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    Glioblastoma multiforme expresses cell surface PCNA, a potential target for NK cell-mediated immunotherapy.
    (2022) Cooksey, Luke; Mathew, Porunelloor A.
    Purpose: Glioblastoma multiforme (GBM) is the most common form of primary brain cancer and carries a dreadful five-year survival rate of 9%. Current treatment options include surgery, chemotherapy, and radiation. Recently, there has been a move to pursue immunotherapy options to improve patient outcomes. These therapies often depend on the identification of molecular markers that are distinctive to the tumor cells. Some markers, such as HER2/Neu and EGFR, are overexpressed on a significant percentage of GBM tumors and are used as targets for immunotherapies. However, to address GBM tumors that do not overexpress HER2/Neu and EGFR, our lab set out to identify novel markers on GBM as future candidates for Natural Killer (NK)cell-mediated immunotherapy. Methods: Previously, our lab demonstrated that Lectin-like Transcript-1 (LLT1), membrane-bound Proliferating Cell Nuclear Antigen (PCNA), NKp44 Ligand (NKp44L) and CS1 (CD319) are targets of NK cell-mediated killing of various cancers. Based on these prior studies, we examined their expression on the well-known GBM cell lines LN-229 and LN-18 by flow cytometry using PE-labeled antibodies specific for each marker. Results: PCNA was the lone marker of our panel identified to be highly expressed on both LN-229 and LN-18 cells. Conclusions: Based on our results, we concluded that cell surface PCNA is an ideal candidate for NK cell-mediated immunotherapy for GBM. Currently, we are evaluating blocking inhibitory signals to NK cells from the PCNA-NKp44 interaction to target GBM for NK-mediated killing. Recent findings demonstrating the ability to transiently open the blood-brain barrier further increase the feasibility of targeting GBM by NK cells with monoclonal antibodies in the future.
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    Cytokine Response in an Endotoxin-Mediated Sepsis Model
    (2022) Martinez, Richard; Aguirre, David Salinas; Warne, Cooper; Dick, Gregory; Mallet, Robert T.; Tune, Johnathan; Hodge, Lisa
    Purpose: Sepsis is a life-threatening condition that develops secondary to infection and can manifest acute organ dysfunction due to the body's overactive systemic response. Sepsis affects approximately 1.7 million US adults and claims 270,000 lives as a result. The long-term goal of our project is to gain a better understanding of the roles of the lymphatic and immune systems in the progression of sepsis. The purpose of this study is to collect pilot data using a translational swine model of endotoxin-mediated sepsis. We chose a swine model because it closely mimics how sepsis progresses in humans. Sepsis was induced by infusion of lipopolysaccharide (LPS) from Escherichia coli. LPS was chosen because it is a key mediator in the activation of the immune system and the development of sepsis. We hypothesized that the administration of LPS would increase the concertation of the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-?) in a dose-dependent manner over time. Methods: Yorkshire pigs (61 � 4 kg, n = 4, 2 male) were sedated, intubated, and ventilated. Thoracotomy was performed under anesthesia to record flow data and sample cardiac blood for use in another study. Femoral artery and venous lines were placed to allow measurement of blood pressure and infusion of LPS. Specifically, LPS was prepared at 1, 5, 25, or 50 ?g/kg (pig body weight) in sterile saline. LPS was infused into anesthetized pigs over a 2-hour period. Blood samples were collected immediately prior to LPS administration and at 30 min intervals during 2 hours of LPS infusion up to 4 hours following LPS infusion. The plasma was analyzed via enzyme-linked immunosorbent immunoassay (ELISA) for the concentrations of IL-6 and TNF-? using commercially available kits. Results: As hypothesized, the infusion of LPS increased the concentration of the inflammatory mediators IL-6 and TNF-? over time compared to pre-LPS infusion. Specifically, the greatest increase in IL-6 was seen at 180 minutes in both the 50 and 25 ?g/kg LPS infused pigs. TNF-? concentration peaked between 30 to 90 minutes during LPS infusion in both the 50 and 25 ?g/kg LPS infused pigs. The lower doses of 1 and 5 ?g/kg LPS produced little to no IL-6 or TNF-?. Furthermore, we discovered that the pigs who received 50 or 25 ?g/kg of LPS died from septic shock within 180 minutes of LPS infusion, whereas the pigs that received 1or 5 ?g/kg of LPS survived longer. Conclusion: In this study, we identified the impact of increasing the doses of LPS on the production of IL-6 and TNF-? in swine. Our preliminary results suggest a dose range of 10-20 ?g/kg of LPS may be ideal to study the inflammatory response in this model. The acquisition of these data are essential to pursue our long-term research objective, which is to identify the role of the lymphatic and immune systems during sepsis.
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    Suppression of Adaptive Immunity by Borrelia burgdorferi: An Investigation of Complex Host-Microbe Interactions
    (2022) Williams, Megan; Zhang, Yan
    Purpose: Infection with Borrelia burgdorferi (Bb), the causative agent of Lyme Disease, induces broad suppression of the host adaptive immune response. It has been shown that short-lived germinal centers form in the lymph nodes shortly after infection, but then collapse approximately one month post-infection. The resulting adaptive immune response is characterized by unusually strong and persistent IgM production and lack of long-lived immunity. Additionally, when a group of Bb-infected mice were given a co-administered influenza vaccine, they mounted a significantly abrogated influenza-specific antibody response when compared to an uninfected group that received the same vaccine. A better understanding of how Bb manipulates host immunity can help enhance serological testing and treatment options for Lyme Disease. We aim to determine the duration of humoral immune suppression by Bb and characterize the events leading up to germinal center collapse. Methods: We will randomly assign mice to 5 groups (n=4). One group will be inoculated with a SARS-CoV-2 spike protein vaccine. The remaining 4 groups will be infected with Bb and will subsequently receive the SARS-CoV-2 vaccine at Day 0, 14, 28, or 42. We have recently tested the safety of the vaccine for use in mice. This protocol has undergone IACUC approval (IACUC-2020-0033). We will assess the SARS-CoV-2-specific antibody response at different stages of infection with Bb, which will be measured using enzyme-linked immunosorbent assays (ELISA). We will also use genetic techniques such as RT-qPCR and single-cell RNA sequencing to interrogate the events underpinning this immune suppression. Results and Conclusion: This study has not yet been completed. Although conclusions cannot yet be drawn, preliminary evidence demonstrates that the SARS-CoV-2 vaccine is safe for use in mice and induces a robust antibody response. We have recently successfully established infection in mice using Bb strain N40 and are currently examining lymph node histology of infected mice to confirm germinal center suppression in our infection model.
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    Aging impairs regulatory T cell (Treg) cells to affect late-onset (aged) multiple sclerosis (MS) - with the model of experimental autoimmune encephalomyelitis (EAE)
    (2022) Wang, Weikan; Thomas, Rachel; Oh, Jiyoung; Su, Dong-Ming
    Purpose: A master immunoregulator FoxP3-expressing CD4+Treg cells play an ameliorative role in the severity of MS/EAE disease. However, it remains to be determined why severe MS/EAE symptoms and pathology in the aged are co-existing with relatively enhanced thymic CD4+Treg generation and accumulated peripheral CD4+Treg cells. Methods: We immunized young and aged mice to induce EAE and investigated the disease courses and Treg cell associated mechanisms. Results: The EAE onset was delayed in aged mice, but the disease severity is increased relative to young mice. Using single-cell (sc)-RNA-Seq assay, we found that CNS-infiltrating CD4+Treg cells in aged EAE mice had increased pathogenic properties, showing co-expression of Infg and Il17a with Foxp3, and reduced suppressive effect, exhibiting increased clonal expansion of pathogenic CD4+ T cells. These indicate pathogenic changes in Treg quality in aged EAE mice. In addition, CNS-infiltrating CD8+ T cells also gained increased Infg and Il17a expressions in the aged EAE mice. Transient inhibition of aged peripheral FoxP3+ Treg cells mitigated the disease severity in the aged mice. The ameliorative effect was more significant when partially inhibiting FoxP3+ Treg cells with a drug P300i than completely depleting FoxP3+ Treg cells in FoxP3DTR transgenic mice. The mitigation is probably attributed to the correction of Treg cell distribution outside and inside the CNS. By inhibiting accumulated FoxP3+ Treg cells adhering to the brain's choroid plexus (outside the CNS), the IFN-γ-producing cells can be restored, thereby, the impediment of immune cell trafficking into the inflamed CNS is released in aged EAE mice. As a result, the proportion of myelin-specific CD4+Treg cells inside the CNS was increased for repairing neuroinflammatory damage. Conclusion: The underlying mechanism of severe MS symptoms in elderly patients is associated with the accumulation of Treg cells outside the CNS, which prevents the reparative antigen- specific Treg cells from entering the CNS during the disease.
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    (2022) Donkor, Michael; Jones, Harlan
    Despite medical advances in the diagnosis and treatment of cancer, metastatic breast cancers remain a leading cause of death in the U.S. Increasingly, novel immune-based treatments which harness the patient's immune system have promise for improving survivorship in metastatic breast cancer patients. Such therapies take advantage of the immune system's natural defense mechanisms to halt progression of breast cancer. This is mainly through the early activation of innate immune cells such as natural killer cells and the subsequent activation of the adaptive immune responses such as T and B lymphocytes which elicits a tumor-specific cytolytic and humoral antibody response, respectively. Researchers have taken advantage of these immune mechanisms of tumor defense as a complementary approach to current radio-chemo treatments, which have shown to be limited by adverse off-target effects on patients. This is particularly problematic for recurrent highly metastatic lung, brain, and bone disease, where the physiological function is a premium. Ongoing research in our laboratory is focused on using nanotechnology to develop immune-based vaccines to target local immune protection against metastatic lung disease. Because the lung is naturally tolerogenic, making it easy for disseminated tumor cells to grow, the expectation is that boosting immune responses at the lungs before seeding tumors from primary organs would mitigate metastasis and reduce mortality risks. Using an experimental murine breast cancer model of metastasis, we sought to examine the effect of intranasal vaccination to induce local and systemic adaptive immune responses as a first step in conceptualizing an immune-based nano-vaccine. We hypothesized that an intranasal vaccine protocol would induce protective lung mucosal immune protection against secondary lung metastasis. Our results demonstrated that intranasal vaccination provides protection against secondary lung metastasis using murine model of experimental lung metastasis. This protection was due to increased accumulation of both CD4+ and CD8+ T cells in the lungs that produced IFN-gamma as shown by flow cytometry and ELISA techniques. Again, our results show that intranasal vaccination produces higher tumor-specific IgG responses across respiratory tissues. These results provide initial findings suggesting the potential for targeted tumor vaccines to produce a local tumor-specific T-cell and antibody response with the potential to prevent tumor metastasis. Future challenge studies using spontaneous model of lung metastasis will test our working hypothesis that intranasal tumor vaccination protects the lung from tumor development in the presence of a primary breast tumor.
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    Mapping the distribution of immune cells in various mouse tissues
    (2022) Park, Daniel Choi
    The immune system is complex machinery where various interactions between immune cells via cell signaling, hormones, and chemokines lead to differentiation of the immune cell. With each interaction that causes immune cells to differentiate, the cellular properties will undergo modification where cells will express different cell surface markers, synthesize different proteins, gain, or lose a function, or start proliferation. By utilizing the changes in cell surface markers, it is possible to categorize immune cells and accurately identify the type of immune cells within a mice tissue which will be the focus of this project. The particular interest in this research is to identify M1 and M2 cells, and myeloid-derived suppressor cells. M1 (CD45+CD11b+F4/80+CD80+CD163-) cells are classically known as activated macrophages, and as a histiocyte, it phagocytoses pathogens and generates reactive oxygenated species. It is activated by pro-inflammatory cytokines such as IFN-y, TNF-a, or LPS, and it is activated by TH1. M2 (CD45+CD11b+F4/80+CD206+) cells are noted for their anti-inflammatory properties and promote tissue repair and wound healing. Myeloid-derived suppressor cells (MDSCs), on the other hand, are noted for their immunosuppressive effects. Mouse G-MDSCs (CD11b+Ly6G+Ly6Clo) suppresses immune responses in an antigen-specific manner via the production of reactive oxygenated species while mouse M-MDSCs (CD11b+Ly6G-Ly6Chi) suppresses immune responses in both antigen-specific and non-specific manners via synthesis of reactive oxygenated species. One of the hallmarks of MDSCs is that it is one of the cells that are found early in tumor progression. During the tumor formation, there is a notable increase in extramedullary hematopoiesis, neutrophilia, presence of abnormal myeloid cells which lack the membrane marks of known immune cells which are now identified as MDSCs. As this research will be a part of multi-part research, mapping the mouse immune cell tissue distribution will be utilized to build a foundation and set up an initial data for comparison for later when we start looking at mice with cancers and ones that are treated with chemotherapy. This will help us to easily identify the recruitment/loss/differentiation of immune cells caused by any systemic changes.
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    Pursuit of an Optimal Murine Model for Early Life Stress: Does Diet Matter?
    (2022) Choe, Jamie Y.; Jones, Harlan
    Early life stress (ELS) is known to have negative effects on long-term human health. Neglect is a significant source of ELS during childhood and accounts for 80% of reported abuse and over 30% of maltreatment-related deaths in the United States. A major challenge of studying the impact of stress on immune competency has been difficulty developing a reliable mouse model with reproducible stress effects. Animal models of ELS emulate the nature of childhood neglect through scheduled separation. Although variations of maternal separation in rodents have been published, the reported results are inconsistent. This may in part be attributed to variations in animal housing conditions between research institutions, such as diet. In the present study, we describe a modified version of the maternal separation with early weaning (MSEW) paradigm using C57BL/6 mice and compare the effects of two commercially available diets (ClearH2O® DietGel® and PicoLab® 5058) on peripheral stress hormones and cytokine profiles of select primary and secondary lymphoid tissues. Pups were produced via in-house breeding procedures and subjected to our modified MSEW protocol. Euthanasia occurred at postnatal day (PD) 21 for tissue harvest and blood collection via cardiac puncture. Cytokines and serum catecholamine and corticosterone levels were detected using commercially available ELISA. This pilot study sheds light on the impact dietary variations have on immune outcomes in the context of stress. We describe an updated MSEW protocol in C57BL/6 mice and demonstrate diet is a critical component of our stress model. Preliminary data suggests diet affects cytokine production within select lymphoid tissues at PD21. This work provides insight into the need for MSEW diet standardization to improve the reliability and reproducibility of murine models designed to study ELS.
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    Investigating the role of interleukin 1 alpha during Listeria monocytogenes infection
    (2022) Kim, Andrew; Berg, Rance E.
    Purpose: Listeria monocytogenes (LM) causes listeriosis, one of the leading causes of death by foodborne illness in the United States. Although generally self-limiting in immunocompetent people, listeriosis can cause meningitis or sepsis in immunocompromised people and spontaneous abortion in pregnant women. Our interest in interleukin 1 alpha (IL-1α), a cytokine historically associated with inflammation and alarmin activity, stemmed from a previous study in our lab showing that mice produced IL-1α when infected with LM. Currently, the role of IL-1α during infection is largely unexplored. Elucidating the role of IL-1α during LM infection will determine if IL-1α can potentially be used as a therapeutic agent and will expand our understanding of this cytokine. Method: Enzyme-linked immunosorbent assay (ELISA) was used to measure IL-1α production by LM infected RAW 264.7 macrophages. Dose response and kinetic experiments were performed to optimize culture conditions. RAW 264.7 macrophages were then infected with LM and the impact of recombinant mouse IL-1α, recombinant interleukin 1 beta (IL-1β), recombinant interferon-gamma (IFN-γ), neutralization of IL-1α, or blockade of the interleukin 1 receptor (IL-1R1) on bacterial burden was determined. Macrophage viability was measured using trypan blue to determine if the culture conditions severely impacted cell viability and to correlate viability with cytokine production and specific treatments. The total LM burden in the cultures was quantified to determine the impact of specific treatments on the ability of macrophages to kill LM. Results: IL-1α production was significantly increased in LM infected RAW 264.7 macrophage cultures compared to uninfected control cultures. The production of IL-1α by RAW 264.7 macrophages increased, plateaued, and then decreased at 6, 12, 18, and 24 hours post LM infection. We determined that infecting 500,000 macrophages at a multiplicity of infection of 1 resulted in significant IL-1α production while maintaining adequate macrophage viability. These culture conditions were then used for our bacterial burden studies. Bacterial burden decreased in LM infected cultures with recombinant IL-1α at 18 hours compared to untreated cultures, but not significantly. Conclusion: Our data suggest that macrophages may contribute significantly to IL-1α production during LM infection. Furthermore, recombinant IL-1α may have the potential to activate macrophages, resulting in enhanced LM killing. The reduction in bacterial burden in macrophages treated with recombinant IL-1α was similar to the reduction in bacterial burden in IFN-γ treated macrophage cultures. Therefore, our data suggest that recombinant IL-1α may contribute to LM resistance. Future experiments include observation of bacterial burden after addition of recombinant IL-1β, neutralization of IL-1α and blockade of IL-1R1 in LM infected macrophage cultures. LM targets the liver, so we will also investigate the impact of recombinant IL-1α on LM infected Hepa 1-6 hepatocytes and cocultures of RAW 264.7 macrophages and Hepa 1-6 hepatocytes.
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    Cardiovascular Response to Endotoxin-Mediated Sepsis: A Dose-Response Study
    (2022) Aguirre, David Salinas; Martinez, Richard; Warne, Cooper; Mallet, Robert T.; Dick, Gregory; Tune, Johnathan; Hodge, Lisa
    Purpose: Our long-term goal is to advance our understanding and treatment of sepsis, a potentially life-threatening condition that occurs when the response to infection causes tissue and organ damage. Sepsis can be caused by lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria. We used a swine model of LPS-induced sepsis to study the impact of the lymphatic and immune systems on the disease progression. Our first experiments were aimed at determining the optimal dose of Escherichia coli LPS in order to study the effect of sepsis on the cardiovascular system. We hypothesized that 2-hour intravenous infusions of 1- 50 µg/kg LPS would reveal dose- and time-dependent changes in hemodynamic parameters that are consistent with sepsis. Methods: Yorkshire pigs (61 ± 4 kg, n = 4, 2 male) were sedated, intubated, and ventilated. Femoral artery and venous lines were placed to allow measurement of blood pressure, infusion of LPS, and blood gas sampling. A thoracotomy was performed in order to secure a Transonic flow probe around the left anterior descending coronary artery and to insert a sampling catheter in the anterior cardiac vein. A dose of LPS (1, 5, 25, and 50 µg/kg) was given to each pig over 2 hours. Blood samples were collected immediately before LPS infusion and for every 30 minutes during and after LPS infusion for blood gas measurements. Vital signs were recorded as the animals developed sepsis. Results: Only the pig given the lowest dose of LPS (1 µg/kg) survived the full 6 hours (mean survival time in remaining 3 pigs was 180 ± 30 min). At 150 min, a ≈55% decrease in mean arterial pressure was observed (107 ± 4 to 48 ± 13 mmHg), resulting in a ≈60% increase in heart rate (91 ± 9 to 146 ± 14 beats/min). Coronary blood flow and myocardial oxygen consumption decreased ≈28% (0.53 ± 0.06 to 0.38 ± 0.02 ml/min/g) and ≈33% (60 ± 6 to 44 ± 2 µl/min/g), respectively. Ventricular fibrillation was the cause of death in the 3 non-surviving pigs. Conclusion: A dose of 1 µg/kg appears to be an optimal dose for future studies, as this dose was survivable in the desired time frame, while causing hypotension and tachycardia. In future studies, this model will allow us to study the effect of novel therapeutics during acute sepsis.
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    NK-cell target immunotherapy for Hepatocellular carcinoma (HCC)
    (2022) Allison, Michaela; Mathew, Stephen O.; Mathew, Porunelloor A.; Cooksey, Luke
    Natural killer (NK) cell immunotherapies have recently been gaining traction for treatment of both hematological and solid tumors due to their innate anti-tumor characteristics. NK cell activity is characterized by a balance of activating and inhibitory receptor interactions rather than antigen recognition, rendering this innate lymphoid cell a promising therapeutic target that does not rely on prior sensitization. Immunotherapies focused on targeting NK cell activity in the form of adoptive transfer, immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR) NK cells have shown some success in combating immunosuppressive effects seen during cancer. Significant suppression of NK cells has been identified in the most common type of liver cancer, hepatocellular carcinoma (HCC). NK cells play a pivotal role in the liver by early recognition and lysis of virally infected and cancerous cells introduced through portal circulation and unsurprisingly, dysfunction of this immune cell subset due to the hypoxic HCC microenvironment has been implicated as strongly correlated with poorer prognosis and decreased survival of HCC patients. Research investigating the effects of NK cell suppression has indicated that targeting NK cell suppressive interactions mediates increased lysis of HCC cells. Our research has shown the upregulation of immunosuppressive NK cell ligands on HCC cells that could potentially lead to immune escape mechanisms in HCC cells. Research further elucidating receptor-ligand interactions involved in suppression of NK cell activity during HCC could provide insight into potential therapeutic targets for patients who are untreatable with conventional therapies.