Browsing by Author "Berg, Rance E."
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Item Aging impairs regulatory T cells to affect the mouse model of late-onset multiple sclerosis(2022-08) Wang, Weikan; Su, Dong-Ming; Berg, Rance E.; Bunnell, Bruce A.; Yang, Shaohua; Jones, Harlan P.; Zode, Gulab S.Although multiple sclerosis (MS) primarily onsets in young adults, it can also develop in the elderly, which is termed late-onset (aged) MS. CD4+ Foxp3 + regulatory T (Treg) cells play an ameliorative role in severity of MS or its animal model experimental autoimmune encephalomyelitis (EAE), and the aged immune system accumulates peripheral Treg (pTreg) cells. However, late-onset MS in the aged patients presents a more progressive disease course. We investigated why the accumulated pTreg cells fail to ameliorate the MS severity in the aged individuals by using an aged EAE mouse model to recapitulate late-onset MS in patients. We observed that the onset of EAE is delayed in aged mice, but disease severity is increased compared to young EAE mice. We found that the distribution of Treg cells in aged EAE mice exhibited an increased proportion of polyclonal (pan-) pTreg cells and a decreased proportion of antigen specific-pTreg cells in the periphery, but decreased proportions of both pan- and antigen specificTreg cells in the central nervous system (CNS). Transiently inhibiting Foxp3 or depleting pTreg cells partially corrected Treg distribution and restored the balance of effector T cells (Teff) and Treg cells in the aged inflamed CNS, thereby ameliorating the disease in the aged EAE mice. Furthermore, in the aged inflamed CNS, CNS-Treg cells exhibited a high plasticity and T effector (CNS-Teff) cells presented a great clonal expansion, disrupting the Treg/Teff balance. These results provide evidence and mechanism that accumulated aged pTreg cells play a detrimental role in neuronal inflammation of aged MS.Item Atrophied thymus, a tumor reservoir for harboring melanoma cells(2018-12-01) Sizova, Olga; Su, Dong-Ming; Berg, Rance E.; Basu, AlakanandaTumor metastatic relapse is the major cause of cancer-associated mortality. Metastatic relapse is believed to arise from quantities of tumor cells that are below detectable thresholds, which are able to resist radio/chemotherapy by obtaining a dormant state and hiding in certain organs, i.e., tumor reservoirs. The thymus, the central T-cell immune organ, has been suggested to be a pre-metastatic tumor reservoir for B-lymphoma cells. However, it remains unknown whether the thymus is able to harbor nonlymphoid solid tumor cells, and whether chemotherapy can thoroughly eliminate cancer cells in the thymus. If chemotherapy is not able to eliminate these cells in the thymus, then what processes allow for this? Melanoma cell–inoculated and genotoxic doxorubicin-treated mouse model systems were used to determine that the thymus, particularly the atrophied thymus, was able to harbor bloodstream–circulating melanoma cells. In addition, chemotherapy-induced DNA-damage response triggered p53 activation in nonmalignant thymic cells, which in turn resulted in thymocyte death and thymic epithelial cell senescence to develop an inflammatory thymic microenvironment. This inflammatory condition induced thymic-harbored minimal tumor cells to acquire a chemo-resistant state.Item Blood-Based Inflammation Biomarkers of Neurocognitive Impairment in People Living with HIV(2020-05) Swanta, Naomi K.; Borgmann, Kathleen; Berg, Rance E.; Cunningham, Rebecca L.; Johnson, Leigh A.; Yan, Liang-JunRace and sex minorities are disproportionately affected by HIV in the United States. Approximately 50% of people living with HIV (PLWH) experience HIV-associated neurocognitive disorders (HAND). ART has decreased incidence of HAD but the less severe forms of HAND has increased. Diagnosis of HAND is challenging as the often-subtle forms of impairment are not as overt as dementia. HIV infection promotes neurocognitive dysfunction through persistent inflammation, which correlates with the severity of impairment. The objective of this study was to identify blood-based cytokines that associate with, and could predict, neurocognitive functioning in a demographically balanced cohort of PLWH. Inflammatory biomarkers of HIV-associated neurocognitive impairment could improve current diagnosis methods and may be specific for populations disproportionately affected by the HIV. Seven neurocognitive domains were evaluated in 121 seropositive African American, Non-Hispanic White and White Hispanic men and women using computerized assessments. A panel of 26 inflammation-associated cytokines were measured in plasma and blood mononuclear cells. Significant associations among neurocognitive functioning and HIV-related parameters, relevant sociodemographic variables and cytokine panel were determined using multivariate and univariate regression analyses. Following corrections for education, CD4 T cell counts, viral load and eliminating outliers. Our results demonstrate that chemokine C-C motif ligand (CCL) 8 significantly correlated with memory, complex attention, cognitive flexibility, psychomotor speed, executive functioning and processing speed. Tissue inhibitor of metalloproteinases-1 (TIMP-1) significantly correlated with the aforementioned domains except memory and processing speed. In addition, interleukin (IL) - 23 significantly associated with executive functioning and processing speed. The biomarkers demonstrated a negative correlation to neurocognitive functioning. Race disparities were identified in memory and CCL8, furthermore, sex disparities were identified in executive functioning and TIMP-1. The plasma biomarkers were evaluated as predictive classifiers of neurocognitive functioning. Decision tree models for NCI and executive functioning predicted visit two at 67.2% accuracy. Collectively, these data identified blood-based inflammatory biomarkers of neurocognitive function with the potential to assist in the diagnosis of HIV-associated neurocognitive impairments in PLWH.Item Characterization of Markers on Pancreatic and Colon Cancer Stem Cells to Enhance Natural Killer Cell Effector Functions(2021-05) Malaer, Joseph D.; Mathew, Porunelloor A.; Mathew, Stephen O.; Berg, Rance E.; Jones, Harlan P.; Prokai, LaszloDue to advancements in technology and medicine, the average human lifespan in the United States has increased to 79 years since the start of the 20th century. This increase in lifespan is also coupled with an increased risk of developing cancer. While cancer detection and initial treatment has increased the survival of patients, it is not a cure and patients wonder how long they will remain in remission. It is estimated that over 90% of cancer related deaths are due to relapse and metastasis. There is currently a growing body of research that indicates cancer stem cells (CSC) are responsible for relapse and metastasis. CSC are a small subpopulation of cells that retain self-renewal and pluripotency. These cells are also described as being quiescent, which may contribute to their chemotherapy resistance. Additionally, CSC are thought to express ligands to aid in immune evasion mechanisms. Natural Killer (NK) cells are lymphocytes of the innate immune system which function to combat infection and cancer. NK cells, unlike T and B cells, do not require prior sensitization. NK cell function is regulated through activating and inhibitory receptors. Recently, Proliferating Cell Nuclear Antigen (PCNA) was identified as an inhibitory ligand for the natural cytotoxicity receptor NKp44. Lectin-like transcript 1 (LLT1) expression is known to facilitate escape of NK cell effector functions in prostate and breast cancer, and now colorectal cancer. In this study we analyzed the expression of molecules on pancreatic and colon cancer cells that could allow escape from NK cell-mediated immune surveillance. The data presented here demonstrates surface PCNA expressing cells as CSC through co-expression of CSC surface markers CD44 and CD133 as well as overexpression of CSC transcription factors NANOG, SOX-2, and Oct-4. Blocking PCNA or NKp44 alters interferon-ℽ secretion by NK cells when cocultured with pancreatic and colon cancer cells. It is further demonstrated that blocking LLT1 or the PCNA-NKp44 interaction led to an increase in specific lysis of tumor cells. This study suggests that cell surface PCNA could function as a biomarker and an immunotherapeutic target for NK cell mediated killing of pancreatic cancer and colon cancer cells.Item Compact NMR Relaxometry of Human Blood and Living Tissues(2018-08) Patel, Vipulkumar R.; Cistola, David P.; Gryczynski, Ignacy; Berg, Rance E.; Lacko, Andras G.; Park, InWooMetabolic syndrome (MetS) is a cluster of metabolic abnormalities. The designation of MetS requires three or more of five clinical criteria: central obesity, high triglycerides, low HDL cholesterol, elevated blood pressure and high blood glucose. The main purpose of the MetS diagnosis is to prevent diabetes. However, the clinical criteria of MetS are poorly calibrated and fail to detect early metabolic abnormalities essential for diabetes prevention. Additionally, the MetS definition lacks a measure of chronic inflammation, an important driver of metabolic dysregulation. Our lab has shown that plasma and serum water T2, measured using benchtop nuclear magnetic resonance (NMR) relaxometry, are better metabolic health indicators and inclusive of inflammation. In Chapter 2 of this dissertation, we describe a broad-based, unbiased proteomic search for new biomarkers that predict plasma and serum water T2. Using a multistep statistical approach, we identified five circulatory proteins that are strongly implicated in metabolic health. In Chapter 3, we investigated whether whole blood T2 can provide similar metabolic information. Mixed blood yielded a single T2, whereas settled blood gave rise to two distinct T2 values for the cell pellet (T2P) and plasma supernatant (T2S). Supernatant T2S showed strong correlations with red blood cell count and hematocrit, and this association was due to paramagnetic relaxation enhancement. In contrast, the pellet T2P exhibited strong correlations with metabolic biomarkers. Hemoglobin glycation (HbA1C, a marker of metabolic health) is responsible for this association, as it provides water binding sites that lead to faster T2 relaxation because of increased binding and chemical exchange. The T2 value for mixed blood revealed strong associations with red blood cell count and hemoglobin. In Chapter 4, we investigated the feasibility of acquiring T2 data non-invasively from living human tissue using a custom-build NMR relaxometry device equipped with a magnet configuration to accommodate the human fingertip. Using healthy volunteers, we showed that three T2 components, corresponding primarily to different mobility domains of adipose tissue, can be measured reproducibly, with significant subject-to-subject biological variation. We propose that the source of variation is adipose tissue fluidity, which varies with lipid composition and the state of connective tissue matrix.Item Comparing Short-Term Radiographic Outcomes of Cementless Primary Reverse Total Shoulder Arthroplasty Implantation With and Without Augmentation by Humeral Matchstick Autograft: A Retrospective Cohort Study(2021-12) Ouseph, Alvin; Berg, Rance E.; Gwirtz, Patricia A.; Lund, Julia; Cohen, ClaudiaReverse Total Shoulder Arthroplasty (RTSA) is an arthroplasty procedure that is increasing in incidence in the United States (26). As the incidence of the procedure increases, the gross number of RTSA complications is also expected to increase. Humeral-sided complications, accounting for up to 21% of RTSA complications requiring revision surgery, are associated with progressive humeral bone loss (10, 28). This bone loss can be explained by Wolff's law, which states that bone will adapt according to the amount of stress placed upon it (6, 27). Per Wolff's law, having a large, stiff humeral construct will lead to humeral adaptations and implant-induced osteopenia, a phenomenon called stress shielding (5, 6). Studies by Raiss et al., have suggested that lowering the size of the humeral implant in relation to the humeral metaphysis of patients can lower the incidence of stress shielding (7-9). Surgeons at The Shoulder Center at Baylor University Medical Center in Dallas, Texas, inspired by Raiss et al., have developed and implemented a new matchstick autografting procedure to reduce the size of humeral implants and combat the risk of stress shielding. This study seeks to evaluate whether the recommendations and results of Raiss et al. hold true with regards to the new matchstick RTSA procedures as well as traditional RTSA procedures performed by surgeons at The Shoulder Center (9).Item Diagnostic Value of Dynamic Ultrasound in Supination-External Rotation Injuries(2018-08) Fisher, Cara L.; Reeves, Rustin E.; Berg, Rance E.; Maddux, Scott D.; Rosales, Armando; Wood, Addison R.Ankle syndesmosis injuries are common and range in severity from subclinical to grossly unstable. Definitive diagnosis of these injuries can be made with plain film radiographs if the injury is severe enough, but often is missed when severity or image quality is low. Computed tomography (CT) and magnetic resonance imaging (MRI) can provide early definitive diagnosis regardless of severity, but are costly and introduce the patient to radiation when CT is used. Ultrasound diagnosis may circumvent many of these disadvantages by being inexpensive, efficient, and able to detect subtle injuries without radiation exposure. This study evaluates the ability of ultrasound to detect subtle supination-external rotation (SER) ankle syndesmosis injuries with a dynamic external rotational stress test. Nine all male fresh frozen specimens were secured to an ankle rig and stress tested to 10 Nm of external rotational torque with ultrasound monitoring at the tibiofibular clear space. The ankles were subjected to syndesmosis ligament sectioning and repeat stress measurements of the tibiofibular clear space at peak torque. Stress tests and measurements were repeated three times and averaged. Data was analyzed using a two-way repeated measures ANOVA.Item Divergent Roles of Extracellular Superoxide Dismutase During Intracellular Bacterial Infection(2016-03-23) Break, Timothy J.; Okunnu, Busola; Swanta, Naomi; Berg, Rance E.; Witter, AlexandraExtracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species (ROS) to protect tissues during infection and inflammation. Using congenic mice with varying levels of ecSOD activity (ecSOD HI, WT and KO), we have previously shown that ecSOD activity enhances neutrophil recruitment to the liver, yet inhibits the innate immune response against Listeria monocytogenes (LM) leading to increased host susceptibility. Additionally, we determined that ecSOD activity protects the extracellular matrix (ECM) from degradation and promotes egress of immature neutrophils out of the bone marrow and into the liver where they are unable to provide protection against LM. Since ecSOD can be produced by cells from the hematopoietic lineage as well as somatic cells, the potential contribution of ecSOD produced by cells from each lineage required further investigation. In order to determine the relative contributions of ecSOD produced from either hematopoietic-derived cells or somatic cells, we generated bone marrow chimera mice using ecSOD KO mice and C57Bl/6 mice. Briefly, host mice were irradiated to eliminate hematopoietic lineage cells and reconstituted with bone marrow cells isolated from donor mice. Control groups consisted of ecSOD KO mice reconstituted with bone marrow from ecSOD KO donors (KO -> KO) or C57Bl/6 mice with bone marrow from C57Bl/6 mice (WT -> WT). Experimental groups consisted of ecSOD KO mice reconstituted with bone marrow from C57Bl/6 mice (WT -> KO) or C57Bl/6 mice with bone marrow from ecSOD KO mice (KO -> WT). All mice were then infected with LM and evaluated for neutrophil recruitment and bacterial burden. We observed that ecSOD produced by hematopoietic cells leads to increased bacterial burden during LM infection, while ecSOD produced from somatic cells is essential for increased neutrophil recruitment. Collectively, our data suggest that ecSOD produced by both hematopoietic cells and somatic cells is involved in our observed phenomena; however, the contribution of ecSOD from each cell lineage is skewed towards either increased neutrophil recruitment or increased susceptibility to LM infection, but not both. These studies highlight the potential therapeutic value of ecSOD inhibitors to enhance immune responses during bacterial infections.Item Does Diversity Matter? Disparities in Diagnostic Delays and Contralateral Risk Factors in Slipped Capital Femoral Epiphysis(2022-12) Purcell, Maureen W.; Reeves, Rustin E.; Mayfield, Matthew; Lovely, Rehana S.; Berg, Rance E.The skeletal pathology slipped capital femoral epiphysis (SCFE) is one of the more common hip diseases that can affect adolescents. Delays in diagnosis and the risk of contralateral SCFE are recognized issues for this patient population. However, SCFE studies often do not include the groups of people most often diagnosed with this pathology, namely Black and Hispanic individuals. This project aimed to address this literature gap by investigating the recognized issues with a sizeable sample of individuals in those ethnic groups, roughly equal to the White patient group. We found that the severity of SCFE measured by Southwick slip angle (SSA) is significantly associated with both insurance type and patient status. Patients who were covered by private insurance or were already an established patient at the medical center were more likely to be diagnosed with mild SCFE, and patients with no insurance had a significantly higher mean SSA than patients with insurance. Posterior sloping angle (PSA) and physeal sloping angle in the anterior-posterior view (PSA-AP) are two of the most often used measurements to estimate contralateral slip risk. They were not predictive of contralateral slip risk in our sample, except for PSA-AP in male patients. When analyzed within each ethnic subgroup, we found significant differences in the PSA and PSA-AP between males and females within the Hispanic patient sample only. This suggests that these angles are not necessarily predictive for all patients groups, and/or that there may be sex differences within patient populations that can affect the utility of these metrics. To the authors' knowledge, the above findings are the first to link SSA to insurance status and patient status, and to the first to analyze the PSA and PSA-AP angles of a Hispanic SCFE patient group.Item Effect of CD44 Expression on T Cell Acute Lymphocytic Leukemia(2014-08-01) Racine, Ronny R.; Mummert, Mark E.; Jones, Harlan; Berg, Rance E.CD44 is a cell surface glycoprotein that serves as a multifunctional receptor aiding in trafficking and adhesion of immune cells. CD44 also serves as a recruitment platform for signaling molecules and has been shown to regulate proliferation. In several types of leukemia the presence or absence of CD44 expression is associated with different clinical outcomes, with patients who have increased expression of CD44 exhibiting a stronger response to conventional chemo- and radiotherapy. By using Jurkat T cells, which do not express CD44, to determine the effects of CD44 expression in a model Acute Lymphocytic Leukemia cell line, we have outlined two major areas of study. Firstly, upon expression of CD44, Jurkat T cells proliferate slowly compared to the control cells. This decrease in proliferation is coupled to an arrest in the cell cycle during the transition from the G1 phase into S phase. The dysregulation of the cell cycle induced by CD44 also leads to the induction of aneuploidy. CD44 expressing Jurkat T cells have reduced mRNA expression for several key regulators of chromosome separation and the mitotic spindle complex. This finding, coupled with decreased EGR-1 expression, which controls the cyclins responsible for transition from G1 into S phase, leads to an unstable cell phenotype which proliferates slowly and accumulates extra chromosomes in daughter cells. The second area of study focuses on the mechanism by which CD44 expression at the cell surface results in the observed decreases in proliferation, Akt activation, and EGR-1 expression. We observed that CD44 expressing Jurkat cells show four to five times higher calcium influx when at rest compared to the vector control cells. This influx in calcium is due to CD44 expression activating a cell surface inducible calcium release activated calcium channel. The excess calcium activates calcium-activated phosphatases and kinases, disrupting EGR-1 expression and inducing a hypophosphorylation of Akt. Together, these findings indicate that CD44 expression can regulate cell proliferation and signal transduction pathways in addition to its role in adhesion. Thus, our data provide a further understanding of how CD44 expression modifies leukemic cells into cells that are favorable for therapeutic intervention.Item Establishment of Animal Models of Mycoplasma pnumoniae pneumonia and Staphylococcus arueus osteomyelitis(2018-12) Chikelue, Calvin I.; Simecka, Jerry W.; Berg, Rance E.; Jones, Harlan P.; Park, InWoo; Reeves, Rustin E.Animal models are useful tools in the study and development of clinical solutions to pathogens. Our focus is on two clinically relevant bacterium: Mycoplama pnumoniae which can lead to community acquired pneumonia and Staphylococcus aureus which can result in osteomyelitis. We formed experimental designs to establish murine models for these pathogens. By testing multiple strains of mycoplasma within mice, infecting both immunocompetent and immunodeficient mice as well as humanized mice, we have begun the preliminary development of a humanized mouse model for M. pneumoniae. As well, by testing multiple strains of S. aureus and their ability to both attach to and from biofilm on orthopedic pins, we've developed the first steps toward a murine model for S. aureus Osteomyelitis.Item EXTRACELLULAR SUPEROXIDE DISMUTASE ENHANCES NEUTROPHIL RECRUITMENT TO THE LIVER BY MODULATING THE EXTRACELLULAR ENVIRONMENT(2014-03) Witter, Alexandra R.; Break, Timothy J.; Indramohan, Mohanalaxmi; Berg, Rance E.Listeria monocytogenes (LM) infection represents one of the leading causes of death from foodborne infection, especially in immunocompromised individuals, and can cause spontaneous abortion in pregnant women; however, it is commonly used as a model to study the host immune response against infection. Our lab utilizes three groups of mice with varying levels of extracellular superoxide dismutase (ecSOD) activity (high, wild-type, and none) to determine the impact that this antioxidant enzyme has on the host immune response during LM infection. We have previously shown that ecSOD activity leads to decreased innate immune response against LM. Our current data shows that ecSOD activity protects the extracellular matrix from degradation, and leads to increased neutrophil recruitment; however, these neutrophils display inhibited function and therefore do not provide adequate protection against LM. This research is relevant due to the potential use of ecSOD inhibitors to enhance immune responses during bacterial infection. In addition, these findings help clarify the impact of the oxidative environment on the immune response and how antioxidants contribute to this dynamic. Purpose (a): Listeria monocytogenes (LM) is an intracellular foodborne pathogen that causes severe disease in immunocompromised individuals, spontaneous abortion in pregnant women, and results in ~25% mortality rate in infected individuals. Extracellular superoxide dismutase (ecSOD) converts superoxide into hydrogen peroxide in the extracellular milieu and protects against oxidative stress. We have previously shown that ecSOD activity inhibits innate immune responses during LM infection leading to increased bacterial burden; however, it is unclear whether ecSOD activity affects neutrophil recruitment and function in a cell-intrinsic manner or by modulating the extracellular environment. Methods (b): Congenic mice with high ecSOD activity (ecSOD HI), wild type ecSOD activity (ecSOD WT), or lacking ecSOD (ecSOD KO), on the C57Bl/6 background were used to perform adoptive transfer experiments after intravenous infection with ~10,000 wild-type LM (WTLM). Either isolated neutrophils or labeled whole bone marrow cells were transferred from ecSOD HI or ecSOD KO mice into ecSOD WT mice and then flow cytometry analysis was performed and colony forming units (CFUs) were calculated. Concentrations of hyaluronan and lymphotoxin alpha were determined by ELISA. Results (c): Whole bone marrow cell transfers indicated that there was no difference in recruitment of neutrophils transferred from ecSOD HI or ecSOD KO mice to the liver when the neutrophils were all in the same environment (ecSOD WT mice). In addition, neutrophils isolated from ecSOD HI or ecSOD KO mice showed no difference in their ability to protect against LM infection, as shown by equivalent CFUs, when in comparable environments (ecSOD WT mice). Analysis of hyaluronan concentrations – a component of the extracellular matrix (ECM) – indicated that ecSOD activity protects the ECM from degradation. Conclusions (d): We observed from adoptive transfer experiments that ecSOD activity does not affect neutrophil recruitment or function in a cell-intrinsic manner. Additionally, we determined that ecSOD activity protects the ECM, which is important for neutrophil trafficking. Overall, we concluded that ecSOD activity enhances neutrophil recruitment yet decreases their function by modulating the extracellular environment.Item Extracellular Superoxide Dismutase Hinders Effective Containment of Listeria monocytogenes by Neutrophils(2018-03-14) Berg, Rance E.; Okunnu, BusolaPurpose: Increased activity of Extracellular Superoxide Dismutase (ecSOD), an enzyme widely regarded as having protective functions during ROS induced inflammation, has been shown to be detrimental to host survival during infection with the intracellular bacteria, Listeria monocytogenes (Lm). Although, we have also demonstrated that neutrophils are essential for protection during Lm infection, a higher percentage of neutrophils are present in mice with high ecSOD activity during Lm infection. However, these mice are still more susceptible to infection in comparison to mice that lack ecSOD activity. These paradoxical findings led to the objective to better understand how ecSOD activity modulates the protective functions of neutrophils during Lm infection. Materials and Methods: For these studies, ecSOD congenic mice: ecSOD HI mice with high ecSOD activity, ecSOD WT mice with normal ecSOD activity, and ecSOD KO mice with no ecSOD activity were utilized. To determine phagosomal containment, we made use of flow cytometry and a strain of Lm, actA:LMGFP, which only fluoresces GFP when the bacteria escapes out of the phagosome into the cytosol. Results: A higher percentage of neutrophils from the ecSOD KO mice took up Lm in comparison to the HI neutrophils. Correlated with this was also a higher percentage of ecSOD KO neutrophils allowing for phagosomal escape in comparison to the ecSOD HI neutrophils. Analysis of the mean fluorescence intensity (MFI) showed that although there were more bacteria present in the ecSOD KO neutrophils, the amount of escaped bacteria was comparable to that in the ecSOD HI neutrophils. Treatment of the ecSOD KO neutrophils with IFN-g, an activator of neutrophils, also led to better phagosomal containment. Though the IFN-g treated neutrophils took up more bacteria, there was no difference in the amount of escaped bacteria in comparison to the non-treated cells. Conclusion: EcSOD activity hinders the ability of neutrophils to keep Lm contained in the phagosome which prevents effective bacterial killing. Additionally, neutrophil activation with IFN-g also makes the cells more effective at bacterial containment. However, the effect of ecSOD activity in conjunction with IFN-g is yet to be determined. Future studies on how ecSOD affects bacterial killing and other functions downstream of phagosomal escape will aid in a better understanding of how ROS can modulate neutrophil function during intracellular bacterial infections.Item Extracellular Superoxide Dismutase Indirectly Enhances the Release of Immature Neutrophils from the Murine Bone Marrow(2016-08-01) Witter, Alexandra R.; Berg, Rance E.; Hodge, Lisa M.; Mummert, Mark E.Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species (ROS) to protect tissues during infection and inflammation. Using ecSOD HI, ecSOD WT, and ecSOD KO mice, we have previously shown that ecSOD activity enhances neutrophil recruitment to the liver, yet inhibits the innate immune response against Listeria monocytogenes leading to increased host susceptibility. Using adoptive transfer experiments, we observed that ecSOD activity does not affect neutrophil recruitment or function in a cell-intrinsic manner. Additionally, we noted that ecSOD activity results in decreased retention of immature neutrophils in the bone marrow without altering granulopoiesis. Furthermore, we determined that ecSOD activity protects the extracellular matrix (ECM) and increases concentrations of neutrophil-attracting chemokines leading to an increase in immature neutrophils in the liver. Since ecSOD can be produced by cells from the hematopoietic lineage as well as non-hematopoietic cells, we used bone marrow chimeric mice to investigate the relative contribution of ecSOD produced by cells from each lineage. Ultimately, it was determined that ecSOD from both hematopoietic and non-hematopoietic cells contributes to the overall phenotype observed in ecSOD congenic mice. Collectively, our data suggest that ecSOD activity inhibits degradation of the ECM and promotes egress of immature neutrophils out of the bone marrow and into the liver where they provide inadequate protection against L. monocytogenes. These studies highlight the potential therapeutic value of ecSOD inhibitors to enhance immune responses during bacterial infections.Item Extracellular Superoxide Dismutase Promotes Immature Neutrophil Egress from the Bone Marrow(2015-03) Witter, Alexandra R.; Break, Timothy J.; Indramohan, Mohanalaxmi; Mummert, Mark E.; Berg, Rance E.Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species (ROS) to protect tissues during infection and inflammation. Using three groups of mice with varying levels of ecSOD activity, we have previously shown that ecSOD activity enhances neutrophil recruitment to the liver, yet inhibits the innate immune response against Listeria monocytogenes (LM) leading to increased host susceptibility. However, it is unclear whether ecSOD activity affects neutrophil recruitment and function in a cell-intrinsic manner or by modulating the extracellular environment. Using adoptive transfer experiments, we observed that ecSOD activity does not affect neutrophil recruitment or function in a cell-intrinsic manner. Additionally, we determined that ecSOD activity protects the extracellular matrix (ECM) and leads to an increase in phenotypically immature neutrophils in the bone marrow and liver. Collectively, our data suggest that ecSOD activity inhibits degradation of the ECM and promotes egress of immature neutrophils out of the bone marrow and into the liver where they provide inadequate protection against LM. These studies highlight the potential therapeutic value of ecSOD inhibitors to enhance immune responses during bacterial infections.Item FUNCTIONAL REGULATION OF PHAGOCYTIC CELLS BY IL-23 DURING LISTERIA MONOCYTOGENES INFECTION(2014-03) Indramohan, Mohanalaxmi; Break, Timothy J.; Witter, Alexandra R.; Berg, Rance E.Listeria monocytogenes (LM) is a Gram-positive intracellular pathogen that causes meningitis and septicemia in immunocompromised individuals, and spontaneous abortion in pregnant women. The versatility of LM makes it a useful tool for immunologists to understand how the immune system responds against harmful microorganisms. Cell recruitment mediated by the IL-23/IL-17 axis is important for protection against infectious diseases, but can cause damage during autoimmune disorders. By utilizing mice lacking IL-23 (IL-23p19 KO), our lab examines the role of this cytokine during a systemic bacterial infection. We have demonstrated that IL-23 promotes resistance against LM infection by increasing the recruitment of neutrophils to the liver, and monocytes to the spleen during LM infection. Interestingly, IL-23 or IL-17A is not required for enhancing phagocytic cell functions including phagocytosis, production of ROS, MPO, and pro-inflammatory mediators during LM infection. Understanding the significance of IL-23/IL-17axis in mediating the recruitment and function of immune cells will aid in the development of effective therapeutics depending on the disease condition. Purpose (a): Listeria monocytogenes (LM) is a Gram-positive intracellular foodborne pathogen that causes meningitis and septicemia in immunocompromised individuals, and spontaneous abortion in pregnant women. LM is widely used as a model pathogen to study host pathogen immune interactions. Cell recruitment mediated by the IL-23/IL-17 axis is necessary for protection against multiple infectious diseases, but can be detrimental during autoimmune disorders. We have previously shown utilizing mice lacking IL-23 (IL-23p19 KO) that IL-23 provides protection against LM infection by promoting the optimal recruitment of neutrophils to the liver, and monocytes to the spleen. The receptors for IL-23 and IL-17A are present on phagocytic cells including monocytes, neutrophils, and macrophages. However, it is not known whether IL-23 or IL-17A can impact the function of phagocytic cells during LM infection. Methods (b): Splenocytes and liver leukocytes were harvested from mice infected intravenously with ~10, 000 LM. Peritoneal wash was performed to isolate resident peritoneal macrophages. Flow cytometry was utilized to determine phagocytosis, production of reactive oxygen species (ROS), and myeloperoxidase (MPO). The concentrations of TNF-α, IL-1, IL-6, and nitric oxide (NO.) were measured by ELISAs/Griess assay. Results (c): Phagocytic cells isolated from control C57Bl/6 (B6) and IL-23p19 KO mice displayed equivalent phagocytic potential. There were no differences in the production of ROS or MPO from splenocytes isolated from both groups of mice. Furthermore, exogenous stimulation with rIL-23 or rIL-17A did not induce or enhance production of ROS or proinflammatory mediators from B6 splenocytes. Conclusions (d): IL-23 does not impact the function of phagocytic cells either by a direct or indirect mechanism during LM infection. Collectively, our data suggest that the lack of efficient recruitment of neutrophils to the liver, and monocytes to the spleen, results in a reduction in the overall levels of TNF-α and NO. and therefore, increases the susceptibility of IL-23p19 KO to LM infection.Item Geographic Analysis of Trauma Readmissions in North Texas(2016-12-01) Sanchez, Derick J.; Gwirtz, Patricia A.; Berg, Rance E.; Singh, MeharvanDue to the high cost and increased risk of mortality associated with unplanned patient readmissions, research has been aimed to identify risk-factors in patients with high hospital utilization and recidivism. The primary aim of this study was to characterize readmissions across multiple institutions in patients initially admitted to a single urban Level I trauma center. Analysis was carried out to test the hypothesis that a patient’s geographic location of residence can be used to predict readmission rates. Data was queried from a regional database that is comprised of more than 150 hospitals in the North Texas region. Patient ZIP code and county of residence were analyzed using binary logistic regression to determine significance of predictability of readmission by patient geography. Additional variables such as demographics, diagnosis, Elixhauser comorbidities, and insurance were also analyzed to create a full clinical and geographic regression model describing patterns in readmissions.Item Investigating the role of interleukin 1 alpha during Listeria monocytogenes infection(2022-05) Kim, Andrew J.; Berg, Rance E.; Fudala, Rafal; Park, InWooListeria 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 where we observed that immune cells isolated from LM infected mice produced IL-1α. 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. Enzyme-linked immunosorbent assay (ELISA) was used to measure IL-1α concentration produced by LM infected RAW 264.7 macrophages and LM infected Hepa 1-6 hepatocytes. Dose response and kinetic experiments were performed to optimize culture conditions. Cell viability of macrophage cultures, hepatocyte cultures, and cocultures of macrophages and hepatocytes was measured using trypan blue to determine if the culture conditions severely impacted cell viability. LM burden of infected macrophage cultures, infected hepatocyte cultures, and infected cocultures of macrophages and hepatocytes were quantified using colony forming unit counting method and compared with control. Infected cultures were treated with recombinant (r-) IL-1α, r-interferon gamma (r-IFN-ℽ), r-interleukin 1 beta (r-IL-1β), or anti-IL-1α. IL-1α production was significantly increased in LM infected RAW 264.7 macrophage cultures compared to uninfected control. The concentration of IL-1α produced by infected macrophage cultures and infected cocultures increased, plateaued, and then decreased at 6, 12, 18, and 24 hours post LM infection. IL-1α was not detected in infected hepatocyte cultures. LM burden of infected macrophage cultures, infected hepatocyte cultures and infected cocultures treated with r-IL-1α was reduced compared to control. Our data suggest that macrophages contribute significantly to IL-1α production during LM infection and r-IL-1α may contribute to LM resistance.Item 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.Item Involvement of Caspase-2 in Cisplatin-Induced Cell Death in 2008 Ovarian Cancer Cells(2008-04-01) Adkins, Brett T.; Basu, Alakananda; Berg, Rance E.; Gryczynski, ZygmuntAdkins, B., Involvement of caspase-2 in cisplatin-induced cell death in 2008 ovarian cancer cells. Master of Science (Molecular Biology and Immunology) April, 2008, 59 pp., 12 illustrations, bibliography, 73 titles. Cisplatin, one of the most effective anticancer drugs in the treatment of ovarian cancer, causes DNA damage and leads to apoptosis. Caspases, a family of cysteine proteases, are essential for the induction of apoptosis. Initiator caspases activate effector caspases to trigger apoptosis. Caspase-2 can function as both an initiator and effector caspase although there are controversies regarding its role in DNA damage-induced apoptosis. Caspase-2 is the only caspase constitutively located in the nucleus, although its function there is unknown. In the present study we have investigated if caspase-2 is important during cisplatin-induced apoptosis and whether cisplatin treatment affects the localization of caspase-2. Caspase-2 depletion suggested that caspase-2 acts upstream of caspase-2 acts upstream of caspase-9 in cisplatin-induced apoptosis. We also made a novel observation that rottlerin, an inhibitor of DNA damage-induced apoptosis, specifically downregulates caspase-2 via the ubiquitin proteamose-mediated pathway. We further show that cisplatin induces caspase-2 translocation out of the nucleus. Moreover, translocation of caspase-2 is more important for cisplatin-induced cell death.