "organoids") in petri dishes. Further, we assess varying levels of NF-κB signaling through the genetic deletions of NIK and RelA to inhibit noncanonical and canonical NF-κB pathways, respectively. Reciprocally, we also examine overactivated signaling via loss of the negative regulatory NLRs, which are proteins that function to impede NF-κB signaling. Clinical relevancy of this work is evaluated using biopsy samples collected from human patients with active disease states. Culminating our work, we find that noncanonical NF-κB signaling levels is both tissue- and cell-type specific in driving disease formation. Finally, we conclude our findings by suggesting the promise of NIK as a potential candidate for disease biomarkers and a target for future drug development.
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    Effect of Salmonella enterica serovar Typhimurium VNP20009 and VNP20009 with restored chemotaxis on 4T1 mouse mammary carcinoma progression
    Coutermarsh-Ott, Sheryl; Broadway, Katherine M.; Scharf, Birgit E.; Allen, Irving C. (Impact Journals, 2017-05-16)
    A variety of bacterial strains have been evaluated as bio-therapeutic and immunomodulatory agents to treat cancer. One such strain, Salmonella enterica serovar Typhimurium VNP20009, which is attenuated by a purine auxotrophic mutation and modified lipid A, is characterized in previous models as a safely administered, tumor colonizing agent. However, earlier work tended to use less aggressive cancer cell lines and immunocompromised animal models. Here, we investigated the safety and efficacy of VNP20009 in a highly malignant murine model of human breast cancer. Additionally, as VNP20009 has recently been found to have a defective chemotaxis system, we tested whether restoring chemotaxis would improve anti-cancer properties in this model system. Exposure to VNP20009 had no significant effect on primary mammary tumor size or pulmonary metastasis, and the tumor colonizing process appeared chemotaxis independent. Moreover, tumor-bearing mice exposed to Salmonella exhibited increased morbidity that was associated with significant liver disease. Our results suggest that VNP20009 may not be safe or efficacious when used in aggressive, metastatic breast cancer models utilizing immunocompetent animals.
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    Effects of Keratin Biomaterial Therapeutics on Cellular and Inflammatory Mechanisms in Injury and Disease Models
    Waters, Michele (Virginia Tech, 2018-06-11)
    Keratins are fibrous structural proteins found in human hair that have been used to develop bioactive and biocompatible constructs for a wide variety of tissue engineering and healthcare applications. Their ubiquity, capacity for self-assembly, ease of use and versatility in blended materials, and ability to modulate cell behavior and promote tissue ingrowth have made keratins well-suited for the development of regenerative therapies. In particular, keratins have demonstrated bioactivity in both in-vivo and in-vitro studies, by altering immune and stem cell phenotype and function and promoting an anti-inflammatory/wound healing environment. This work seeks to build on previous research by investigating the ability of low and high molecular weight keratins to augment anti-inflammatory primary macrophage phenotypes and examining the influence of keratin biomaterials on cellular and inflammatory mechanisms in two models of injury and disease. Rodent models of blast induced neurotrauma (BINT) and severe osteoporosis were used to inform the development of 2D and 3D in-vitro models of macrophage/endothelial cell injury and osteogenic differentiation respectively. Keratin biomaterials exhibited some potential to alter macrophage and endothelial cell dynamics following blast, specifically by promoting anti-inflammatory (M2c-like) macrophage polarization and diminishing endothelial cell injury responses (i.e. endothelial glycocalyx shedding). A more clinically relevant model of osteoporosis found that stem cells harvested from older, osteoporotic animals demonstrated limited proliferative and bone differentiation potential compared to healthy cells. However, 3D constructs (especially keratin-based materials) were able to enhance calcification and osteogenic gene expression of diseased cells. These results highlight the complexity of macrophage phenotypic switching and cellular dynamics in these systems. However, keratin-based therapeutics may prove useful for facilitating tissue regeneration and limiting detrimental inflammatory and cellular responses in various models of injury and disease.