Browsing by Subject "Cytokines"
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Item Cytokine detection in eiav-infected equine monocyte-derived macrophages using quantitative real-time polymerase chain reaction(2009-05-15) Allen, Charlotte AnnetteThe replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1?, IL-1?, IL-6, IL-8 and TNF-? were validated using QPCR primers and probes which were generated for the aforementioned equine genes.Item The effect of quercetin on exercise induced cytokine response in trained cyclists(2012-12) Chou, Ting-Heng; Coyle, Edward F., 1952-; Brothers, Robert MQuercetin is a flavonoid found in commonly consumed fruits and vegetables that has exhibited powerful antioxidant and anti-inflammatory properties in rodents and in vitro. In humans, the effect of quercetin on exercise induced oxidative stress and inflammation is still equivocal and need to be further investigated. A powerful antioxidant such as quercetin may inhibit the high levels of oxidative stress and inflammation associated with the high volume and intensity of exercise training seen with endurance-trained individuals. PURPOSE: To determine the effect of 28 days of daily quercetin supplementation on intensive endurance exercise induced cytokine response. vi METHODS: Thirteen trained cyclists (VO2peak 58.8 ± 3.9 ml/kg/min) were recruited for this study from the University of Texas at Austin and the local Austin, Texas community and participated in this placebo controlled, randomized, crossover designed study. After initial assessment of baseline data (VO2peak, lactate threshold, and two familiarization time trials), participants began daily supplementation with either an antioxidant supplement containing vitamins and quercetin (Q-VIT: 1000mg quercetin, 820mg Vitamin C, 40mg Vitamin B3) or the same vitamin supplement without quercetin (VIT: 820mg Vitamin C, 40mg Vitamin B3). A simulated time trial using an electromagnetically braked cycle ergometer in which subjects had to complete a set amount of work (kJ) as fast as possible was performed on the last day of supplementation. Blood collection was performed at three time points of the time trial days: before exercise (PRE), after warm up (MIN 20), and immediately after time trial exercise (POST). Measured plasma markers were Interleukin-6 (IL-6), C-Reactive Protein (CRP), and Interleukin-10 (IL-10). RESULTS: Q-VIT compared to VIT had no effect on pre, min 20 and post exercise plasma IL-6, CRP, and IL-10 ( P= 0.7, 0.08, and 0.32 respectively). However there was a trend that Q-VIT lowered plasma CRP compare to VIT ( P = 0.08). CONCLUSION: Chronic supplementation for 28 days with a quercetin based antioxidant supplement did not affect plasma cytokine before during or after exercise. The results of the current study suggest that chronic supplementation with quercetin does not influence plasma cytokine and exercise induced cytokine response in endurance-trained athletes.Item Genetics of plasma cytokine variation in healthy baboons and humans(2005) Proffitt, John Michael; Freeland-Graves, Jeanne H.The goal of this project was to investigate the genetic regulation of plasma cytokine variation in healthy baboons and humans. The first objective was to estimate the heritabilities of plasma levels of inflammatory cytokines in healthy baboons. In Study I, levels of the inflammatory cytokines TNF-α, GM-CSF, IL-1β, and IL-6, as well as sTNFR1, and sTNFR2 were measured in 370 related baboons. All of the traits exhibited significant evidence for genetic regulation. The heritabilities ranged from 61% for TNF-α to 14% for sTNFR1. The second objective was to identify chromosomal regions governing the production of resistin in healthy baboons. In Study II, resistin levels were measured in 416 related baboons. Variation in the plasma concentration of resistin was significantly linked to chromosome 18, between the markers D18S475 and D18S172 on the cytoband 18q12 (LOD = 4.0). The third objective of this study was to locate quantitative trait loci (QTLs) that affect circulating levels of plasma TNF-α and IL-1β levels in healthy humans. Study III examined plasma levels of TNF-α and IL-1β in a population of Caucasian Americans from the Midwestern United States. Quantitative trait loci were identified for TNF-α and IL-1β (LOD = 3.0 and LOD = 4.0, respectively) on chromosome 18 between markers ATA82B02 and D18S1371. The fourth objective was to detect QTLs affecting plasma levels of CRP, and to investigate its association with obesity phenotypes in healthy humans. Study IV revealed that chromosome 12 harbors a QTL regulating circulating CRP levels between markers D12S375 and D12S1052 (LOD = 4.1). CRP was genetically correlated with parameters of adiposity. The results from these baboon and human studies suggest that circulating cytokine levels in healthy animals are under significant genetic control. Chromosomes 12 and 18 appear to contain genetic differences that influence inflammation. Future work should aim to resolve the specific genetic elements through fine mapping and the positional candidate gene approach.Item Nanoparticles as a carrier for protein and plasmid DNA vaccines in microneedle-mediated transcutaneous immunization(2013-08) Kumar, Amit, active 21st century; Cui, ZhengrongSkin is the largest immune organ and an ideal site to administer vaccines. However, by nature, skin is not permeable to antigens, which are macromolecules. The major hurdle in skin permeation is the outermost stratum corneum layer. Microneedles have proven feasible to create micron-sized channels in the epidermis of the skin, through which protein and plasmid DNA antigens can penetrate into the viable skin epidermis and dermis. However, the immune responses induced by microneedle-mediated transcutaneous immunization with protein or plasmid DNA alone are generally weak, and a vaccine adjuvant is often required to induce strong immune responses. Data from numerous previous studies have shown that nanoparticles as a vaccine carrier can significantly enhance the immunogenicity of antigens, but the feasibility of utilizing nanoparticles as a vaccine carrier to enhance the immune responses induced by microneedle-mediated transcutaneous immunization has rarely been studied. In this dissertation, using protein antigen (OVA) chemically conjugated onto the surface of solid-lipid nanoparticles and plasmid DNA (pCMV-beta, pVax/opt-BoNT/C-Hc50, and pCI-neo-sOVA) physically coated on the surface of cationic polymeric nanoparticles, we showed that the immune responses induced by microneedle-mediated transcutaneous immunization with protein antigens or plasmid DNA vaccines are significantly enhanced by delivering the proteins and plasmid DNA with nanoparticles. Importantly, microneedle-mediated transcutaneous immunization with proteins or plasmid DNA induces not only systemic immune responses, but also mucosal immune responses. In addition, it is generally believed that microneedles are safe. However, it remained unclear whether the micropores created by microneedles on the skin will also facilitate the permeation of microbes such as bacteria into the skin. In this dissertation, we also designed an unique ex vivo model to evaluate the permeation of live bacteria through mouse skin pretreated with microneedles. The results demonstrated that the risk of potential bacterial infection associated with microneedle treatment is not greater than that associated with a hypodermic needle injection.Item New Insights Into the Role of Equine Infectious Anemia Virus S2 Protein in Disease Expression(2011-08-08) Covaleda Salas, Lina M.Equine infectious anemia virus (EIAV) is an important animal model to study the contribution of macrophages in viral persistence during lentiviral infections. EIAV is unique amongst the lentiviruses in that it causes a rapid, rather than the very slow disease progression, characteristic of other lentiviral infections. The accessory gene, S2, unique to EIAV, is an important determinant in viral pathogenesis. A functional S2 gene is required to achieve high-titer viremia and the development of disease in infected horses. Despite its essential role, the mechanisms by which S2 influences EIAV pathogenesis remain elusive. The goal of this research was to gain insight into the role of S2 in pathogenesis. To accomplish this goal we: (i) Examined the effects of EIAV and its S2 protein in the regulation of the cytokine and chemokine responses in macrophages, (ii) Assessed the influence of EIAV infection and the effect of S2 on global gene expression in macrophages and (iii) Identified host cellular proteins that interact with S2 as a starting point for the identification of host factors implicated in S2 function. The results from this study provide evidence for a role of S2 in enhancing a proinflammatory cytokine and chemokine response in infected macrophages. Specifically, S2 enhances the expression of IL-1 alpha, IL-1 beta IL-8, MCP-2, MIP-1 beta, IP-10 and a newly discovered cytokine, IL-34. Involvement of S2 in cytokine and chemokine dysregulation may contribute to disease development by optimizing the host cell environment to promote viral dissemination and replication. Microarray analyses revealed an interesting set of differentially expressed genes upon EIAV infection. Genes affected by EIAV were involved in the immune response, transcription, translation, cell cycle and cell survival. Finally, we used the yeast two-hybrid system to identify S2 host cellular interacting proteins. We identified osteosarcoma amplified 9 (OS-9) and proteasome 26S ATPase subunit 3 (PSMC3) proteins as interacting partners of S2. Additional evidence is needed to demonstrate the physiological relevance of these interactions in vivo. In summary, the results from this study contribute towards our understanding of the role S2 in disease expression and allow the formulation of new hypotheses as to the potential mechanisms of action of S2 during EIAV infection.Item Systematic Approach to Compare the Inflammatory Response of Liver Cell Culture Systems Exposed to Silver, Copper, and Nickel Nanoparticles(2011-10-21) Banerjee, NiveditaAlthough nano-sized metal colloids are used in industrial and medicinal applications, little is known about the potential liver toxicity of these materials after occupational or intentional exposures. To begin to resolve some outstanding hepatotoxicity concerns, the inflammatory response of hepatocytes after exposure to metal colloids was assessed. Four ~30-nm-sized metal colloids, including silver (nano-Ag), copper (nano-Cu) and nickel (nano-Ni) were examined in an effort to understand the induced cytokine expression in a murine liver cell line (AML12). Here we also utilized another system, co-cultures of hepatocytes, Kupffer?s cells, and lymphocytes isolated from C57BL6 mice. Cells were exposed to the materials over dose-response (0.1mg/L to 1000mg/L) and time-dependent (4 h, 48 h, and 1-week) studies. Cytotoxicity was measured via metabolism of resazurin and validated via MTT assay and cell counts. Inflammatory response was determined by cytokine profiles (TNF-a and IL-6), as well as by mRNA and protein expression of heat shock protein (Hsp70). Results from cells exposed to nano-Ag to doses of up to 100mg/L exhibited no significant changes in cytotoxicity, IL-6, or TNF-a production, or Hsp70 expression. Both nano-Cu and nano-Ni exposed cells exhibited decreased metabolism, increased Hsp70 induction, and increased inflammatory responses (IL-6 and TNF-a). Dynamic light scattering and electron microscopy were used to characterize particle size and surface charge. All three metal colloidal systems demonstrated different particle size distributions, agglomerated sizes, and surface zeta potentials. Furthermore, each metal colloid system elicited different inflammatory biomarker responses and stress protein expression.Item The effect of a cognitive behavioral exercise program on the pro-inflammatory and anti-inflammatory cytokines(2012-12) Panke, Rutika; McComb, Jacalyn J. R.; Norman, Reid L.; Park, YoonjungObjective: The purpose of this study is to investigate the effects of Mindfulness-Based Exercise Program (MBEP) on pro-inflammatory cytokines (Interferon –γ and Tumor Necrosis Factor - α) and anti-inflammatory cytokines (Interleukin – 4 and Interleukin - 10). Methods: After completing a measure of psychological stress, 19 female college students (23 ± 3.7 years old) were assigned to either the control (CTRL) or intervention (TC) group on the basis of their resting levels of cortisol values. The MBEP (experimental) group practiced MBEP sessions twice a week for 8 weeks with each session of total 2 hours. Subjects were analyzed through Trier Social Stress Protocol for assessing subject to quantify the physiological stress response. Blood samples for the quantification of pro-inflammatory cytokines (Interferon –γ and Tumor Necrosis Factor - α) and anti-inflammatory cytokines (Interleukin – 4 and Interleukin - 10) and subjective stress measures were obtained before and after the MBEP sessions. Two separate 2 x 2 repeated measures ANOVA’s were used to analyze pro-inflammatory cytokines and anti-inflammatory cytokines. Separate 2 x 2 repeated measures ANOVA’s were used to analyze subjective levels of stress pre-post intervention. Results: No significant differences were found between groups for pro-inflammatory cytokines and anti-inflammatory cytokines; however slight increase was observed in trends of anti-inflammatory cytokines. Significant differences were observed in Spielberger’s Trait and Spielberger’s State Anxiety scale, Perceived stress scale and Stress Vulnerability scale.Item The role of neutrophil recruitment in the pathogenesis of salmonella enterica serotype typhimurium-induced enteritis in calves(2009-05-15) Nunes, Jairo SantosThe role of neutrophils in the pathogenesis of Salmonella typhimurium-induced ruminant and human enteritis and diarrhea remains incompletely understood. To address this question, the in vivo bovine ligated ileal loop model of non-typhoidal salmonellosis was used in calves with the naturallyoccurring Bovine Leukocyte Adhesion Deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from BLAD calves were compared to those from genetically normal calves negative for the BLAD mutation. Morphologic studies showed that the absence of significant tissue influx of neutrophils in intestine infected by S. typhimurium resulted in less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion compared to regular calves. Study of gene expression profile of cytokines by quantitative Real-Time PCR (qRTPCR) revealed that the massive tissue influx of neutrophils during acute infection is mainly driven by the CXC chemokine GRO- ? especially in the last stages of acute infection and to a lesser extent, IL-8. In contrast, the pro-inflammatory cytokines IL-1 ? and TNF- ? were not significantly correlated with the presence or absence of tissue neutrophils. The precise in situ localization of gene expression of these major cytokines and chemokines was investigated by qRTCPR from specific groups of intestinal cells captured by Laser Capture Microdissection in S. typhimuriuminfected ileal loops from BLAD animals. Our data confirmed that gene expression of IL-8, GRO- ?, and IL-1 ? was predominantly localized to enterocytes of crypts with less expression in enterocytes of villi tips and cells that form the domed villi were not an important source of TNF- ? gene expression. Microarray technology was used to determine the global transcriptional profile of bovine intestinal loops inoculated with S. typhimurium. The host samples were hybridized on a 13K bovine-specific oligoarray and microarray data was analyzed using a suite of gene expression analysis and modeling tools. Analysis of our data revealed that the tissue influx of neutrophils in ileal loops greatly influenced the host gene expression. Major differences in gene expression in relevant fields of Salmonella research including inflammation and immune response, Toll-like receptor signaling, cytokine profiles, apoptosis, and intracellular defense against infection are discussed.