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    (2014-03) Bowen, Kelly E.; Mathew, Porunelloor A.
    Purpose (a): Natural Killer (NK) cells are lymphocytes that play a vital role in defense against cancer and infections. NK cell function is regulated by a balance between activating & inhibitory signals transmitted through NK cell surface receptors upon interaction with their ligands. Identification of NK cell receptors and their ligands on tumor cells allow targeted therapy for the specific tumor. CD48 is the only known natural ligand for NK receptor 2B4. Previous studies indicate an in vivo role of 2B4 in B16 melanoma tumor rejection. B16 melanoma cells do not express CD48. We hypothesize that 2B4 interacts with a melanoma specific ligand and that this interaction regulates NK cell function. Methods (b): We generated m2B4-Fc fusion protein, consisting of the extracellular domain of mouse 2B4 and human IgG1 Fc portion. To produce the soluble m2B4-Fc, the p2B4-Fc plasmid was transiently transfected into HEK cells. The purified protein was tested for binding by flow cytometry to mouse CD48 of P815 cells, which express CD48, and for binding to mouse 2B4 melanoma cells, which do not express CD48. The melanoma specific ligand was isolated by immunoprecipitation. Results (c): We show that NK cell receptor 2B4 binds to a melanoma specific ligand on B16 melanoma cells. Conclusions (d): Identification and molecular characterization of the melanoma specific ligand for 2B4 will help in developing new strategies to target melanoma with NK cells.
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    (2014-03) Chikelue, Calvin I.; Mize, Maximillion T.; Simecka, Jerry
    The contribution of Mycoplasma pneumoniae to infections leading to community-acquired pneumonia (CAP) makes it a prime bacterium to study for the development of protective vaccines against it. Despite this, the scant few vaccines researched and tested offer little to no immune protection. In order to further along development of a protective vaccine, the human immune response to M. pneumoniae infection needs to be appropriately deduced. Our lab seeks to develop a humanized mouse model for M. pneumoniae which can be used for observing the most likely human immune response to this bacterium. These preliminary experiments revolve around the investigating the infectivity and pathogenicity of three different strains: S1, M129, and UABPO1. Whichever strain exhibits the ability to cause severe disease within our test animals in comparison to the others will be the strain we utilize in our future studies. These strains were used to infect multiple groups of Balb/c mice which were observed over 14 day time period. At the conclusion of the time period, the mice were sacrificed and the lungs were scored for lung lesions and afterward, depending on the experimental group, the lungs were either homogenized for dilution plating or stored in fixative for histological staining. As of this point in our precursory experimentation, the UABPO1 strain has shown the most promising results in terms of its pathogenicity in comparison to the other experimental strains and may be the strain our lab will utilize in the future studies for developing the humanized mouse model. Purpose (a): Community-acquired pneumonia (CAP) is a lung disease caused by infection with a respiratory bacterium. M. pneumoniae, a major contributor to CAP infections, is a bacterium that invades and attaches to the airway epithelium causing damage to the host through the production toxic substances or Community Acquired Respiratory Distress Syndrome (CARDS) toxin. No commercial vaccine currently exists for the bacterium and tested vaccines have shown to be more immunopathologic rather than protective. To develop a protective vaccine, the immune response against M. pneumoniae must be better understood. Our research is focused on the development of a humanized mouse model for the study of the immune mechanisms that occur during and infection with M. pneumonia. Methods (b): Three strains are used in these preliminary experiments: S1, M129, and UABPO1. Whichever strain causes severe disease within mice will be used in the future experiments with the model. Groups of Balb/c mice were inoculated intranasally with 20 to 40 uL of bacteria from cultured stocks. A fourth control group was included. Infected animals were observed for 14 days and clinical signs were documented. At the end of period, the mice were sacrificed and lungs harvested and scored for lung lesions. Harvested lungs were either homogenized for dilution plating on agar or stored in fixation solution for histological staining. Results (c): Studies were done using older mice, different dosages of mycoplasmas, and incubating thawed bacterial stocks to allow the organism to recover from their frozen state prior to inoculation. Each of these changes appeared to increase disease severity. In particular, mice infected with UABPO1 strain of mycoplasma developed more severe disease compared with the other tested strains of mycoplasma. Conclusions (d): At this point in our studies, we have seen that UABPO1, in comparison with the strains of mycoplasma, may be the most promising for use in further experiments developing the humanized mouse model. Future experiments will observe bacterial load within infected animals at several different time points since it is possible that bacterial clearance is occurring during by the end of the 14 day time course. As well as repeat studies in immunodeficient and humanized mice.
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    (2014-03) Racine, Ronny R.; Mummert, Mark E.
    CD44 is a cell surface glycoprotein that serves as the major receptor for hyaluronan, 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. We have shown that CD44 expression in Jurkat T cells causes a decrease in proliferation. In our current study, we have observed that CD44 expression greatly increases the influx of calcium from extracellular sources. Calcium influx is necessary for the proliferation of T cells, but CD44 expressing Jurkat cells show a disrupted calcium homeostasis. Through use of calcium channel inhibitors we have shown that Jurkat T cells rely on calcium release activated calcium channels for influx. We have observed that CD44 induced excess calcium influx negatively regulates early growth response protein 1 expression, which is responsible for the decrease in proliferation. Our findings show for the first time that CD44 can influence the calcium signaling of leukemic T cells, impacting their proliferation and potentially making a less aggressive cancer cell. Purpose (a): CD44 is a cell surface glycoprotein that serves as the major receptor for hyaluronan, 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. We have shown that CD44 expression in Jurkat T cells causes a decrease in proliferation. Methods (b): In our current study, we have observed that CD44 expression greatly increases the influx of calcium from extracellular sources. Calcium influx is necessary for the proliferation of T cells, but CD44 expressing Jurkat cells show a disrupted calcium homeostasis. Through use of calcium channel inhibitors we have shown that Jurkat T cells rely on calcium release activated calcium channels for influx. Results (c): We have observed that CD44 induced excess calcium influx negatively regulates early growth response protein 1 expression, which is responsible for the decrease in proliferation. Conclusions (d): Our findings show for the first time that CD44 can influence the calcium signaling of leukemic T cells, impacting their proliferation and potentially making a less aggressive cancer cell.
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    (2014-03) Burnley, Brittney N.; Burnley, Preston I.; Su, Dong Ming; Jones, Harlan P.
    This research aims to identify a better approach in the treatment of bacterial pneumonia. Current treatment uses steroids to decrease tissue damage, however evidence has yet to determine if this treatment leads to increased survival. Our studies uses the stress hormone CRH to enhance this therapeutic approach. Purpose (a): Complications from respiratory pneumonia, mainly caused by Streptococcus pneumoniae (S. pneumoniae), account for the majority of deaths due to respiratory disease worldwide. Treatment with antibiotics is the standard protocol in eliminating S. pneumoniae during infection. However, suppression of inflammatory responses, through glucocorticoids, is also used as an adjunctive therapy during severe infection. Glucocorticoids (GCs) are central mediators of immune suppression and are the primary pharmacologic treatment used to reduce inflammatory responses (IR) in patients with severe bacterial pneumonia. GC treatment however, remains controversial due to inconclusive evidence in reducing mortality amongst at risk populations. Cortisol is an important human GC, whose release is regulated by Corticotropin Releasing Hormone (CRH), a neuropeptide produced primarily by the hypothalamus that mediates adaptive physiological responses. Regulation by CRH is typically involved in controlling immune and IR through cortisol, but has been found to have direct impact within inflamed tissues through ligation with two cellular receptors, CRH-R1 and CRH-R2.Though they provide total immune suppression preventing extensive tissue damage, a problem arises with treatment because they leave the host prone to secondary infections which they are unable to combat due to the actions of the GCs. Therefore, a gap remains in the ability to maintain host immune defenses to facilitate clearance of the pathogen, particularly in instances of ineffective antibiotic treatment caused by bacterial resistance. Therefore, the development of approaches that dampen excessive inflammatory responses without jeopardizing the host may hold promise for reducing risk of mortality due to sepsis. Methods (b): The current study tested the effects of CRH and CRH-R1 antagonist, Antalarmin (ANT) administration, in ICR (CD1) mice subjected to 1 x105 colonies forming units (CFUs) of S. pneumoniae. All experimental groups were compared to the experimental group receiving Dexamethasone (DEX), which is the pharmacological glucocorticoid analogue currently used in clinical settings. Survival studies were conducted to determine the effect that treatment has on overall survival, after treatment at 18 hours. Lung tissue and total blood was analyzed by CFUs to determine bacterial load 24 hours after infection and treatment (18 hours). Additionally lung tissue was analyzed via H&E to determine changes in lung pathology. Results (c): Results of survival indicated that when mice were administered CRH, they had a significant increase in survival when compared to the infection only, ANT, and DEX groups. Conclusions (d): These findings suggest that CRHR1 plays a role in host defenses against pulmonary S. pneumoniae infection and may hold promise as a target to control disease mortality as an alternative approach to glucocorticoids.
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    (2014-03) Joshi, Chaitanya R.; Ghorpade, Anuja
    Purpose (a): More than 50% of the human immunodeficiency virus type 1 (HIV-1) infected individuals exhibit some form of HIV-associated neurocognitive disorders (HAND). Several studies reported that HIV-1 transactivator of transcription (Tat) protein was associated with HAND pathophysiology. HIV-1 Tat induces apoptosis and dysregulates cytokine/chemokine profiles leading to neurotoxicity. Previous studies have studied the in vivo HIV-1 Tat regulation using transgenic animal models. Although animal models have helped determine the in vivo disease pathology, application of in vitro neural cell models will be critical to decipher cellular and molecular mechanisms associated with HAND. Here, we report an in vitro model system developed by transfecting human astrocytes with a full-length (101 AA) HIV-1 Tat protein expressing plasmid (pTat). HIV-1 Tat expressing in vitro system was used to evaluate HIV-1 Tat regulation of astrocyte inflammatory responses and altered neuroprotective function i.e. glutamate uptake from synapse. Methods (b): Primary human astrocytes were transfected with pTat by nucleofection and HIV-1 Tat expression was evaluated by immunocytochemistry. Effects of HIV-1 Tat on cell viability and replication were determined with metabolic activity and cell proliferation assays. Proinflammatory cytokines and chemokines were assayed using ELISAs. HIV-1 Tat regulation of glutamate clearing ability of astrocytes was determined using a modified amplex red glutamic acid/glutamate oxidase assay. Additionally, mRNA and protein expression of excitatory amino acid transporter-2 (EAAT-2), the major glutamate transporter in astrocytes, was measured by RT-PCR and western blot analysis respectively. Results (c): The immunostaining confirmed HIV-1 Tat expression in transfected astrocytes, while glial fibrillary acidic protein (GFAP) staining indicated morphological alterations. The pTat transfection did not significantly change cell metabolism as compared to controls. However, HIV-1 Tat expression altered chemokine and cytokine levels; specifically HIV-1 Tat increased CCL2 levels significantly (P. Conclusions (d): HIV-1 Tat expression upregulated inflammatory biomarkers and altered glutamate clearing ability of astrocytes, implicating a direct role for astrocyte-expressed HIV-1 Tat in HAND neuropathogenesis.
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    (2014-03) Burnley, Preston I.; Su, Dong Ming
    The thymus is a vital organ necessary to fight infections. Unfortunately, it shrinks with age resulting in a compromised function. Our lab uses mice to study the events leading to the shrinkage, hoping to one day develop therapeutic agents to reverse the compromised function. Purpose (a): The postnatal thymic epithelial progenitor (TEP) pool is proposed to be regulated by the p63 and FoxN1 genes through proliferation and differentiation, respectively. However, the combined role of these two genes in the aging TEP is still a mystery. Evidence from murine models has elucidated contrasting roles of the p63 isoforms during the aging process. Methods (b): Wild type and FoxN1 cKO mice were used throughout the experiment to evaluate the amount of p21, p63, and FoxN1 present. Immunofluorescence and senescence staining was performed on frozen sections harvested from euthanized mice. PEI intrathymic injections were performed with either TAp63 or FoxN1 cDNA. Results (c): We found that TAp63+, but not ΔNp63+, thymic epithelial cells (TECs) were increased with age, accompanied with increased senescence associated β-gal clusters and p21+ TECs. Senescent clusters also developed after intrathymic infusion of exogenous TAp63 cDNA into young wild-type mice. Using our conditional FoxN1 gene knockout mouse model to disrupt TEP differentiation accelerated this senescent phenotype to early middle age. However, upon infusion of exogenous FoxN1 cDNA into aged wild-type mice resulted in only an increase in ΔNp63+ TECs, but no change in TAp63+ TECs in the partially rejuvenated aged thymus. Interestingly, using a novel FoxN1 transgenic mouse model to enhance TEP differentiation, ΔNp63+ TECs were decreased in young thymus. Additionally, the TAp63+ population contained a high percentage of phosphorylated-p53 and apoptotic TECs, but showed no changes in BrdU-labeled proliferation. Conclusions (d): FoxN1 controlled TEC differentiation as a bottleneck to determine TEP pool via affecting TAp63 and DNp63 levels. Thus, TEC homeostasis during aging has been determined through the p63-FoxN1 regulatory axis.
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    (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.
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    (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.
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    (2014-03) Mize, Maximillion T.; Simecka, Jerry
    Mycoplasma are bacterial organisms that can cause disease within the airways. Specifically, Mycoplasma pneumoniae causes pneumonia in humans which accounts for 30% of all cases of pneumonia worldwide with 100,000 cases per year resulting in hospitalization. This disease is characterized by a persistent infection, often leading to the initiation of chronic inflammation thus damaging the lungs and respiratory tract. In addition, it can also potentiate the disease severity of other respiratory conditions, such as asthma. Infection is often associated with close-quarter communities (i.e. dormitories, military barracks, etc.) allowing for quick spread of the disease. Furthermore, mycoplasma have also been found in livestock demonstrating that this bacteria has infiltrated important aspects of human society. Due to its resistance to antibiotics, such as penicillin, research has been forced to focus on alternative methods to treating disease. Interleukin-17A (IL-17A), a molecule secreted by immune cells, promotes inflammation and has also been implicated in the development of chronic inflammatory conditions. However, the role that IL-17A plays in chronic airway inflammation associated with Mycoplasma infection is currently unknown. Here, we use a mouse model of human pneumonia to determine if IL-17A contributes to tissue damage associated with disease. We demonstrate that targeting IL-17A with antibodies that block its biological function decreases disease severity within a susceptible BALB/c mouse strain. This alleviation was associated with decreased weight loss and lung damage when compared to identical mice not given antibody treatment. However, this method did not decrease bacterial numbers indicating that IL-17A is only associated with inflammation and not clearance of the organism. Interestingly, the opposite affect was seen in the more resistant C57BL/6 mouse strain leading our lab to believe that a genetic predisposition may be the underlying cause of IL-17A associated airway inflammation. A particular genetic background may dictate the amount of IL-17A released during infection, with high amounts predisposing an individual to severe and irreversible tissue damage, thus initiating a state of chronic inflammation. Purpose (a): Mycoplasma cause 30% of all cases of pneumonia worldwide. Infections induce chronic airway inflammation and has also been associated with the exacerbation of other respiratory diseases, like asthma. Due to a prevalence tied to close-quarter communities (dorms, military barracks, etc.), and the possession of a reservoir in livestock, it has infiltrated human society. Furthermore, the lack of a cell wall prevents this organism from being treated with beta-lactam antibiotics forcing research to focus on alternative treatment methods. While Interleukin-17A (IL-17A) has been linked to the induction of chronic inflammatory diseases, its role in mycoplasmosis is currently unknown. Here, we hypothesize that IL-17A leads to chronic inflammation, exacerbating disease pathogenesis. Methods (b): Female BALB/c and C57BL/6 mice, aged 6-12 weeks, were obtained from HARLAN Laboratory, Inc. Mice were housed in sterile microisolator cages supplied with sterile bedding, food, and water all given ad libitum. The UAB CT strain of M. pulmonis was administered intra-nasally to induce infection. Murine anti-IL-17A (αIL-17A) neutralizing antibody was administered intraperitoneal at a concentration 0.150 mg/mL daily. Results were analyzed via one-way and two-way ANOVA. Results (c): After administration of αIL-17A, BALB/c mice infected with M. pulmonis lost less weight when compared to identical mice given Phosphate Buffered Saline (PBS). In addition, infected mice given antibody displayed a reduction in gross lung lesions, however, bacterial burden was not affected. In contrast, infected C57BL/6 mice given αIL-17A antibodies demonstrated increased disease susceptibility associated with an increase in bacterial burden and gross lung lesions when compared to controls given PBS. Conclusions (d): While the presence of αIL-17A in infected BALB/c mice appears to exacerbate disease, this cytokine may play a protective role in C57BL/6 mice infected with the same pathogen. Lung damage decreased, independent of bacterial burden, in infected BALB/c mice administered antibody. This demonstrates that IL-17A does not play a role in pathogen clearance, but does potentiate chronic inflammation. In contrast, infected C57BL/6 mice given antibody had a slight increase in both lung damage and bacterial numbers indicating that IL-17A is needed to decrease disease pathogenesis. Thus, genotypic differences may play a role in how IL-17A influences an immune response through favoring the development of chronic diseases during bacterial infection.
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    (2014-03) Coder, Brandon; Wang, Hongjun; Su, Dong-Ming
    The thymus is the organ responsible for developing a type of white blood cell called T cells. However, with age comes an increased susceptibility to T cell derived autoimmune disease. Additionally, the thymus progressively shrinks with increased age due to the progressive loss of the gene FoxN1. We want to determine if the loss of FoxN1leads to the thymus being unable to either delete or suppress autoimmune T cells. We utilize a mouse model that has a progressive loss of the FoxN1 gene. Our findings are significant because knowledge gained about the role of the FoxN1 gene with age-related autoimmune disease may lead to novel evidence-based gene therapy that targets the FoxN1 gene to help treat a wide range of autoimmune diseases associated with aging. Purpose (a): The thymus protects against autoimmune disease by generating immunotolernace to self-tissues. This is accomplished through the process of negative selection where self-reactive T cell clones are deleted and also by the generation of natural regulatory T cells (nTregs), which help suppress autoimmunity in the periphery. However, natural aging is associated with thymic atrophy driven by the progressive loss of the gene FoxN1. We wanted to determine if thymic aging impairs immunotolerance, either by disrupting negative selection or altering the generation of suppressive nTreg cells. Methods (b): We answered this question by utilizing a FoxN1 conditional knockout (FoxN1 cKO) mouse model that mimics natural thymic aging through the progressive loss of FoxN1. Results (c): We found that the loss of FoxN1 is associated with the impairment of negative selection characterized by increased single positive T cells and a decrease in Aire+ medullary thymic epithelial cells. Recent thymic emigrants from the FoxN1 cKO thymus have increased proliferation and are more often CD44+, indicating that they are antigen experienced and may be self-reactive T cells. Furthermore, we found that the frequency of nTregs was increased in the FoxN1 cKO thymus, but was normal in the spleen. Additionally, nTregs from the FoxN1 cKO thymus retained normal suppressive function. We adoptively transferred aged wild-type splenocytes, in which there are a higher proportion of Treg cells, into young Rag2-/- mice. We found that the young periphery was able to reverse the accumulation of Tregs. Additionally, the adoptive transfer led to an increase in infiltrating lymphocytes to the salivary gland, which was independent of peripheral age. Conclusions (d): We conclude that the loss of FoxN1 impairs negative selection, which may lead to an escape of self-reactive T cells. However, rather than being cell-intrinsic, the age-related accumulation of Tregs depends on the age of the peripheral microenvironment. These results indicate that the increased susceptibility to autoimmune disease observed with aging is likely due to defects in negative selection rather than changes in nTregs.
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    (2014-03) Carter, KiahRae J.; Orlowski, Ashley; Hodge, Lisa
    Breast cancer is the leading cause of cancer-related deaths. More research needs to be done to examine the role of the lymphatic system during metastasis and therapies directed at the lymphatic system. Our rat model was used because it closely mimics human breast cancer. These results will allow for the future studies of therapies targeting the lymphatic system and if they will prevent metastasis. Purpose (a): Breast cancer is the leading cause of cancer-related morbidity and mortality. New research suggests the lymphatic vessels play a key role during the metastasis of breast cancer and therapies directed at the lymph system may aid in the treatment of breast cancer. MTLn3 is a mammary adenocarcinoma that is commonly used to study the effects of tumor metastasis in Fischer 344 rats. MTLn3 closely mimics human breast cancer pathogenesis, making it ideal for the study of breast cancer disease; however, little is known about the role of the lymphatic and immune systems in this disease model. The purpose of this study was to identify the type of immune response generated during MTLn3 disease. Specifically, we proposed that natural killer cells (NK), T cells, B cells and macrophages (MO) would increase in response to disease. Methods (b): To test our hypothesis, rats were randomized into control group or were sub-cutaneously injected in the right mammary fat pad with 1x106 MTLn3 tumor cells/mL on day 0. At days 0, 7, 14, 21 and 25 post-injection, lungs, tumor-adjacent lymph nodes (ALN), tumor–opposite lymph nodes (OLN) and spleens were removed and the concentration of leukocytes was determined. Primary tumors were excised and measured to calculate tumor volume. Blood was analyzed for the complete blood count and serum was measured for cancer-specific biomarkers. Results (c): All animals gained weight until day 14 post-injection. However, rats injected with MTLn3 suffered weight loss between days 14-25 post-injection. Furthermore, primary tumor size significantly (p < 0.05) increased during this time, suggesting weight loss may be related to disease. CD4+ T cells, B cells and MO in the spleen at day 21 decreased by day 25. Tumor adjacent lymph nodes experienced an increase in all cell populations, T cells, B Cells, MO, dendritic cells and NK. There were no differences in cell populations between ALN and OLN, except MO were significantly (p < 0.05) increased in ALN at Day 25. There was no change in pulmonary leukocytes by day 25. Neutrophils, monocytes and lymphocytes in the blood were significantly (p < 0.05) increased between control and 25 days post-injection rats, suggesting there is an immune response against MTLn3 tumor cells. Conclusions (d): Collectively, our results suggest MTLn3 initiates an immune response mediated by T cells, B cells, macrophages and NK cells between days 14-25 of disease. Of interest, these cells increase in the ALN at day 25 post-injection, suggesting they migrate into the lymph nodes in response to disease. In future studies, we will determine if MTLn3 metastasizes to the sentinel lymph nodes and the lung and determine if therapies targeting the lymphatic system inhibit this process.