Browsing by Subject "Other Immunology and Infectious Disease"
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Item A Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes Required for DNA Damage-Induced Mutagenesis(2008-07-01) Gong, Jinjun; Siede, Wolfram; Sheedlo, Harold; Reeves, RustinA Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes required for DNA Damage-Induced Mutagenesis. Jinjun Gong Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107. Summary. Deoxyribonucleic acid (DNA) damage is common in a cell’s lifetime. DNA can be damaged by endogenous factors such as reactive oxygen species (ROS) or exogenous agents such as ultraviolet (UV) or industrial chemicals. DNA damage will trigger cell responses including cell cycle arrest, transcription activation, DNA repair or apoptosis. In addition to various DNA repair mechanisms including damage reversal, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining, translesion DNA synthesis is an important DNA damage tolerance pathway that can bypass the lesion on template DNA to finish the replication for cell survival but at the risk of potential mutation in the daughter cells. Accumulation of mutation may lead to cancer occurrence. Translesion DNA synthesis components are highly conserved from yeast to humans. Important players in trans-lesion synthesis pathway such as Rev1, Rev3 and Rev7 were first discovered in budding yeast. Saccharomyces cerevisiae. Homologues were found later in human cells. I used the Saccharomyces cerevisiae deletion mutant collection to do a systematic screen to search for novel genes required for DNA damage induced mutagenesis in yeast. After CAN1 forward mutation assay for the systematic screen and reverse mutation assay for further confirmation, two candidate genes SWI6 and DOA4 were detected. Deletion of SWI6 and DOA4 decreases mutagenesis of cells. At the molecular level, Swi6, a transcription cofactor, is involved in mutagenesis by regulating expression of REV7 at the mRNA and protein levels. Rev7 is a regulatory subunit of DNA polymerase zeta, which is essential for DNA damage induced mutagenesis as well as spontaneous mutagenesis. Rev7 is not UV inducible or cell cycle regulated. The regulation of Rev7 at the transcriptional level by Swi6 is essential. Future experimental approaches are planned to address the mechanism by which DOA4 is involved in mutagenesis.Item Characterization of a Novel Receptor CS1 in Human Lymphocytes; Studies in Natural Killer Cells and B-Lymphocytes(2005-06-01) Lee, Jae Kyung; Porunelloor Mathew; Ming-Chi Wu; Hriday DasThe purpose of this study was to investigate the roles of CS1 on human lymphocytes. The molecular and functional characterization of CS1 receptor in the natural killer (NK) cells and B lymphocytes was investigated. CS1 (CRACC, novel Ly9) is a novel member of the CD2 family receptor expressed on natural killer (NK), T cells, activated Bcells and dendritic cells. To examine the existence of isoform of CS1, library from NK cells was screened based on wild type of CS1 (CS1-L). A splice variant form of CS1 (CS1-S), which lacks immunoreceptor tyrosine-based switch motifs (ITSMs) in cytoplasmic domain, was identified. To demonstrate the function of CS1 on human NK cells, transfectants that stably express each isoform were generated. CS1-L was able to mediate redirect cytotoxicity of P815 target cells as well as intracellular calcium influx in the presence of monoclonal antibody against CS1 suggesting that CS1-L is an activating receptor. CS1-S showed no effect on the cytolytic function and calcium influx suggesting that CS1-L and CS1-S may differentially regulate human NK cell functions. Although CS1 was also cloned from cDNA library of human B-lymphocytes as well as of NK cells, very little is known regarding its biology on human B-lymphocytes. Here I investigated the expressions and functions of CS1 in human B cells. Human B cells expresses only CS1-L isoform and the levels of CS1 expression are upregulated after activation in vitro. Importantly, monoclonal antibody of CS1 (1G10 mAb) strongly enhances proliferation of both freshly isolated and activated B cells. The enhanced proliferation effects of CS1 were most prominent on B cells activated by anti-CD40 mAbs and/or IL-4. Human cytokine microarray results indicated CS1 enhanced mRNA transcripts of fms-line tyrosine kinase 3 ligand, lymphotoxin A, tumor necrosis factor, and IL-14 which are related with mostly growth promoting activity. These results suggest that autorine cytokines might be the mediators for the function of CS1 on B cell in which it can induce proliferation of activated B cells. This study suggests that CS1 plays important role in human NK cells and B-lymphocytes.Item Effects of Serine Protease-Like F (SPLF) on Alpha-Toxin Expression in Staphylococcus aureus(2003-05-01) Pulse, Mark E.; Mart Hart; Jerry Simecka; Ming-Chi WuPulse, Mark E., Effects of Serine protease-like F (Sp1F) on Alpha-Toxin Expression in Staphylococcus aureus. Master’s of Science (Microbiology). May 2003. Pages-67. Table-5. Figures-9. Transposon mutagenesis (Tn551) was used to generate agr-suppressor mutations in the agr-null Staphylococcus aureus strain RN6911 (Δagr;;tmn). Firty-four suppressor mutants displaying changes in hemolysin, protease, and lipase activities were isolated, and only twenty-six mutants contained Tn551 within their chromosomes. Transposon insertion sites for seven mutants were determined by sequencing amplicons generated by arbitrary-PCR. One of the insertion sites was within the serine protease-like F (spʅF) gene. Alpha-toxin message levels for the spʅF mutant were similar to RN6911. However, alpha-toxin activity in spent media isolated from the spʅF mutant were similar to RN6911. However, alpha-toxin activity in spent media isolated from the spʅF mutant was increased ten-fold as compared to RN6911. Transduction of the spʅf:Tn551 mutation back into the parental strain verified the link between the phenotype and the mutation. Whole cell lysates from Escherichia coli cells containing a plasmid copy of spʅF displayed protease activity on casein. These data suggest that SpʅF may be post-translationally modifying alpha-toxin through proteolysis.Item Health Risk Associated with Microbial Contamination in Healthcare Facilities(2007-07-01) Palmer, Eboni D.; Larranaga, Michael; Gratton, Terry; Ramphal, LilyPalmer, Eboni D., Health Risk Associated with Microbial Contamination in Healthcare Facilities. Master of Public Health (Occupational Health Practice), July 2007, 96 pp., 20 tables, 8 illustrations, bibliography, 140 titles. This study developed a model assessing the risk associated with indoor microbial contamination in health care facilities. A semi-quantitative model resulting in numerical scores was used to describe the severity of risk associated with given levels of contamination. The hospital used in this study had problems with water intrusion. There were 99 locations from 3 air handler unit (AHU) service area locations examined. The final results produced a health risk rating for all three AHUs of medium risk. There is an increased risk of adverse health outcomes due to exposure from environmental microbial contamination. Immunocompromised patients and patients with allergies are not protected from the risk of developing a nosocomial infection or allergic reaction. Remediation of the contaminated areas must be performed in order to reduce the risk.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 Interleukin-8: Baculovirus Expression and the Receptor Signal Transduction Pathway(1994-06-01) Kang, Xiaoqiang; Stephen R. Grant; Rafael Alvarez; Paula SumstomXiaoqiang, Kang., Interleukin-8: Baculovirus Expression and the Receptor Signal Transduction Pathway. Doctor of Philosophy (Biomedical Sciences), June, 1994, 150 pp., 4 tables, 36 illustrations, bibliography, 212 titles. The cDNA for human interleukin-8 (IL8) was subcloned from a bacterial source into the eukaryotic baculoviral vector expression system. Recombinant human IL-8 (rhIL-8) was synthesized and secreted from SF9 cells following infection of a recombinant virus harboring the full-length IL-8 structural gene. Recombinant human interleukin-8 was purified ([greater than] 600 fold) to homogeneity using preparative HPLC. The rhIL-8 preparation retained all of the physical, immunological, and biochemical properties of the natural product (monocyte-derived IL-8). Baculovirus vector expression coupled to preparative HPLC proved to be a very efficient method for large-scale recombinant interleukin production. Biochemical mechanisms that mediate IL-8 receptor-stimulated activities are poorly understood. In this study, I have explored the intracellular mechanism(s) induced by IL-8 in differentiated HL-60 cells. IL-8 induced a rapid and transient activation of phospholipase A2 in differentiated HL-60 cells. A consequence of phospholipase A2 activation was the release of arachidonic acid and the generation of lysophospholipids from membrane phospholipids. The IL-8 stimulated-arachidonic acid release was pertussis toxin and phospholipase A2 inhibitor sensitive, and protein kinase C independent. In contrast to another neutrophil chemotactic factor, fMLP, IL-8 did not stimulate the activation of phospholipase C and phospholipase D. When comparing the phosphorylation events induced by IL-8 and fMLP, I found that these two chemotactic factors triggered different protein phosphorylation profiles. Tyrosine phosphorylation of proteins was not detected following IL-8 stimulation in HL-60 cells. However, IL-8 stimulated the rapid autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaM kinase II). These results strongly suggest that the IL-8 receptor is closely coupled to the activation of PLA2 and that CaM kinase II is an integral component of IL-8 receptor signal pathway.Item Lethality of Staphylococcus in Murine Pneumonia is Due to Alpha-Toxin and Other Secreted Factors Regulated by AGR and SAR(2003-08-01) Overheim, Katie A.; Dan Dimitrijevich; Glenn Dillon; James CaffreyOverheim, Katie A., Lethality of Staphlococcus aureus in Murine Pneumonia is Due to Alpha-Toxin and Other Secreted Factors Regulated by agr and sar. Doctor of Philosophy (Biomedical Sciences), August, 2003, 91 pp, 6 Tables, 9 illustrations, bibliography, 106. The purpose of these studies was to determine if the S. aureus global regulators agr and sar play a role in staphylococcal pneumonia and if the virulence factors regulated by them contributed to the severity of staphylococcal pneumonia. To determine this, we established a pneumonia model in mice in order to identify if S. aureus global regulators agr and sar play a role in the pathogenesis of staphylococcal pneumonia. As well, we took steps to identify the extracellular factors responsible for the lethality in a murine model of staphylococcal pneumonia and determine if these factors involved in disease process could be used as targets for immune therapy. My work revealed that lethal pneumonia in a mouse model is dependent on the S. aureus global regulators agr and sar. This study also revealed that the lethality associated with our model is due to secreted factors, regulated by S. aureus global regulators agr and sar. Further investigation demonstrated the alpha-toxin is a major virulence factor involved in the lethality in our model. By generating an alpha-toxin deficient strain in S. aureus RN6390, we show a reduced virulence in our disease model. As well, antiserum to alpha-toxin, when administered with a lethal dose of S. aureus RN6390, we show a reduced virulence in our disease model. As well, antiserum to alpha-toxin, when administered a lethal dose of S. aureus RN6390, we show a reduced virulence in our disease model. As well, antiserum to alpha-toxin, when administered with a lethal dose of S. aureus RN6390 protected animals from death. By evaluating the role of alpha-toxin’s ability to contribute to lethality, we assessed numerous strains of S. aureus in our pneumonia model. We discovered that there was a correlation to alpha-toxin production levels and lethality in our pneumonia model. However, our study also demonstrated that alpha-toxin is not the only factor involved in the disease process.Item Regulation and Characterization of Cardiac Phosphoinositide-Specific Phospholipase C (PLC) Isoenzymes(1997-12-01) Wang, Juan; Eugene E. Quist; Thomas Yorio; Ming-Chi WuWang, Juan, Regulation and Characterization of Cardiac Phosphoinositide-Specific Phospholipase C Isoenzymes. Master of Biomedical Science, Dec., 1997, 79 pp., 20 illustration, bibliography, 62 titles. It is hypothesized that myocardial phosphoinositide-specific phospholipase C (PLC) isoenzymes are regulated by physiological intracellular Ca2+ and by cytosol-membrane translocation. The regulation and identification of PLC isoenzymes in rat and dog ventricular subcellular fractions were studied. PLC-β1, PLC-β3 and PLC-δ1 were identified in rat and dog cytosol and microsomal membranes by chromatographic separation, enzyme assays and western blotting. Truncated PLC-β isoforms with molecular weights of 69 kDa and 114 kDa were isolated from rat and dog cytosol, respectively. Species differences in the relative distribution of PLC isoenzymes were evident as PLC-δ dominant in rat whereas PLC-β isoenzymes were dominant in dog. A 91 kDa cytosolic protein which did not contain PLC activity alone markedly led to PLC activation when combined with microsomes. The activator protein was immunoprecipitated with an anti-PLC-δ identifying this activator as an inactive PLC-δ isoenzyme. These studies indicate that cytosolic PLC-δ may be activated by translocating to membranes. In addition, proteolysis may be involved in long term activation of cytosolic PLC isoenzymes. Further studies will be required to resolve the physiological significance of these modes of cardiac PLC activation.Item Signaling in Natural Killer Cells: NK Cell Activation by LLT1 Receptor(2008-05-01) Bambard, Nowland D.; Jerry Simecka; Richard Easom; Harlan JonesBambard, Nowland D., Signaling in Natural Killer Cells: NK Cell Activation by LLT1 Receptor. Doctor of Philosophy (Microbiology and Immunology), May 2008, 162 pp., 1 table, 30 illustrations, bibliography, 179 titles. Natural Killer (NK) cells are large granular lymphocytes of the innate immune system that constitute the first line of defense against viral pathogens and cancer. Unlink cells of the adaptive immune response, NK cells do not recognize specific antigens expressed on MHC receptors, rather they recognize tumorgenic and virally infected cells through a complex balance of activating and inhibiting receptors expressed on the surface of human NK cells. LLT1 is expressed on numerous immune cells and subsequent functional analysis indicates that LLT1 plays an activating role on NK cells by way of stimulating interferon-gamma (IFN-G) secretion. LLT1 has also been shown to have a role on non-immune cells, inhibiting the formation and function of osteoclasts. Additionally, the natural ligand of LLT1 has been identified as NKR-P1A (CD161), an NK cell inhibitory receptor known to play an important role in immune regulation. We hypothesize that LLT1 employs multiple signaling pathways to accomplish its activating functions on human NK cells, and may be associated with one of four known transmembrane accessory proteins associated with NK cell activating receptors. We activated LLT1 on NK92 cells with target cells expressing its natural ligand CD161 and analyzing IFN-G production in the presence of pharmacological inhibitors specific for various signaling mechanisms. These results indicate that LLT1 employs Src-PTK, p38 and ERK signaling pathways, but not PKC, P13K or calcineurin. These results were followed up with phosphorylation analysis, which confirmed that the ERK signaling pathway is associated with LLT1 IFN-G production. Finally, by analyzing IFN-G mRNA we found that LLT1 activation is not associated with any detectable change in IFN-G mRNA levels, suggesting that LLT1 stimulates NK IFN-G production by modulating post transcriptional or translational events. Identification of the signaling pathways associated with LLT1 is of great medical significance as this may provide us with novel insights into activating NK cells to counter infection and cancer.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 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 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 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.