Browsing by Subject "cytokines"
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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 Mechanisms of increased microvascular permeability during acute Rickettsiosis(2007-11-02) Michael Edward Woods; Juan P. Olano; J. Stephen Dumler; Doug S. DeWitt; David H. WalkerRickettsial diseases represent some of the most severe bacterial infections in man including Rocky Mountain spotted fever and epidemic typhus. Rickettsiae primarily target the microvascular endothelium leading to increased microvascular permeability, the mechanisms of which are completely unknown. We sought to determine the impact of host responses to infection on increasing microvascular permeability both in vitro and in vivo. Our work has revealed a role for TNF-á, IL-1â, and IFN-ã as mediators of anti-rickettsial immunity that contribute to increased microvascular permeability in a dose-dependent manner by modulating the function of interendothelial adherens junctions. The permeability-inducing effects of these cytokines appear to occur independently of nitric oxide production since inhibition of iNOS does not prevent cytokine-mediated increases in permeability. Additionally we have shown that iNOS expression in vivo is associated with sites of rickettsial invasion, which also correlates with the leakage of endogenous serum protein. The lack of significantly higher levels of serum cytokines suggests this is primarily a localized response confined to areas of leukocyte infiltration. Likewise we have demonstrated a role for innate endothelial cell responses in modulating adherens junctions following rickettsial invasion. Human endothelial cells infected with rickettsiae produced significantly higher levels of VEGF and IL-6, two cytokines which can have a profound impact on adherens junction stability. This was associated with increased kinase activity in the form of protein kinase C, Src, and focal adhesion kinase. Inhibition of Src during R. rickettsii infection led to a decreased rate of endothelial permeability however this did not prevent rickettsiae-mediated cell death. Finally, we have identified several novel pathways modulated after rickettsial infection that were not previously thought to be important to rickettsial pathogenesis. Future work will be aimed at determining the relative contribution of these pathways to the endothelial dysfunction accrued during rickettsial infection. The work generated here provides a solid foundation for future endeavors aimed at alleviating the vascular dysfunction experienced during severe rickettsial infection.Item Psychological Well-Being and Spinal Cord Injury Recovery: A Two-Way Street?(2014-08-26) Maldonado, SiouiSpinal cord injury (SCI) leads to increased anxiety and depression in as many as 60% of patients. Yet despite extensive clinical research focused on understanding the variables influencing psychological well-being following SCI, risk factors that decrease psychological well-being remain unclear. We hypothesized that excitation of the immune system, inherent to SCI, may contribute to the decrease in well-being. We used a battery of established behavioral tests to assess depression and anxiety in contused rats and (1) characterized psychological well-being as a function of SCI severity, (2) examined peripheral (serum) and central (hippocampi and spinal cord) inflammation in relation to psychological well-being post SCI, and (3) explored whether social enrichment, as a modulator of psychological well-being, could improve overall recovery post SCI, by housing contused animals either alone, or with an injured or an intact cagemate. Following SCI, the contused subjects showed one of three profiles: depression-like, depression- and anxiety-like, or no signs of decreased psychological well-being. Subjects exhibiting a purely depression-like profile showed higher levels of pro-inflammatory cytokines peripherally, whereas subjects exhibiting a depression- and anxiety-like profile showed higher levels of pro-inflammatory cytokines centrally (hippocampi and spinal cord). These changes in inflammation were not associated with injury severity; suggesting that the association between inflammation and the expression of behaviors characteristic of decreased psychological well-being was not confounded by differential impairments in motor ability. Social enrichment, in the form of group housing, did not improve psychological well-being post SCI. Depression- and anxiety-like signs were found in all group housing conditions. Unexpectedly, we found that the intact animals housed with contused subjects showed depression- and anxiety-like signs similar to those of contused subjects, indicating that their psychological well-being was affected by the presence of an injured cagemate. This is reminiscent of the caregiver effect in humans, specifically the manifestation of symptoms of depression in individuals who care for patients suffering with a chronic illness, such as SCI. These experiments demonstrate that the depression and anxiety patients experience following spinal cord injury is not due solely to psychosocial factors, but may also, in part, result from increased immune activation following the injury.Item Social stress exacerbations on acute Theiler's virus infection: a role for Interleukin-6(Texas A&M University, 2006-10-30) Johnson, Robin RaneeNeurodegenerative diseases, such as multiple sclerosis (MS), are adversely affected by both stress and inflammation. Theiler's murine encephalomyelitis virus infection is an excellent animal model of MS, allowing examination of central nervous system inflammation during the acute phase of infection. Social disruption stress exacerbates acute Theiler's virus infection. Both social disruption stress and Theiler's virus infection elevate the proinflammatory cytokine, Interleukin-6 (IL-6). The current study examined the necessity and sufficiency of IL-6 in mediating the negative effects of social disruption stress in acute Theiler's virus infection. Experiment 1 blocked IL-6 function with a neutralizing antibody administered simultaneously with social disruption stress. All mice were then infected, and measures of illness, motor impairment and physiological signs of disease were collected up to 21 d postinfection. Experiment 2 administered exogenous IL-6 for one week (replacing social disruption with the cytokine treatment), followed by infection. Measures identical to those collected in Experiment 1 were collected for up to 21 d postinfection. Results indicate that IL-6 is necessary for the development of the sickness, motor impairment, and immunological effects of social stress in acute Theiler's virus infection. In contrast, IL-6 alone can induce some, but not all, of the sickness behavior exacerbations, and was not sufficient for the development of either motor impairment or immunological effects previously associated with social disruption stress. These results have many important implications for further research in the effects of social stress on Theiler's virus infection, as well as clinical implications for both MS and other inflammatory mediated diseases, such as Alzheimer's disease and Parkinson's disease.Item The effects of Shiga toxin 1 on cytokine and chemokine production and apoptosis in a human monocytic cell line(Texas A&M University, 2004-11-15) Harrison, Lisa MargaretSevere bloody diarrhea and subsequent serious post-diarrheal illnesses, including the hemolytic uremic syndrome and central nervous system complications, may develop following infections with Shiga toxin (Stx)-producing bacteria. The cytotoxic actions of Stxs destroy the microvasculature of organs, preventing function. A role for the cytokines tumor necrosis factor-alpha (TNF-[alpha]) and interleukin-1 beta (IL-1[beta]) in exacerbating disease may lie in their ability to up-regulate the Stx receptor, Gb3, on endothelial cell surfaces. A main source of proinflammatory cytokines is the macrophage, thus leading us to utilize the monocytic/macrophage-like cell line, THP-1, as a model for cytokine production in Stx pathogenesis. In addition to treating THP-1 cells with purified Stx1, cells were also treated with lipopolysaccharides (LPS), since bacterial LPS are known to be potent inducers of cytokines, and may be present during infection. Undifferentiated THP-1 cells are sensitive to Stx1 and do not produce TNF-[alpha] or IL-1[beta], while differentiated THP-1 cells, a better model for resident tissue macrophages, are less sensitive to Stx1 and produce TNF-[alpha] and IL-1[beta]. Prolonged expression of TNF-[alpha] mRNA over a 12 h time course experiment led us to inquire whether the extended elevation of transcripts involved Stx1induced mRNA stability. Our data suggest that the presence of Stx1 increases the stabilities of TNF-[alpha] and IL-1[beta] transcripts. In contrast to TNF-[alpha], the level of secreted IL-1[beta] protein does not correlate with the level IL-1[beta] mRNA, suggesting an alteration of post-translational processing and/or secretion of IL-1[beta]. Differentiated THP-1 cells produce chemokines in response to Stx1 and/or LPS treatments. Chemokines may enhance the destruction of tissue cells during an infection by mediating an inflammatory cell influx. Comparison of cytokine and chemokine mRNA and protein kinetics suggests that the regulation of expression may differ between individual cytokines and chemokines. Extension of experimental time courses demonstrated THP-1 cell sensitivity to killing by Stx1, especially in the presence of LPS. Further experiments revealed that undifferentiated and differentiated THP-1 cells were induced to undergo apoptosis following treatment with Stx1, LPS, and Stx1+LPS, and that caspase activation was involved. Collectively, these results allowed us to propose a model of the role of macrophages in Stx1 pathogenesis.Item The role of CCL5 (RANTES) in the immune response against Mycobacterium tuberculosis in the guinea pig(Texas A&M University, 2005-02-17) Skwor, Troy ArthurTuberculosis is the second leading cause of morbidity and mortality worldwide due to an infectious disease. Development of a new tuberculosis (TB) vaccine would be facilitated by a better understanding of the mechanisms of protection induced by the current TB vaccine, Mycobacterium bovis BCG. Recombinant guinea pig (rgp)CCL5 and anti-rgpCCL5 were developed and characterized. The biological activity of rgpCCL5 was determined in a chemotaxis assay using T lymphocytes and pleural exudate cells. The specificity of rabbit anti-rgpCCL5 polyclonal antibody was confirmed by Western blot. RgpCCL5 was used to stimulate alveolar and peritoneal macrophages in vitro. and cytokine/chemokine gene expression was evaluated using real-time PCR. RgpCCL5 stimulated TNFα, IL-1β, CCL2, and CXCL8 mRNA expression and TNFα protein production (as assessed in the L929 cell bioassay) in macrophages. The effect of BCG-vaccination on CCL5 expression and production in leukocytes infected with M. tuberculosis was examined in vitro and in vivo. Polyclonal anti-rgpCCL5 was used to develop an ELISA assay to quantify gpCCL5 protein levels, and real-time PCR was used to detect CCL5 mRNA. Leukocytes isolated from BCG-vaccinated guinea pigs and infected in vitro with virulent M. tuberculosis demonstrated significantly elevated gpCCL5 mRNA and protein compared to cells from naive animals. The response of gpCCL5 to M. tuberculosis in vivo was studied in tuberculous pleural effusions, where peak levels of CCL5 mRNA and protein were reached at day 4 post-induction. Disease severity, cellular differentiation, histology, and cytokine/chemokine mRNA levels in pleural cells and granulomas were analyzed on day 4 in guinea pigs induced with tuberculous pleurisy and treated with either rgpCCL5 or anti-rgpCCL5 by direct intra-pleural injection. In these studies, neutralizing CCL5 resulted in reduced macrophage accumulation, diminished levels of IFNγ, TNFα, and CCL5 mRNA in pleural effusion cells, and reduced spontaneous lymphocyte proliferation. Together these studies suggest an important role for gpCCL5 in activating leukocytes during M. tuberculosis infection.