Life Sciences Commons^™

Characterization Of The Immunoglobulin Lambda Chain Across Diverse Human Populations., William Gibson

Electronic Theses and Dissertations

The adaptive immune system relies on a diverse set of over one hundred immunoglobulin (IG) genes across three genomic loci that are variably combined to form antibodies (Ab). The IG Lambda locus is one of two loci which encodes the IG light chain. The complexity of the IGL locus severely limits the effective use of standard short-read sequencing, limiting our knowledge of population diversity in these loci. We leveraged single molecule real-time (SMRT) long-read sequencing in conjunction with IGL-targeted DNA capture to develop the method IG-Cap for accurate and high-throughput sequencing of the IGL locus. We benchmarked this method using …

Investigating Host Defenses Of North American Salamanders Against The Recently Emerged Chytrid Pathogen, Batrachochytrium Salamandrivorans, Kenzie Pereira

Electronic Theses and Dissertations

A recently emerged chytrid fungal pathogen, Batrachochytrium salamandrivorans (Bsal) threatens salamander biodiversity. Bsal susceptibility varies between and within salamander species, but little is known about the mechanisms underlying these differences. Susceptibility is likely influenced by numerous interacting factors, but my dissertation studied the role of host immune responses.

My first aim investigated between species differences by studying the bioactive properties of salamander skin peptides against Bsal and the related pathogen, B. dendrobatidis (Bd). Skin peptides were collected from five salamander species, used for in vitro assays, and analyzed by RP-HPLC. While skin peptides from one …

The Effect Of Wild Blueberry Anthocyanins And Phenolic Acids On Innate Immunity: The Compliment System, Vasiliki Papakotsi

Electronic Theses and Dissertations

Ingestion of berries containing polyphenols is associated with lower risk of inflammatory, metabolic, cardiovascular and degenerative diseases. Diet has been shown to modulate the activation of the complement system, a set of over 50 proteins present in the circulation and tissues that reacts in response to damage or microbial encounter and is critical for the maintenance of homeostasis. Imbalanced activation is tightly correlated with inflammation and various pathologies. Wild blueberries are a rich source of anthocyanins and phenolic acids, which can be found in plasma shortly after consumption. Given the involvement of both complement and polyphenols in the modulation of …

Microbiota Activated Cd103+ Dcs Stemming From Oral Microbiota Adaptation Specifically Drive Γδt17 Proliferation And Activation., Christopher Jay Fleming

Electronic Theses and Dissertations

IL-17 producing γδ T cells (γδT17) promote numerous autoimmune diseases such as psoriasis and arthritis, as well as, cancers of the colon, lung and breasts. Yet γδT17 peripheral regulation has yet to be thoroughly explored. In mice deficient in IL-17 signaling, we observed expansion of γδT17 in all major tissues. However, γδT17 expansion was not uniformly distributed systemically and was most prominent in oral draining cervical lymph nodes (LNs) with monoclonal expansion of Vγ6 γδT17. In vitro proliferation assays of these cervical LNs showed endogenous proliferation by γδT17 dependent on cell-to-cell contact with CD103⁺ DCs. CD86⁺ and CD80 …

Role Of Mitogen-Activated Kinases In Cd40-Mediated T Cell Activation Of Monocyte/Macrophage And Vascular Smooth Muscle Cell Cytokine/Chemokine Production, Denise M. Milhorn

Electronic Theses and Dissertations

This dissertation represents efforts to determine the functional consequences acquired by vascular smooth muscle cells (SMC) in response to CD40 ligation by activated CD154+ T cells, and to elucidate components of the signaling pathway(s) activated in response to CD40 signaling in both monocytes and SMC. To study the consequences of CD40 stimulation, primary human monocytes and aortic SMC were treated with plasma membranes purified from CD154 + , CD4+ T cells. The results presented in this dissertation demonstrate that SMC, like monocytes/macrophages, are capable of interacting with T cells in a manner that results in reciprocal activation events. SMC were …

Tnf(Alpha)-Dependent And Tnf(Alpha)-Independent Activation Of Macrophage Effector Function, Annette R. Clemons-Miller

Electronic Theses and Dissertations

Tumor necrosis factor α (TNFα) is a pleiotropic cytokine that is predominantly produced by activated macrophages. The effects of TNFα are as diverse as the cells with which it interacts, e.g., stimulating fibroblast growth, exerting cytotoxic/cytostatic; activity against various human and murine cell lines, promoting inflammation through upregulation of endothelial adhesion molecules and IL-8 production. Yet TNFα is best known, and in fact was originally described, for its role in the bacterial-induced hemorrhagic necrosis of tumors and exacerbation of septic shock in which aberrant TNFα production leads to vascular collapse, cachexia, multiple organ failure, and ultimately death in as many …

Cd40-Mediated Signaling Of Interleukin-1(Beta) Synthesis And Rescue From Apoptosis In Monocytes: Modulation By Il-4 And Il-10, Jonathan C. Poe

Electronic Theses and Dissertations

To date, the cellular mechanisms involved in the progression of diseases characterized by chronic inflammation, such as rheumatoid arthritis (RA), remain largely unknown. However, cell-to-cell contact interactions between CD4+ helper T (Th) cells and monocytes have been implicated in the induction and maintenance of pro-inflammatory cytokine synthesis that is characteristic to the pathogenesis of RA. One such cytokine produced during monocyte-Th cell contact is interleukin (IL)-1 β, a mediator directly involved in the characteristic tissue destruction that occurs in the synovia of individuals with RA. Previous studies in our laboratories have shown that ligation of CD40 on monocytes with CD40 …

Role Of The Cd40-Cd40 Ligand Interaction In Cd4(+) T Cell Activation Of Monocyte Interleukin-1 Synthesis, David H. Wagner

Electronic Theses and Dissertations

Most studies of the induction of cytokine synthesis in monocytes have used an exogenous triggering agent such as Lipolpoysaccharide (LPS). However, during nonseptic chronic inflammatory responses (e.g., rheumatoid arthritis) monocyte activation occurs as a result of T cell generated signals. This report demonstrated that plasma membranes from anti-CD3 activated peripheral CD4$\sp{+}$ T cells (Tm$\sp{\rm A}$) but not from resting CD4$\sp{+}$ cells (Tm$\sp{\rm R}$) induced monocytes to synthesize IL-1 in the absence of costimulatory cytokines. The expression kinetics of the molecule(s) unique to activated T cells which interact with monocyte receptors to induce IL-1 demonstrated that optimal expression occurred at 6h …

Nitric Oxide Production: A Mechanism For Inhibition Of Chlamydia Trachomatis Replication, Bojun Chen

Electronic Theses and Dissertations

Chlamydia trachomatis (CT) replicates in macrophages, but is inhibited by IFN-$\gamma$ or LPS. IFN-$\gamma$ and/or LPS induced nitrite production in mouse peritoneal macrophages, macrophage cell lines (RAW264.7 and J774A.1) and McCoy cells. Kinetic studies indicated that peak production occurred 48 hours post-treatment. CT infection itself was insufficient to induce nitrite production, but resulted in enhancement of nitrite production in IFN-$\gamma$-treated cells. Treatment with IFN-$\gamma$ or LPS resulted in significant inhibition of CT replication in these cells. Strong correlation between nitrite production and inhibition of CT replication was observed in RAW264.7 and J774A.1 cells (correlation coefficients: $-$0.93 and $-$0.94, p $<$ 0.001). N$\sp{\rm g}$- monomethyl-L-arginine (L-NMMA) specifically inhibited nitrite production and partially reversed inhibition of CT replication in macrophage cell lines. NOS mRNA was measured in RAW264.7 cells by Northern blot and Dot blot hybridization. Strong correlation between NOS mRNA expression and inhibition of CT replication (correlation coefficient: $-$0.97, p $<$ 0.05) was observed. Anti-TNF-$\alpha$ antibody completely neutralized the biological activity of TNF-$\alpha$ secreted by LPS-treated RAW264.7 cells, yet the antibody neither reduced nitrite production nor restored CT replication. Combination of the antibody and L-NMMA significantly enhanced restoration of CT replication. In peritoneal macrophages, inhibition of CT replication induced by IFN-$\gamma$ was partially restored by L-NMMA or anti-TNF-$\alpha$ antibody. In McCoy cells, inhibition of CT replication induced by IFN-$\gamma$ and LPS was not significantly restored by L-NMMA. Great restoration of CT replication by 1 mM L-NMMA was observed in LPS-treated J774A.1 cells (31%), but not in IFN-$\gamma$-treated cells (5%). Our data indicate that (1) NO production is one of the mechanisms for inhibition of CT replication in IFN-$\gamma$-activated peritoneal macrophages and RAW264.7 cells; (2) NO plays a significant role in CT inhibition in LPS-treated macrophage cell lines, but not peritoneal macrophages; (3) TNF-$\alpha$ may be associated with inhibition, but the mechanism(s) may not involve NO production; (4) NO production may not be the mechanism for CT inhibition in McCoy cells treated with IFN-$\gamma$ and LPS.

Mechanisms Of T Cell-Mediated Macrophage Activation: Role Of Antigen Specific And Antigen Nonspecific Cognate Interactions, Xiang Tao

Electronic Theses and Dissertations

Macrophages play an important role in host antimicrobial immunity and in non-septic inflammatory reactions. Most studies on macrophage activation have focused on the roles of the T cell-produced cytokine, interferon-$\gamma$ (IFN$\gamma)$ and bacterial product, lipopolysaccharide (LPS). T cell-macrophage interaction is a critical step in initiating both specific and nonspecific immune responses to antigenic stimulation. The current study examines the role of cognate T cell-macrophage interaction in activation of macrophage effector functions and induction of macrophage early activation gene expression. Viable resting T$\sb{\rm H}$2 clone cells can activate IFN$\gamma$-primed macrophages to produce reactive nitrogen intermediates (RNI) or express cytostatic activity. The …

Probing Protein-Protein Interactions Among Proteins Of A Nonaggregated Fatty Acid Synthetase From Euglena Gracilis Variety Bacillaris, Sande G. Williams

Electronic Theses and Dissertations

Enoyl-acyl carrier protein (ACP) reductase from chloroplast nonaggregated fatty acid synthetase (FAS) of Euglena gracilis variety bacillaris was purified to a single band on a denaturing polyacrylamide gel. The enzyme was partially characterized with respect to substrate specificity, reduced nucleotide requirement, and the effect of ACP and Ca$\sp{++}$ on enzyme activity. Antibodies against the purified protein were raised in hens and isolated from eggs. ACP was purified from Euglena in yields of about 1mg/100g (wet weight) of cells. Antibodies were raised against the purified protein. ACP antibodies inhibited the Euglena chloroplast FAS using Euglena or E. coli ACP as a …

Generation, Isolation And Assay Methods For Human Lymphocyte Mitogenic Factor, Thomas E. Seay

Electronic Theses and Dissertations

Activated lymphocytes secrete many products including the lymphokine human lymphocyte mitogenic factor (HLMF). In preliminary experiments lymphocytes from peripheral blood and palatine tonsils were evaluated as possible sources of HLMF by evaluating their level of activation through screening their spontaneous and concanavalin A (con A)-induced blastogenic responses. Tonsil lymphocytes (TL) were found to have high spontaneous proliferation as compared to peripheral blood lymphocytes (PBL). Cells from both sources responded to con A by undergoing a typical blastogenic response. Because TL must be obtained septically, they are frequently cultured in the presence of the antimycotic agent, Amphotericin B (Am B). Since …

Tonsil Cell Products Which Modify In Vitro Proliferation Of Blood Lymphocytes, Thomas W. Hodge

Electronic Theses and Dissertations

Human palatine tonsil lymphocytes, when compared to peripheral blood lymphocytes (PBL), were in an activated state even though there was no in vitro stimulation. When these tonsil lymphocytes were cultured in the absence of serum and polyclonal mitogens or antigens, the supernatant fluid often inhibited the proliferative response of target PBL to con A. The extent of this suppression ranged from 22% to 84%, and target cell viability was 90% or greater. There was no evidence for the presence of immunoglobulins or (alpha)2-macroglobulin in whole supernatant fluids. The suppressor was partially denatured at 80(DEGREES)C and was rendered completely inactive upon …