Browsing by Author "Dervisis, Nikolaos"
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- Dysregulation of Noncanonical NF-κB Signaling in Gastrointestinal DiseasesMorrison, 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.
- Histotripsy ablation for the treatment of feline injection site sarcomas: a first-in-cat in vivo feasibility studyRuger, Lauren N.; Yang, Ester; Coutermarsh-Ott, Sheryl; Vickers, Elliana; Gannon, Jessica; Nightengale, Marlie; Hsueh, Andy; Ciepluch, Brittany; Dervisis, Nikolaos; Vlaisavljevich, Eli; Klahn, Shawna (Taylor & Francis, 2023)Purpose Feline soft tissue sarcoma (STS) and injection site sarcoma (fISS) are rapidly growing tumors with low metastatic potential, but locally aggressive behavior. Histotripsy is a non-invasive focused ultrasound therapy using controlled acoustic cavitation to mechanically disintegrate tissue. In this study, we investigated the in vivo safety and feasibility of histotripsy to treat fISS using a custom 1 MHz transducer. Materials and Methods Three cats with naturally-occurring STS were treated with histotripsy before surgical removal of the tumor 3 to 6 days later. Gross and histological analyses were used to characterize the ablation efficacy of the treatment, and routine immunohistochemistry and batched cytokine analysis were used to investigate the acute immunological effects of histotripsy. Results Results showed that histotripsy ablation was achievable and well-tolerated in all three cats. Precise cavitation bubble clouds were generated in all patients, and hematoxylin & eosin stained tissues revealed ablative damage in targeted regions. Immunohistochemical results identified an increase in IBA-1 positive cells in treated tissues, and no significant changes in cytokine concentrations were identified post-treatment. Conclusions Overall, the results of this study demonstrate the safety and feasibility of histotripsy to target and ablate superficial feline STS and fISS tumors and guide the clinical development of histotripsy devices for this application.
- Mechanical High-Intensity Focused Ultrasound (Histotripsy) in Dogs with Spontaneously Occurring Soft Tissue SarcomasYang, Ester (Virginia Tech, 2023-09-06)Background: Histotripsy is a non-thermal, non-invasive high-intensity focused ultrasound (HIFU) ablative technique that causes mechanical fragmentation of tissue, resulting in liquefied acellular debris with histologically clear demarcated boundaries between treated and non-treated tissues. The acellular debris may include tumor antigens with preserved immunogenicity and the potential to generate systemic immune response against tumor cells. Soft tissue sarcomas (STS) are a common form of cancer in dogs with biological behavior similar to STS in humans. Long-term tumor control requires complete removal with extensive surgical resection, which in many cases is not feasible. As a result, there is need for alternative therapies. Objectives: The primary objective of this study was to demonstrate safety and feasibility of histotripsy in a small animal model of spontaneous STS. The secondary objective was to characterize the impact of histotripsy on the immunologic response. Materials and methods: Pet dogs diagnosed with spontaneous STS were recruited. CT scan of the chest, abdomen, and the tumor was performed for staging and treatment planning. Pretreatment biopsies were obtained. Safety was monitored with physical examinations, owner reports, and CBC/serum biochemistry. Partial tumor ablation was performed using a 500 kHz prototype histotripsy system. A spherical treatment zone of up to 3 cm diameter in each tumor was treated with histotripsy according to the patient-specific treatment plan using 1-2 cycle pulses applied at a pulse repetition frequency (PRF) of 500 Hz. Anatomical ablation zones were evaluated with contrast CT at 1- and 4-days post-treatment, with tumor resection at 4-6 days post-treatment. Tumor microenvironment (TME) gene expression was evaluated with the Nanostring Canine IO panel, and the systemic immune response was evaluated using multiplex serum cytokine levels. Results: Ten dogs were recruited and treated. Tumor histologies included 3 grade III STS, 4 grade II STS, 2 grade I STS, and 1 malignant mesenchymoma. Six dogs were alive, three dogs were euthanized due to disease progression, and one dog was lost to follow up. Histotripsy-related complications were generally self-limiting, with only one patient having increased cutaneous injury score from 1 to 2 (scale 1-5) post-treatment, likely due to prefocal cavitation at the skin. No significant adverse events impacting patient outcome were noted in any of the patients. Visible histotripsy cavitation bubble clouds were seen on real-time ultrasound imaging in nine of ten treatments. Post-treatment histopathology indicated sharply defined regions of ablation that were clearly identifiable grossly and histologically in all samples. Treatment zones were characterized by loss of cell viability, hyalinization, and acute hemorrhage. Post-treatment contrast-enhanced CT images revealed clear, demarcated regions of histotripsy ablated tissue in seven of ten patients. Differential gene expression analysis identified 79 genes with at least 2-fold change following treatment. Genes associated with inflammation, immune cell migration, and immune cell interactions were the highest upregulated. Amongst the gene set analyses, the myeloid compartment gene sets obtained the highest significance score. There were no statistically significant differences between pre- and post-treatment cytokine concentrations for any of the analytes. Conclusions: Histotripsy can achieve safe and effective tumor ablation in dogs diagnosed with STS. Histotripsy induced pro-inflammatory changes within the tumor microenvironment. Histotripsy as an immunotherapeutic treatment option needs to be further investigated. Histotripsy has a potential to be a precise, non-invasive treatment for canine STS.
- NF-kB Inducing Kinase (NIK) Influences Eosinophil Development, Survival, and PlasticityTrusiano, Briana Lynn (Virginia Tech, 2024-04-22)Hypereosinophilic (HES) syndrome is an umbrella term encompassing several disease subsets that affects humans and veterinary species, ultimately resulting in >1,500 eosinophils/uL circulating in the blood documented over six-months. This eventually culminates in end-organ infiltration and increased patient morbidity and mortality. In mice where the gene Map3k14 encoding NF -kB inducing kinase (NIK) is knocked out, a HES-like syndrome develops that is dependent on Th2 cells and cytokines. NIK is the upstream regulator of the noncanonical NF-kB pathway and is involved in lymphoid organ development, B cell lymphopoiesis, and myelopoiesis. In addition to regulating the noncanonical NF-kB pathway, NIK is also involved in regulation of kB dimers of the canonical NF-kB pathway and can function independent of NF-kB signaling by regulating lipid and glucose metabolism, mitochondrial, and RIP1 binding to influence cell survival and death. Despite previous studies performed in the Nik-/- model, the mechanisms underlying eosinophil development, plasticity, and fitness in conjunction with the bone marrow and splenic microenvironments have not been fully elucidated. In the present work, we reviewed current data exploring the influence of the noncanonical NF-kB pathway and NIK specifically on the development of acute myeloid leukemias (AMLs) and Myelodysplastic Syndrome (MDS) with a focus on how these mechanisms might induce subvariants of HES. We next examined the effect of NIK loss on eosinophilopoiesis within hematopoietic tissues in vivo and in various cell culture environments in vitro via cytology, histology, flow cytometry, FACS, positive cell selection, MTT assay, BrDU assay, and protein microarray analysis. Overall, our findings suggest that NIK influences eosinophil maturation, proliferation, metabolism, survival, and potentially plasticity in vivo and in vitro under different environmental conditions and Th2 cytokine influence. NIK loss was also associated with altered free and bound TNFR1 levels on day 13 in vitro. TNFR1 acts upstream of RIP1 and suggests that these differences may be due to NF-kB independent functions of NIK. Overall, these results provide further insight into the potential mechanisms underlying eosinophilopoiesis in the Nik-/- murine model. This information may prove useful in discovering new treatment options underlying subvariants of HES in both human and veterinary patients.