Browsing by Subject "innate immunity"
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Item Cytokine patterns in a comparative model of arenavirus infection: Implications for virulence and control of viral replication(2005-07-12) Erin P. Scott; Judith Aronson; Thomas K. Hughes; Lynn Soong; David N. McMurray; Clarence J. PetersGuinea pig infection with the arenavirus Pichinde provides an animal model for human Lassa fever, a disease that affects 300,000 to 500,000 people a year in western Africa. Low passage Pichinde virus (P2) induces a mild disease with low viremia, while high passage Pichinde (P18) induces a severe disease with high viremia, ending in terminal shock. We hypothesized that severe disease would be associated with a suppression of potentially antiviral cytokines early in infection, and high levels of potentially pathogenic pro-inflammatory cytokines late in infection. Cytokine responses to P2 and P18 infection were measured from primary guinea pig peritoneal macrophages (PM) in vitro when measured by real time RT-PCR. In general, neither P2 nor P18 infection altered cytokine production from unstimulated PM. P18 infected PM did have lower mRNA levels of IL-1beta, IL-12p40, and MCP-1 after LPS addition when compared to P2 infected PM. During experimental guinea pig infection, P18 infection was associated with markedly increased IFN-gamma and MCP-1 mRNA levels from the initial peritoneal target cells relative to P2. P18 infected peritoneal cells had slightly decreased TNF-alpha, IL-8, and IL-12p40 transcripts relative to mock infected peritoneal cells. Late in infection, P18 infected spleens and livers had similar cytokine patterns relative to P2, but P18 infected PBL had decreased TNF-alpha, IFN-gamma, and RANTES transcripts. We also examined the ability of a decoy AP-1 thioaptamer, XBY-S2, to alter morbidity, mortality, and cytokine expression during P18 infection of guinea pigs. After two doses of XBY-S2, 50% (p=.024) of treated guinea pigs survived infection and had undetectable viremia. XBY-S2-treated P18 infected guinea pigs over time had overall increased cytokine mRNA expression of TNF-alpha, IL-8, IL-1beta, and IL-10 compared to PBS-treated P18 infected guinea pigs. A suppression of PBL IL-1beta and RANTES mRNA at day 12 of P18 infection was repeatedly observed. Conclusions from these experiments are 1) macrophage-derived cytokines do not explain the differential replication of P2 and P18 viruses, 2) high levels of IFN-gamma and MCP-1 may contribute to virulence of P18 virus, 3) over-expression of pro-inflammatory cytokines in PBL, liver, or spleen is not associated with terminal shock, 4) boosting of pro-inflammatory cytokines by an AP-1 aptamer correlates with reduced viremia and survival of P18 infection.Item Genetic and Phylogenetic Studies of Toll-Like Receptor 5 (TLR5) in River Buffalo (Bubalus Bubalis)(2012-08-21) Jones, BrittanyRiver buffalo are economically important to many countries and only recently has their genome been explored for the purpose of mapping genetic variation in traits of economic and biologic interest. The purpose of this research is to characterize the genetic and evolutionary profile of Toll-like receptor 5 (TLR5), which mediates the mammalian innate immune response to bacterial flagellin. This study is comprised of three parts: 1) generating a radiation hybrid (RH) map of river buffalo chromosome 5 (BBU5) where the TLR5 gene is located and building a comparative map with homologous cattle chromosomes; 2) conducting a single-nucleotide polymorphism (SNP) survey of the TLR5 gene to reveal variation within river buffalo and other species; and 3) performing an evolutionary study by inferring phylogenetic trees of TLR5 across multiple taxa and determining the possible evolutionary constraints within the TLR5 coding region. River buffalo chromosome 5 is a bi-armed chromosome with arms corresponding to cattle chromosomes 16 and 29. A BBU5 RH map was developed using the previously published river buffalo RH mapping panel and cattle-derived markers. The RH map developed in this study became an integral part of the first river buffalo whole genome RH map. Genetic variation of the TLR5 gene was evaluated in a small domestic herd of river buffalo. Sequencing of the TLR5 coding region and partial associated 5'- and 3'-untranslated regions yielded 16 novel SNPs. Six SNPs were identified as non-synonymous with one predicted to potentially code for a functionally altered product. For the evolutionary study of the TLR5 coding region, phylogenetic trees were inferred based on TLR5 variation across multiple orders and another for artiodactyla. Species that are closely related to river buffalo appear to have undergone negative selection in TLR5 while those that diverged from river buffalo earlier may be retaining alleles that river buffalo are removing from the population. In conclusion, putative chromosomal rearrangements were identified between river buffalo and cattle, the variation that was uncovered in the TLR5 coding region could potentially lead to differential immunity across species, and there appears be some evolutionary flexibility in the DNA sequence of the TLR5 coding region.Item Innate immunity to Rhodococcus equi: the response of adult and juvenile equine neutrophils(2009-05-15) Nerren, Jessica RachelBlood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 ?g of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNF?, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFN? or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFN?, TNF?, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.Item Solid phase peptide synthesis and TLR-5 activity analysis of pertide fragments from the Salmonella muenchen flagellin protein(2005-08-18) Joseph Richard Karam; Dr. Scott R. Gilbertson, Ph.D.; Dr. Richard B. Pyles, Ph.D; Dr. Cornelis Elfernik, Ph.D.Drug design strategies begin by determining the simplest ligand necessary to activate a receptor of interest. The Toll-Like Receptor-5 (TLR-5) is an attractive target for pharmaceutical modulation because it initiates an innate immune response. A TLR-5 agonist or antagonist could help remedy a variety of disorders. Flagellin, the primary component of bacterial flagella, is the only known TLR-5 ligand. Three short regions within this protein are suggested to activate TLR-5: Peptide-N1, LQRVRELAVQ; Peptide-N2, LAVQSANGTNSQSD; and Peptide-C1, QNRFNSAITNLGNT. Here, we report the synthesis of these peptides and their activity against TLR-5 expressing HEK-293 cells. Our goal was to resolve the minimal region of flagellin necessary to bind and/or activate TLR-5. Results showed significant agonist activity (P<0.01) with peptide N2-b (LAVQSANGTN), and peptide N2-f (LAVQSANGTNSQ). Peptide N2-c (ANGTN) and N2-d (LAVQS) showed significant (P<0.05) antagonistic properties for TLR-5. These peptides could make interesting lead compounds to modify for optimal TLR-5 activity.