Browsing by Subject "Antigens"
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Item Assessment of CD4+ T Cell Depletion And Monocyte Function During Nonpathogenic SIV Infection of Sooty Mangabeys(2011-02-01T19:36:48Z) Mir, Kiran D.; Sodora, DonaldSIV-infected sooty mangabeys maintain low levels of chronic immune activation and do not progress to AIDS, making them an important model for elucidating the mechanisms contributing to AIDS pathogenesis. The studies presented here utilize the mangabey model of nonpathogenic SIV infection to assess the impact of CD4+ T cell depletion on immune activation in SIV-infected mangabeys and to assess the contribution of monocytes to nonpathogenic SIV infection. The Sodora laboratory previously identified a cohort of SIV-infected sooty mangabeys that experienced a virally-mediated severe decline in CD4+ T cells yet did not develop opportunistic infections or AIDS. Here, we assessed the immune competence of three mangabeys following viral passage from one CD4-low mangabey that resulted in a dramatic decline in CD4+ T cells within 21 days-post-infection. Despite the rapid depletion of CD4+ T cells, all mangabeys maintained low levels of chronic immune activation and mounted adaptive immune responses to SIV and influenza vaccination. To investigate the contribution of monocytes to the low levels of immune activation, we assessed the effector function of monocytes in SIV-infected CD4-low and CD4-healthy sooty mangabeys. We found that, compared to SIV-negative mangabeys, monocytes from SIV-infected mangabeys produced significantly less TNF-alpha upon stimulation with lipopolysaccharide (LPS). In contrast, hosts of a pathogenic infection, including SIV-infected macaques and HIV-infected humans, displayed no change in monocyte TNF-alpha responses relative to uninfected controls. In mangabey PBMC cultures, stimulation with LPS led to increases in CD8+ T cell activation that could be inhibited in a dose-dependent manner by TNF-alpha –blocking antibodies. Taken together, these results suggest that TNF-alpha production from monocytes can contribute to increases in immune activation and that SIV-infected sooty mangabeys regulate the monocyte response to LPS as one means to avoid chronic immune activation during SIV infection. These studies expand the current knowledge of the mechanisms by which SIV-infected natural hosts avoid progression to AIDS and underscore the importance of controlling immune activation during lentiviral infection, which may inform the next generation of therapies and vaccines for HIV patients.Item B Cell Modulation of T Cell Responses in Multiple Sclerosis(2010-01-12T18:52:03Z) Harp, Christopher Todd; Monson, Nancy L.Until recently, a definitive role for B cells in the pathogenesis of the autoimmune neurological disorder multiple sclerosis (MS), had not been widely accepted, and remains poorly understood. B cells have multiple functions in the immune system and can both positively and negatively modulate immune responses through the production of antibody, cytokine secretion, and/or antigen presentation. Several studies indirectly suggest that B cell-T cell cooperation may be paramount in MS disease pathogenesis, although this interaction has not been well studied in MS. Therefore, the focus of my thesis project was to test the hypothesis that B cells could be efficient neuro-antigen presenting cells in the context of MS. My work has demonstrated that the cerebrospinal fluid (CSF) B cell population in MS shows characteristics of both auto-reactivity and antigen driven selection in a germinal center reaction. These findings suggest that neuro-antigen driven selection had occurred in the periphery and prompted investigation of B cells as neuro-antigen presenting cells. Examination of CD40 ligand (CD40L) and interleukin-4 (IL-4) activated peripheral B cells demonstrated for the first time that B cells could efficiently elicit myelin basic protein (MBP) specific CD8+ and CD4+ T cell proliferation from resting T cells in vitro through a mechanism that was partially dependent on presentation through HLA-DR. Further inquiry into the antigen presentation capacity of specific subpopulations of resting B cells revealed that memory B cells from MS patients (but not healthy donors (HDs)) were significantly better neuro-antigen specific presenting cells than their na?ve counter parts. This data indicated that a specific peripheral immune response had been generated in response to neuro-antigens in RRMS patients but not HDs. Taken together, these data provide a model where antigen experienced peripheral B cells from MS patients (but not HDs) provide important T cell support through antigen presentation and add to our understanding of the role of B cells in the pathogenesis of this autoimmune disease of the CNS. [Keywords: multiple sclerosis; B cells; autoimmunity; antigen presentation; human immunology; T cells]Item Biochemical characterization of cell surface components of Candida albicans(Texas Tech University, 1982-12) Mendel, Susan Mari NegaardExtracellular material (ECM) was isolated from media in which yeast phase Candida albicans of unknown serotype was grown. Cold 95% ethanol precipitated the ECM and upon Sephadex G-50 chromatography, high and low molecular weight components were identified. They consisted of ECM 1 (30,000 Mr), ECM 2A (4,500-6,000 Mr), and ECM 2B (3,800 Mr). The ECM 2B was separated into two components upon ion exchange chromatography. Glucose and mannose are its carbohydrate moieties and proline, lysine and glutamic acid are its major amino acids. It appears to have more protein content than ECM 2B2 as evidenced by absorbance at 2 80 nm. ECM 2B2 contains only mannose and some protein composed mostly of proline, threonine and alanine. The components are neither strain-specific, nor produced only during yeast phase growth. The ECM were compared to material released from the cell surface upon treatment of whole cells with the nonspecific protease papain, and the disulfide bond reducer, dithiothreitol. An ECM 2A is produced upon both treatments. ECM 2B1 closely resembles a low molecular weight component produced upon DTT treatment, and ECM 2B2 is similar to the papain released molecule. The ECM appears to be present either on or within the cell surface of Candida albicans. The ECM did not appear to be strongly antigenic, although unpurified media did induce an antibody response.Item Delineating the Role of CD244 in NK Cell Cytotoxicity and the Contribution of Ly108 to Thymocyte Development(2010-11-02T18:20:55Z) Westcott, Jill Michelle; Schatzle, JohnGenetic analysis of the a murine model of lupus has implicated polymorphisms in the SLAM family of receptors (CD244 (2B4), CD229 (Ly9), CS-1 (CRACC), CD48 CD150 (SLAM), CD84, and Ly108 (NTB-A)) as causative for a breach in tolerance of both T and B cells leading to the production of self reactive antibodies. Analysis of common strains of laboratory mice revealed the existence of two stable haplotypes (b and z) of the SLAM family gene cluster. Given recent studies identifying polymorphisms in the SLAM family in humans, we have determined how polymorphisms in the SLAM family can affect lymphocyte function in mice to model how similar mechanisms may be involved in human pathologies. For these studies, we used mice congenic at the SLAM family locus (B6 –b haplotype and B6.Sle1b–z haplotype) to study two major questions: 1.) how do polymorphisms in CD244 affect NK function? And 2.) how do polymorphisms in Ly108 affect T cell tolerance? In the studies presented herein we demonstrate that CD244 functions largely as an inhibitory receptor in NK cells from B6 mice and as an activating receptor in B6.Sle1b mice. We demonstrate that allelic polymorphisms contribute to this differential function by altering receptor isoform usage, cell surface densities, baseline phosphorylation levels and subsequent adaptor association and receptor downmodulation. We also demonstrate that differential isoform usage of the receptor, Ly108 in B6 and B6.Sle1b mice alters thymocyte differentiation and negative selection events leading to a break in tolerance of T cells in B6.Sle1b mice. This is due to differential affects of the isoforms of Ly108 on thymocyte cell cycle progression and sensitivity to apoptosis. These studies highlight how naturally occurring polymorphisms in SLAM family genes can profoundly affect receptor function and potentially result in pathologic outcomes in certain genetic contexts.Item Engineering anti-infective antibodies(2009-12) Rani, Mridula; Iverson, Brent L.; Georgiou, George; Brown, Katherine A.; Maynard, Jennifer A.; Ren, PengyuIn the past 15-20 years, advances in antibody engineering have facilitated the generation and isolation of monoclonal antibodies (mAbs) to a wide array of antigens. Consequently, mAbs have become essential therapeutic tools and currently dominate the global protein therapeutics market. The engineering of anti-infective antibodies, however, has proven quite a challenge, despite the fact that antibodies were naturally evolved to fight infections. The identification of suitable antigens, the mode of administration and the high cost associated with the production of antibody therapeutics are some of the major hurdles for the progress of anti-infective antibodies. This dissertation addresses issues concerning the development of anti-infective antibodies against two different pathogens: SARS coronavirus (CoV) and two pathogenic species of Burkholderia bacteria. To investigate the role of affinity in viral neutralization and evolution of escape mutants, we first sought to isolate an antibody with high affinity towards the receptor binding domain (RBD) of SARS-CoV. Following high-throughput screening of a library of random mutants via the APEx display system, we isolated antibodies with affinities in the range of 0.8 nM - 0.1 nM. The affinity was further improved by additional mutagenesis and DNA shuffling, and a high affinity variant (45pM) with ~300-fold improvement over the parental antibody was isolated. Evaluation of these antibodies in an in vitro assay demonstrated that neutralization of wild-type Urbani strain of SARS-CoV correlates well with the affinity of the antibody, with higher affinity leading to greater neutralization. Moreover, the antibody exhibiting the highest affinity could neutralize SARS-CoV escape mutants that evaded neutralization by both parental and lower affinity antibodies. Another important aspect for the development of anti-infective antibodies concerns the identification of suitable antigen targets to be used in the isolation of antibodies. In an effort to develop a high-throughput screening method for the isolation of antibodies to a wide array of antigens, we used a synthetic antibody (Fab) library constructed by a minimalist approach and displayed on the surface of filamentous bacteriophage. The library was screened against antigens from Burkholderia pseudomallei and Burkholderia mallei. After only three rounds of selection and enrichment against five different antigens, we obtained Fabs specific to four of the antigens as confirmed by ELISA. These results not only demonstrate the use of a synthetic antibody library for the isolation of antibodies against infectious pathogens, but also its feasibility, and potential applicability as a high-throughput screen for a variety of antigens.Item Role of Nlc Cells in Murine Models of T Cell-Dependent Responses(2007-12-17) Jennings, Paula Alessandra; Yuan, DorothyNK cells are part of the innate immune system, yet they can also modulate the acquired immune response. Activated NK cells, for instance, can increase antigen specific IgG2a production in response to T independent responses, mostly through IFN-gamma secretion. Previous experiments examining the effect of NK cells on T cell- dependent antigens in various laboratories have yielded inconsistent conclusions. Therefore attempts were made to further investigate this question. Whereas depletion of NK cells had no detectable effect on the response to a TD antigen in Ribi adjuvant injected intraperitoneally, the secondary IgG1 response can be significantly reduced. This result suggested a role for NK cells on the generation of memory T cells. Therefore experiments were initiated to investigate the effect of NK cells on T cell proliferation. The absence of NK cells during immunization was found to reduce primary T cell proliferation. Such effect was not observed when B cell antigen presentation was absent, which is known to be important for memory T cell generation. A direct effect of NK cells on B cell antigen presentation was assessed in vitro. These experiments showed that NK cells can upregulate B cell antigen presentation of ovalbumin to na?T cells expressing a transgene specific to the ovalbumin derived peptide, OVAp. This increase is contact dependent and can occur in the absence of IFN-gamma . Moreover, the NK cell enhancement of B cell presentation of intact protein was greater than the presentation of OVAp, which requires no processing. These experiments suggest that the upregulation involves both processing and presentation of antigen. These experiments show that NK cells have a direct effect on B cell antigen presentation and provide a mechanistic basis for the role of NK cells in modulating in vivo T cell- dependent antibody responses.Item Studies on the mechanism of protodyne induced resistance(Texas Tech University, 1972-12) Duncan, James ByronNot availableItem The Salmonella flagellar antigen g ... as a potential carrier of "foreign epitopes"(Texas Tech University, 1993-12) Masten, Barbara JeanSalmonella flagella filaments are polymers of a protein termed, 'flagellin'. We are interested in expressing medically important epitopes at the site(s) of naturally occurring B-cell epitopes expressed by flagellin. To determine the molecular basis for expression of the epitopes by which the Salmonella phase-1 g... series flagellin antigens are distinguished, 17 members (S. adelaide [fliC9^, S. berta [fliC9% S. budapest [fliC9^, S. califomia [fUCd^rJt]^ S. chaco [fliCdrrt]^ S. danysz [fliCd"^, S. derby [fliC9f\, S. dublin [fliC9P], S. enteritidis [fliCQrrf], S. essen [fliCd'T^, S. jena [fiiCdn^, S. monschaui [fliC^^, S. montevideo [fliCd'T^^, S. moscow [fliC9Q], S. oranienberg [ftiC^, S. mstock [fliC9P% and S. senftenberg [fliC9S^) of this series were selected and their fliC (the stmctural gene for phase-1 flagellin) genes sequenced. Comparison of the flagellin amino acid sequences showed complete homology in the N-terminal (region I, II and III) and C-terminal (region VIII) segments of the proteins. Differences in amino acids were found throughout the central portion (regions IV, V, VI and VII) of the flagellins. No localized area substituted to specify subfactor epitopes could be identified, suggesting the subfactors of the g... series are conformational at the molecular level. The amino acids comprising each of the subfactor epitopes were not definable by sequence analysis. Results of epitope mapping, by two approaches, support the view that the g... series phase-1 flagellin B-ceil epitopes are conformational. Based upon amino acid comparison, the fliC gene of S. califomia, S. monschaui and S. oranienberg may be the gene of choice for substitution(s)since it may tolerate amino acid interchanges in region V better than the fliC gene of other members of the g... series. Hydrophobicity and surface exposure plots suggest three potential sites, two within region IV and one within region VI, for epitope substitution within the flagellin of any g... series member Phylogenetic analysis and comparative analysis shows S. california, S. monschaui and S. oranienberg to be the most diverse g... series serovars. A combination of spontaneous mutation, insertion and deletion most likely generated the antigenic polymorphism seen with salmonellar g... series flagellins.