Browsing by Subject "Medical Immunology"
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Item A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichia Coli(2002-12-01) Weilbacher, Thomas; Jerry SimeckaWeilbacher, Thomas S., A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichi coli. Master of Science (Microbiology & Immunology), December, 2002, 57 pp., 2 tables, 12 illustrations, bibliography, 44 titles. Small untranslated RNAs (sRNAs) perform a variety of important functions in bacterial systems. The 245 nt sRNA of Escherichia coli K-12, CsrC, was uncovered using a genetic screen for genes that regulate glycogen biosynthesis. CsrC RNA binds multiple copies of CsrA, a protein that post-transcriptionally regulates central carbon flux, biofilm formation, and motility in E. coli. CsrC antagonizes the regulatory effects of CsrA, presumably by sequestering this protein. The discovery of CsrC is intriguing, in that a similar sRNA, CsrB, performs essentially the same function. Both of these sRNAs possess similar imperfect repeat sequences (18 in CsrB, 9 in CsrC), primarily localized in the loops of predicted hairpins, which may serve as CsrA binding elements. Transcription of csrC increases as the culture approaches the stationary phase of growth and is activated by CsrA and the response regulator UvrY. Complementation and in vitro transcription-translation experiments reveal that CsrA effects on csrC are mediated indirectly, through UvrY. Because CsrB and CsrC antagonize the activity of CsrA and are dependent on CsrA for their synthesis, a csrB null mutation causes a modest compensatory increase in CsrC levels and vice versa. An updated model for the signaling circuitry of the Csr system is discussed.Item Aging of the Thymic Epithelial Progenitor Pool is Determined by the p63-FoxN1 Regulatory Axis(2015-05-01) Burnley, Preston I.; Dong-Ming SuThe immune system is composed of various effector cells and molecules that must work in concert in order to protect the body against infections, auto-reaction, and tumor occurrence. These responses can be divided into two categories – innate and adaptive immunity. The innate response is the host’s first line of defense towards a pathogen by providing a physical and chemical barrier against infection. Once activated, innate cells such as macrophages and dendritic cells can engulf the bacterium, degrade it, and secrete proteins to destroy the pathogen. Although this response occurs immediately after an encounter with a pathogen, the innate immunity is neither long-lasting nor specific. In contrast, the adaptive immune response is initiated when the innate immune response is unsuccessful in eliminating the infection, allowing for recognition and response tailored for a particular pathogen. The cells that make up the adaptive response all originate from a common lymphoid progenitor found in the bone marrow. From this precursor arise natural killer (NK) cells (part of the innate response) and T and B lymphocytes. The T lymphocytes originate from the bone marrow but undergo development in the thymus, hence the name T cells. B lymphocytes, on the other hand, originate and develop in the bone marrow. With the exception of NK cells, these adaptive immune cells require antigen presentation in order to become activated. Once the T and B cells have matured and become activated they can work together to clear the infection by secreting cytokines and antibodies. The most important aspect of the adaptive immune response is its ability to produce immunological memory. Memory T and B cells are able to ensure a rapid and effective response to a second encounter, providing long-last immunity. Unfortunately, this well-ordered process, specifically the development of T cells, becomes compromised during aging. This is due to the fact that thymic involution (or shrinking of the thymus) occurs at the onset of puberty and continues throughout the lifespan, which is primarily resulted from age-related defect in thymic epithelial cells (TECs). The thymus is crucial for the generation of T cells so any compromise to the organ results in changes in the T cells, which can possibly lead to immune insufficiency and autoimmunity [1]. Additionally, these conditions are exacerbated with age [2, 3]. This research project will focus on the molecular mechanism(s) responsible for thymic involution. To do so, we focused on TECS and two genes associated with the homeostatic maintenance of the thymic microenvironment, p63 and FoxN1. These genes regulate the proliferation and differentiation, respectively, of thymic epithelial cells (TECs), thereby maintaining a properly functioning thymus. For this study we will utilize our mouse model (FoxN1 conditional knockout, FC) extensively because it mimics an aged thymus. This model allows us to study the thymic microenvironment of a mouse with a defect in the FoxN1 gene.Item Anti-Tumor Immune Responses Against MTLn3 Mammary Adenocarcinoma(2014-05-01) Carter, KiahRae J.; Hodge, Lisa M.Lymphatic pump treatment (LPT) is used as a lymph enhancing therapy to treat edema. In animals, LPT enhanced lymphatic flow, released leukocytes and inflammatory mediators into lymph, and inhibited pulmonary tumor formation. Therefore, we hypothesized the administration of LPT would enhance immunity and inhibit primary breast tumor growth. Rats were subcutaneously injected with MTLn3 and divided into MTLn3, MTLn3+Sham-LPT and MTLn3+LTP group. Sham group received light touch under anesthesia and LPT group received treatment under anesthesia. There were no changes in tumor growth between groups. Administration of Sham-LPT resulted in an increase in tumor-adjacent lymph node weight. Collectively, our data suggests LPT did not enhance primary tumor growth and may also protect against the pathogenesis exhibited by sham-LPT.Item Approaches to Cloning and Identification of the Ligand for Natural Cytotoxicity Receptor NKp44(2008-07-01) Horton, Nathan C.; Harlan Jones; Stanley Stevens; Raghu KrishnamoorthyHorton, Nathan C., Approaches to Cloning and Identification of the Ligand for the Natural Cytotoxicity Receptor, NKp44. Masters of Science (Microbiology & Immunology), July 2008, 64 pp., 22 illustrations, 37 titles. Natural Killer (NK) cells represent a specialized lymphoid population that mediate innate immune responses against tumor or virally infected cells. NK cell cytotoxicity is regulated by inhibitory and activating receptors. Activating receptors include the Natural Cytotoxicity Receptors (NCRs), 2B4, and NKG2D. The NCRs play a key role in recognition and killing of tumor cells and include the receptors NKp30, NKp46, and NKp44. The ligands for the NCRs are not yet known. NKp44 is of particular interest because it is only expressed on activated NK cells, and is implicated in increased cytotoxicity and HIV infection. To identify and clone the ligand for NKp44, a recombinant fusion protein containing the extracellular domain of NKp44 was constructed and used to identify a cell line, DB, expressing a ligand for NKp44. A directional complimentary DNA (cDNA) library was constructed from this cell line and screened by mammalian expression cloning, resulting in the isolation of several putative cDNA clones of NKp44 ligands.Item Characterization of MRSA Infection at Childrens Medical Center, Dallas, January 2005-June 2005(2006-05-01) Okoro, Ngozi M.; Raghbir Sandhu; Claudia S. Coggin; Sejong BaeOkoro, Ngozi M., Characterization of MRSA infection at Childrens Medical Center, Dallas, January 2005-June 2005. Master of Public Health (Epidemiology), May 2006, 33p., 14 tables, 10 illustrations, bibliography, 13 titles. MRSA infection is increasingly emerging in patients without the established risk factors hence the term CAMRSA. This study is a descriptive secondary data analysis from an ongoing study at UTSM/CMCD and describes the CMCD patients with MRSA infection. Data analysis showed a consistent increase in the incidence rate of the infection with slight female preponderance. Race distribution showed that blacks were the majority. Most children were less than 2years, used Medicaid, had superficial infections and community-acquired infections. All (100%) isolates were susceptible to Vancomycin and Linezolid while many (92.2%) were resistant to Erythromycin. The increasing incidence in CAMRSA infection remains a challenge for public health professionals and the resistant pattern a potential problem to the pharmaceuticals.Item Diverse Immune Responses Mediated by Beta-Adrenergic and Corticotropin-Releasing Hormone Receptors in a Model of Pneumococcal sepsis(2010-08-01) Kim, Byung-Jin; Harlan JonesNeuroendocrine stimulation can impact disease states by regulating immune function. The purpose of our studies was to define the functional role of stress-induced neuroendocrine factors, catecholamines and corticotropin-releasing hormone (CRH) on immune responses involved in the pathogenesis of Streptococcus pneumoniae (S. pneumoniae) infection implementing both in vitro and in vivo methodology. Dendritic cells play a pivotal role in antigen presentation and cytokine production, influencing both innate and adaptive immunity. Initial studies examined the potential immunomodulatory effect of epinephrine and CRH on DC cytokine production in response to the bacterial pathogenic ligand, lipopolysaccharide (LPS). In addition, the ability of DC to dictate CD4+ T cell activation as a consequence of CRH or epinephrine pre-treatment was examined using an in vitro co-culture system. Epinephrine and CRH pre-treatment resulted in a preferential increase in IL-23 and IL-10 cytokine production. In contrast, IL-12p70 was significantly attenuated in response to epinephrine and CRH pre-treatment. Preferences in IL-23 and IL-10 cytokine production by DC pre-treated with epinephrine and CRH corresponded with an increase in IL-4 and IL-17A, but not IFN-y cytokine production by CD4+ T cells. These results suggest that exposure to stress-derived epinephrine/CRH dictates dendritic cells to generate a dominant Th2/Th17 phenotype in the context of subsequent exposure to a pathogen. Our second study examined the functional properties of IL-23 during pulmonary S. pneumoniae infection. IL-23 plays a crucial role in establishing host defenses against extracellular pathogens. Further investigation is still required to define the impact of IL-23 on acute pulmonary S. pneumoniae infection. Utilizing IL-23p19 genetic deficient mice, we determined bacterial load, cytokine production and the contribution of neutrophils against S. pneumoniae infection using monoclonal antibody-mediated systemic neutrophil depletion. The absence of IL-23 induced a higher bacterial load in lung and blood as compared to IL-23 competent counterparts. In the absence of IL-23, production of proinflammatory cytokines such as IL-6, IL-12p70 as well as IL-17A and IFN- were dampened as compared to wild type mice. In addition, neutrophil distribution was also altered in IL-23-deficient mice, suggesting impaired neutrophil recruitment into lung. Interestingly, neutrophil depletion did not impact bacterial load in lung and blood in both IL-23 competent and deficient mice. These findings, suggest a novel role of IL-23 in pulmonary S. pneumoniae infection, potentially independent of neutrophil function. We next examined the possible impact of CRH and catecholamines as regulators of immune function against acute bacterial infection in response to stress. Utilizing a murine model of acute pulmonary S. pneumoniae infection and restraint stress, we selectively blocked CRH receptors (CRHR1 and CRHR2) as well as the 2 adrenergic receptor prior to restraint stress followed by intranasal pulmonary S. pneumoniae infection. Antagonist administration did not impact restraint stress-induced physiological responses as compared to restraint stressed mice, which did not receive receptor antagonists. However, following S. pneumoniae infection, physiological changes including weight and temperature were altered in response to administration of selective CRH receptor and β2 adrenergic receptor antagonists. Survival rate, bacterial load and cytokine production corresponded with physiological differences observed in response to selective CRH receptor and 2 adrenergic receptor antagonists. Importantly, preferential differences in bacterial colonization and survival corresponded with distinct differences in inflammatory cytokine production and immune cell distribution along pulmonary airways. In particular, opposing effects in IL-17A and neutrophil accumulation was found among mice administered the CRHR1 versus the CRHR2 antagonists. Together, these findings indicate that activation of each receptor can influence immune responses against S. pneumoniae infection. Thus, our findings provide further understanding of how stress-derived neuroendocrine factors directly impact immune responses related to immunopathology and immunoprotection.Item Epidemiologic Assessment of a Targeted Tuberulosis Screening and Treatment Program Based on Geographic and Molecular Clustering(2005-08-01) Moonan, Patrick KevinMoonan, Patrick K., Epidemiologic Assessment of a Targeted Tuberculosis Screening and Treatment Program Based on Geographic and Molecular Clustering. Doctor of Public Health (Disease Prevention and Control), August 2005, 93 pp., 12 tables, 7 illustrations, bibliography, 148 tables. One of the primary goals of the tuberculosis elimination strategy is to interrupt the transmission of mycobacterium tuberculosis (TB). The most effective way to accomplish this goal is to identify and treat individuals who have active tuberculosis. However, even in highly effected tuberculosis control programs, M. tuberculosis continues to be transmitted to others, largely because most transmission occurs before diagnosis and initiation of therapy. Under the current recommendations, testing should be targeted at specific high-risk populations. While a strategy of targeted testing and treatment of persons most likely to develop tuberculosis is attractive, it is uncertain how best to accomplish this goal. This is the first study to assess the use of geographic and molecular surveillance in guiding a targeted tuberculosis screening and treatment of active tuberculosis and latent tuberculosis infection that monitors potential transmission in a defined high risk geographic area. The results of this geographically targeted program demonstrate significant yield for discovering active cases, latent tuberculosis infection, and recent transmission (TST converters). In this setting, geographically targeted screening identified as many as 19.8 tuberculosis cases per 1,000 persons screened and as many as 292.4 latent tuberculosis infections per 1,000 persons screened. Additionally, successful treatment of these individuals reduced the number of both cases and latent infection identified. Over a three-year period the case detection rate, latent infection detection rate, and TST conversion rate was reduced by 335%, 171% and 285% respectively.Item Immune and Inflammatory Responses Differ Between the Upper and Lower Respiratory Tract(2001-05-01) Hodge, Lisa M.; Simecka, Jerry; Goldfarb, Ronald H.; Mathew, Porunelloor A.The purpose of these studies was to evaluate the role of upper and lower respiratory immune responses during immunization against respiratory disease antigens, and to characterize which immune responses during immunization against respiratory disease antigens, and to characterize which immune responses contribute to protection in the respiratory tract during infection. After nasal immunization, antigen-specific IgA antibody forming cells dominated throughout the respiratory tract. However, IgG responses were significant in lungs, but not in nasal passages. Furthermore, parental immunization did not enhance humoral immunity in the upper respiratory tract even after a nasal challenge, whereas extrapulmonary lymphoid responses enhanced responses in the lung. After nasal immunization, inflammatory reactions developed within the lungs of mice, but not in nasal passages. Lowering dosages of CT reduced, but did not eliminate, these adverse reactions without compromising immunogenicity. Serum IgE responses were also enhanced in a dose dependent manner by inclusion of CT. During infection, mRNA expression for IL-4 was greater in the nasal passages, while both mRNAs for IL-4 and IFN-y were increased in the lungs. As well, we found increased mycoplasma organisms in the lungs of IFN-y-/- mice, suggesting a protective role for cell-mediated immunity in the lung. In contrast, IL-4-/- mice had greater mycoplasma organisms in the nasal passages, indicating IL-4 responses are crucial for upper respiratory tract protection. Consistent with antigen deposition, nasal inoculation with 10 μl volume of antigen plus CT resulted in significant IgA responses in the nasal passages compared to mice given 24 μl immunizations; however, lower respiratory tract immunizations generated antibody responses in both nasal passages and lungs. In addition, both immunizations resulted in equivalent serum antibody responses. Upper and total respiratory tract immunizations provided protection in the nasal passages when CT was added. However, in the lung, all immunizations resulted in protection against mycoplasma infection, regardless of the inclusion of CT, suggesting a different role for CT as an adjuvant in upper and lower respiratory tract immune protection. In conclusion, we found immune responses generated during immunization and infection are different between the upper and lower respiratory tracts, and the contribution of these responses to clearance of respiratory infection differs.Item Molecular Basis for 2B4-CD48 Interactions(2001-08-01) Huynh, Van T.; Mathew, Porunelloor A.; Goldfarb, Ronald; Das, HridayHuynh, Van T., Molecular Basis for 2B4-CD48 Interactions. Master of Science, Molecular Biology and Immunology, August 2001, 93 pp., 3 tables, 19 illustrations, bibliography, 51 titles. Natural killer cells are lymphocytes that play a role against cancer and viral infections. 2B4 is a membrane glycoprotein expressed on natural killer cells. In the present study we characterized 2B4 from mice strains BALB/c, 129/svj and A.CA. Nucleotide and peptide analysis revealed that polymorphyic residues in 2B4 are located in the variable domain. My second project was to determine the amino acids involved in the binding between 2B4 and CD48. Twelve mutations were made in human 2B4 to disrupt their interaction. In the last part of the study, an attempt has been made to elucidate the role of tyrosine and threonine amino acids found in the novel tyrosine motifs (TxYxxI/V) that reside in the cytoplasmic domain.Item Predictors of Complicated Staphylococcus Aureus Bacteremia: A Retrospective Validation Study(2008-04-01) Krishnamurthy, Pramod; Fischbach, Lori; Cardarelli, Roberto; Coggin, Claudia S.Krishnamurthy, P., Predictors of Complicated Staphylococcus aureus Bacteremia (SAB): A Retrospective Validation Study. Master of Public Health (Epidemiology), April 2008, 57 pp, 9 tables, 1 illustration, bibliography, 39 titles. SAB often has a complicated clinical course and it is important to identify those at risk for complications to guide management. We conducted a validation study of a clinical prediction tool that uses a scoring system to predict the likelihood of developing complicated SAB. Chapter I is a review of background literature and rationale for our study. Chapter II has sections describing the study design, methods, eligibility criteria, statistical analysis and a summary of the results. We observed significantly higher complications among patients with SAB in our validation study. The prediction tool is not a valid predictor of complicated SAB and we recommend better prediction models to accurately predict complications of SAB.Item Setting Us Up to Fail: Pulmonary Dendritic Cells Promote Immunopathology during Mycoplasma Respiratory Disease(2010-08-01) Dobbs, Nicole A.; Jerry SimeckaThe purpose of these studies was to define the contributions of T helper 2 cells and dendritic cells toward the development of immunopathology during mycoplasma respiratory disease. IFN-γ+ CD8+ T cells, IFN-γ+ Th1 cells and IL-13+ Th2 cells developed over the course of mycoplasma infection. By day 14 post-infection, the results demonstrated a significant and preferential increase of an IL-13+ Th2 cell sub-population in the LRNs. Additional studies using STAT4-/- animals, which have a Th2 polarized environment, demonstrated no difference in disease compared to the wild-type animals. Absence of STAT6, which strongly contributes to a Th1 polarized environment, conveyed significantly more protection from mycoplasma disease in immunized mice compared to STAT4-/- and WT mice. By day 14 post-infection, all mice had significantly more IL-13+ Th2 cells than IFN-γ+ Th1 in the LRN compared to STAT6-/- immunized mice, thus suggesting that the reduction in the IL-13+ Th2 population leads to protection, while an increase in Th2 is pathogenic. Additional studies demonstrated that pulmonary dendritic cells support the mycoplasma-specific CD4+ and CD8+ T cell activation when stimulated with mycoplasma antigen. Knowing that T cells and DCs have an intimate relationship during mycoplasma disease, sub-classes of cytokine differentiated BMDCs were created to attempt to skew to the protective arm of immunity against mycoplasma disease. However, in vivo adoptive transfer studies demonstrated antigen pulsed DCs accelerated and exacerbated the pathological effects of mycoplasma disease. The exacerbation was antigen-specific and lymphocyte dependent. Mice that received antigen pulsed DCs demonstrated a significant increase in IL-13+ Th2 cell sub-population in the LRNs with a similar trend found in the lungs prior to infection. The same exacerbation was seen when antigen pulsed pulmonary DCs were adoptively transferred into mice, but not with antigen pulsed splenic DCs. Prior to infection, mice that received antigen-pulsed pulmonary DC, not splenic DC, had a significant increase in a IL-13+ Th2 population in the LRNs. Taken collectively, these studies demonstrate two key players in the development of the detrimental response against mycoplasma disease. This knowledge will assist in the development of targeted vaccines that will promote protection over pathology.Item Studies of Protein F1 (GAP-43) Expression and Function in Spinal Neuronal Cultures(1994-08-01) El-Badawy, Hassan M.E. Azzazy; Ming-Chi Wu; Guenter W. Gross; Scott NortonEl-Badawy, Hassan M. E. Azzazy, Studies of Protein F1 (GAP-43) Expression and Function in Spinal Neuronal Cultures. Doctor of Philosophy (Biochemistry and Molecular Biology), August 1994, 167 pp., 32 illustrations, References, 194 titles. Protein F1 (GAP-43, B-50, neuromodulin) is a membrane-bound phosphoprotein that has been studied mainly in neurons and is implicated in synaptic plasticity, axonal growth and regeneration, and neurotransmitter release. In this study, a 21 amino acid polypeptide that corresponds to the C-terminus sequence of protein F1 and contains a potential PKC phosphorylation sequence (SXR) was synthesized. The synthetic peptide was phosphorylated by rat PKC in a concentration-dependent manner suggesting that this site in the intact protein may be phosphorylated by PKC in vivo. Polyclonal antibodies against the peptide were produced in a rabbit and used to: (i) recognize native non-phosphorylated protein F1 purified from rat brain, (ii) immunoprecipitate phosphorylated protein F1, and (iii) stain the cell bodies and neuritis of cultured neurons. Electron microscopic studies revealed intracellular protein F1 immunoreactivity but no specific subcellular association of the gold label could be demonstrated. The antibodies were also used to compare protein F1 levels during the development of spinal neurons in culture and in vivo. The highest levels of protein F1 were detected by ELISA, at 2 days in culture. These results are in accordance with previous reports that correlate high expression of protein F1 to neurite outgrowth. In vivo, however, protein F1 reached maximal level at one day after parturition. Two approaches were utilized to investigate the potential physiological functions of protein F1 in spinal neurons networks. First, interaction of positively charged, rhodamine-labeled liposomes with spinal neurons was characterized by fluorescence microscopy and electrophysiological recording. Uniform, non-toxic, and preferential interaction of liposomes with spinal neurons over glia was established. These liposomes were used to deliver anti-protein F1 antibodies into spinal neurons but did not affect neurite formation by these cells. Second, antisense oligodeoxynucleotides internalized into spinal neurons in order to interfere with protein F1 expression had no effect on the development of these cells in culture. Data from this study suggest that Ser-210 at the C-terminus of protein F1 may be a substrate for PKC phosphorylation in vivo. Antibodies raised against F1 peptide revealed protein F1 immunoreactivity that outlined cell bodies and neuritis of cultured spinal neurons. Positively charged liposomes were characterized as a potential delivery system for macromolecules into spinal neurons. Protein F1 levels were shown to be developmentally regulated in mouse spinal neurons in culture and in vivo. Finally, the use of antisense oligodeoxynucleotides against protein F1 mRNA revealed that protein F1 may not be essential for neurite outgrowth of mouse spinal neurons in culture.Item Synergy 2011: Annual Research Report(2011-01-01)Item T-Helper Cell Responses in Lungs After Immunization and Chronic Respiratory Disease; And Their Association With Pulmonary Inflammation(2001-05-01) Jones, Harlan P.; Simecka, Jerry; Dimitrijevich, S. Dan; Goldfarb, Ronald H.The purpose of these studies was to characterize T helper cell responses in the lungs of mice after immunization and chronic respiratory infection. CD4+ T cells were the major population of T cells resident in the lung in comparison to CD8+ T cells. Polyclonal activation of resident CD4+T cells produced abundant levels of IL-4 in comparison to IFN-γ, indicating that Th2 cells were the major sub-population of CD4+ T cells. In contrast, resident CD8+ T cells were the sole producer of IFN-γ by naïve T lymphocytes. Furthermore, the distribution of T cells was similar between BALB/c, C3H/HeN, C57BL/6 and DBA/2N strains of mice. However differences in the distribution of CD8+T cells, as well as the levels of IL-4 and IFN-y production produced by resident T cells were found between C57 and the other strains of mice tested. These results demonstrate that host genetic factors may be involved in determining host susceptibility to respiratory disease. Differences in the intensity of antigenic stimulation provoke changes in the type of T cell response generated. Intranasal immunization with influenza (FLU) vaccine antigen alone initiated solely an antigen-specific Th2-like response. In contrast, the addition of the potent mucosal adjuvant cholera toxin (CT) in combination with FLU antigen induced not only resident Th2 responses, but also induced antigen-specific Th1-like responses. This change corresponded with a dramatic increase in the number of CD4+ T cells in the lung. Thus, intense immunization of respiratory T cells enhanced resident T helper cell responses, but also promoted the activation of Th1 responses. Chronic respiratory infection also elicited changes in the resident population of T cells consistent with pulmonary inflammatory immune responses. At early stages of infection, CD4+, but not CD8+ T cells increased in number within inductive respiratory lymphoid tissues (lower respiratory nodes [LRNs]). Between day 7 and 14 however, there was a dramatic increase in the number of CD4+ T cells in the lung. Interestingly, CD8+ T cells also increased in the lungs, suggesting their activation along mucosal sites during mycoplasma infection. Mycoplasma-specific IL-4 and IFN-γ production also increased in a tissue-specific/time-dependent manner. IL-4 production was initially observed in the LRNs, whereas significant levels of IL-4 and IFN-γ was produced in both tissues 14 days after infection. In comparison, IFN-γ was the predominate cytokine, produce at 14 days coinciding with pulmonary inflammation. Suggesting that intense activation promoted changes in the resident pulmonary Th2 environment, and possible is a major component of pulmonary inflammatory immune responses. Both CD4+ and CD8= T cells were shown to have a role in modulation of disease severity during mycoplasma disease. Observation of gross pulmonary lesions reveal that mycoplasma infected mice treated with anti-CD8 antibody showed increase clinical signs of disease and pronounced gross pulmonary lesions. Additionally the number of total mononuclear cells increased dramatically in the absence of CD8+ T cells. Thus, CD8+ T cells may have a regulatory role in controlling resident CD4+ T cells that increased 14 days after infection. Chemokine production is known to mediate the recruitment of lymphocytes to enhance the initiation of immunity as well as be responsible for modulating inflammatory responses. We find that mycoplasma increase the number of dendritic cells in the lung 14 days after infection, and stimulated the production of dendritic cell-derived ABCD-1 chemokine. Also, β-chemokine MIP-1α and MIB-1β production was observed during intense immunization as well as during mycoplasma infection. These results provide evidence for a potential mechanism through which changes in resident pulmonary T cell responses occur given the intensity of the immune response generated.Item The Characterization of T Cell Responses Along the Respiratory Tract(2004-05-01) Ivey Jr., James A.; Brown, Mary B.; Fling, John; Wu, Ming-ChiIvey, James A., The Characterization of T Cell Responses Along the Respiratory Tract. Masters (Immunology). May, 2004. 63 pages. 2 tables, 10 figures. 55 titles. This research converged on T cell responses to respiratory agents, tobacco smoke and allergens in humans, and T cell responses to an extracellular bacterium, M. bovis, in calves. In the first chapter, we will discuss the basic functions of the immune system. In the second chapter, we will discuss the basic functions of the immune system. In the second chapter, we characterized the impact of tobacco smoke on T cell responses in atopic smokers and atopic nonsmokers. The results were inconclusive and the study was postponed until the proper allergens were found. We also characterized the T cell responses of calves infected with Mycoplasma bovis in the third chapter. The results showed an increase in T lymphocytes in the upper respiratory tract of infected calves, which correlated with sites of infection.Item The Effect of CsrA on Biofilm Development in Escherichia coli(2001-05-01) Jackson, Debra White; Julian Borejdo; Richard Easom; Jerry SimeckaJackson, Debra W., The Effect of CsrA on Biofilm Development in Escherichia coli. Doctor of Philosophy (Biomedical Sciences), May 2001, 127 pp., 2 tables, 15 illustrations, bibliography, 138 titles. CsrA, carbon storage regulator, is a small RNA-binding protein that acts as a global regulator and modulates specific mRNA stability in Escherichia coli. CsrA regulates central carbon metabolism in addition to flagella biogenesis. In this study, the phylogenetic distribution of csrA and its role in Escherichia coli biofilm development were examined. CsrA homologs were examined using Southern hybridization experiment and by analyzing existing sequencing data and was found to be widespread among eubacteria. CsrA was shown to be capable of acting as a genetic switch for biofilm formation and dispersal. A csrA mutant of E. coli was shown to increase biofilm formation and exhibit apparent pillars and channels characteristic of a mature biofilm. Over-expression of csrA completely inhibited biofilm formation in E. coli K-12 and decreased biofilm formation in related enteric pathogens. Induction of csrA expression from a multicopy plasmid caused dispersal of a pre-formed biofilm. Gene expression studies revealed that csrA expression is dynamically regulated during biofilm formation. Several outer-membrane factors and global regulators that have been implicated in biofilm formation were examined for effects on biofilm formation in a csrA mutant. Crystal violet adherence assays revealed that flagella and type I pili affect biofilm formation in a scrA mutant strain; however, colonic acid and curli fimbriae did not exhibit quantitative effects on biofilm formation in the csrA mutant, but the stationary phase sigma factor, RpoS, had no quantitative effect on csrA mutant biofilm formation. Therefore, a csrA mutant will form a biofilm in the absence of each of these outer-membrane factors and global regulatory factors of biofilm formation. The effects of csrA on biofilm formation were found to be mediated in part through its effects on intracellular glycogen metabolism. Thus the redirection of carbon flux, in response to environmental and/or physiological cues, is important for biofilm development.Item The Functional Role of Human 2B4 (CD244) Isoforms in Natural Killer Cells(2007-05-01) Rao, Krithi K.; Porunelloor Mathew; Rance Berg; Harlan JonesRao, Krithi K., Functional role of human 2B4 (CD244) isoforms in natural killer cells. Master of Science (Immunology), July, 2007, 66 pp., 15 illustrations, bibliography. Natural killer (NK) cells are a subpopulation of lymphoctyes that play an important role against tumor metastasis and various viral and bacterial infections. NK cell functions are controlled by a balance between positive and negative signals through various receptors. We have identified, cloned, and characterized the 2B4 (CD244) receptor in mice and human. 2B4 is involved in killing cancer cells and virus-infected cells by NK cells. 2B4 is involved in killing cancer cells and virus-infected cells by NK cells. 2B4 is a counter-receptor for CD48 and recent findings show that 2B4-CD48 interactions plan an important role in NK, T and B cell functions. In humans, two isoforms of 2B4, h2B4-A and h2B4-B, are expressed that differ in the extracellular domain. In the present investigation, we have studied the functions of h2B4-A and h2B4-B. Our data demonstrate that these two isoforms differ in their binding affinity for CD48, resulting in differential cytolytic function as well as cytokine production by NK cells. Thus, differential expression of 2B4 isoforms by NK cells may regulate immune responses mediated through 2B4-CD48 interactions.Item The Role of Calcium in Light-Induced Photoreceptor Apoptosis(2002-05-01) Krueger, Darrel Scott; Collier, Robert; Agarwal, Neeraj; Wordinger, Robert J.Krueger, Darrel Scott, The Role of Calcium in Light-Induced Photoreceptor Cell Apoptosis. Doctor of Philosophy (Biomedical Sciences), May, 2002; 305 pp., 15 tables, 31 illustrations, bibliography, 421 titles. Retinal degenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) result in loss of photoreceptors by apoptosis. Photo-oxidative stress accelerates the rate of photoreceptor apoptosis in models of retinal dystrophies. Thus, a better understanding of light-induced apoptosis is important for developing preventative and treatment options for persons with these blinding diseases. The goals of this dissertation were to investigate the effect of photo-oxidative stress on [Ca2+]I in rat photoreceptor cells and determine whether cell death could be prevented by altering cells’ abilities to manage [Ca2+]i. Thirty minutes of light exposure resulted in significant elevation of [Ca2+]i, determined using Fura-2 ratiometric imaging, which increased with additional light exposure. At 120 minutes, the F340/380 ratio was 3-times the beginning baseline ratio. Using multiple techniques indicative of early and late phases of apoptosis, changes consistent with apoptosis were observed, including early labeling (30 Min) with Annexin-V, activation of caspase-3 (2IIr), TUNEL labeling and Propidium Iodidc staining. TUNEL and Propidium labeling were more intense at 3Hrs light exposure, reflecting late phase changes. Apoptosis was confirmed using electron microscopy (TEM). TEM showed mitochondrial swelling, indicative of permeabilization, prior to chromatin condensation. Pharmacological agents were utilized to mediate participation of cellular calcium sources or storage sites in the maintenance of intracellular calcium homeostasis during photo-oxidative stress. Agents were separately added to the media prior to light exposure and their effects on cell viability were assessed using the Formazan assay. Mitochondria were confirmed to be the site of action affected by elevated [Ca2+]I, since prevention of calcium uptake by ruthenium red (1-100-μM) provided significant protection of cell viability in a dose-related manner. The 100- μM concentration resulted in complete maintenance of viability. BAPTA-AM also demonstrated some protection (50%) indicating that reduction of [Ca2+]I independent of source is beneficial is maintaining cell viability. Identification of mitochondrial uptake and cytosolic buffering of calcium as key viability determinants in light-induced apoptosis is a significant discovery for targeting future research for preventing or inhibiting photoreceptor cell apoptosis associated with retinal dystrophies such as RP and AMD.Item uPAR Interaction and Regulation of Natural Killer Cell Integrins: Implications for the Modulation of NK Cell Migration and Invasion(2003-05-01) Gellert, Ginelle C.; Goldfarb, Ronald H.; Roque, Rouel; Hart, MarkGellert, Ginelle C. uPAR Interaction and Regulation of Natural Killer Cell Integrins: Implications for the Modulation of NK Cell Migration and Invasion. Doctor of Philosophy (Biomedical Sciences), May 2003; pp. 118, 2 tables; 12 figures; bibliography 163. The urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored receptor, devoid of an intracellular domain, but nevertheless initiates signaling, possibly through lateral interactions with integrins. Since adoptively transferred interleuking-2 (IL-2) activated natural killer (A-NK) cells can accumulate within established cancer metastases, these A-NK cells may integrate components of adhesion and proteolysis to facilitate their infiltration into tumors. The work in this dissertation investigates the hypothesis that uPAR directly interacts with and regulates the expression of integrins on the surface of NK cells in the potential modulation of NK cell migration and invasion. Crosslinking studies have revealed a relationship between the integrins and uPAR on the surface of the human NK cell line, YT. Crosslinking uPAR, which mimics uPAR clustering at focal adhesion sites, caused an increase in the expression of the αM, αv and β2 integrins. Although uPAR is GPI-linked to the plasma membrane and has no direct means of initiating intracellular signaling, crosslinking uPAR activated the MEK/ERK signaling cascade, as phosphorylation of both MEK ½ and ERK ½ occurred following receptor clustering. The MEK-specific inhibitors PD98059 and U0126 blocked MAP kinase phosphorylation, and PD98059 inhibited the increase in integrin expression induced by uPAR crosslinking. Furthermore, the binding of urokinase plasminogen activator (uPA) to uPAR also activated the MEK/ERK signaling pathway. Fluorescence microscopy revealed the cocapping of uPAR with the αv integrin, a process inhibited N-acetyl-D-glucosamine, which abrogates the lectin-like interactions that have been suggested to exist between uPAR and integrins. The work presented herein indicated that signaling initiated either by uPAR crosslinking, leading to increased integrin surface expression, or by uPAR occupancy with uPA may depend on the physical association of uPAR with integrins. These studies will enhance our understanding of the mechanisms utilized by NK cells for their adhesion to tumor vasculature and accumulation within established cancer metastases, thereby potentially identifying targets for enhancing their effectiveness during adoptive immunotherapy.