Browsing by Author "Slayback-Barry, Denise"

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    Biofilm and Virulence Regulation of the Cystic Fibrosis Associated Pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa
    (2020-05) Ramos-Hegazy, Layla; Anderson, Gregory; Perrin, Benjamin; Slayback-Barry, Denise
    Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in S. maltophilia, isogenic deletion of phosphoglycerate mutase (gpmA) and two chaperone-usher pilin subunits, S. maltophilia fimbrae-1 (smf-1) and cblA, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δsmf-1 and ΔcblA showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in P. aeruginosa, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when polB, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in S. maltophilia and genetic switches during the acute infection lifestyle in P. aeruginosa.
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    Inflammation in the early pathogenesis of diabetic retinopathy
    (2018-06) Wang, Shukun; Belecky-Adams, Teri; Dai, Guoli; Slayback-Barry, Denise
    Introduction – Diabetes is a growing health concern. Diabetic retinopathy (DR), is a complication resulting from long-term diabetes and is currently the leading cause of blindness in the US. The cause of pericyte loss, an early indiciator of DR progression, is currently unknown. Inflammation, increased in diabetes, could play a role in the progression of DR and be one of the causes of pericyte loss. Methods and Materials- Retinas from 3, 6, 12, 18, and 24 week Akita (AK) and DB mice were taken and cell counts, vasculature, and mRNA expression were examined. Pericytes treated with IFN-γ and PDGF-BB in chronic and acute conditions and PDGFRβ signaling was determined using Western blot analysis. Apoptosis and proliferation were examined using western blots and immunohistochemistry of IFN-γ treated pericytes. Results- Pericytes numbers were changed as 3 weeks in DB and Akita models. Proinflammatory markers increased at 6 weeks and displayed their maximum expression at 12 and 18 weeks. Chronic treatments with IFN-γ changed AKT and PKCδ activation and increases pericytes apoptosis. Discussion- Pericyte loss appears to begin prior to an increase in pro-inflammatory factors indicating that perhaps retinal inflammtion may not initiate pericyte loss or that loss of pericyte numbers at 3 weeks may be due to reduced numbers of neural crest cells migrating to vasculature or a reduced number of neural crest cells differentiating into pericytes. Proinflammatory changes occur between 3-6 weeks in both Akita and DB models of diabetes; however, the peak levels in expression of proinflammtory markers differs between Akita and DB models. One of the markers that increased very early in the progression of diabetic retinopathy, IFN-γ, triggered apoptosis in isolated retinal pericytes by increasing protein kinase Cδ activation, which then reduced activity of AKT.
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    Meningeal Foam Cells and Ependymal Cells in Axolotl Spinal Cord Regeneration
    (Frontiers, 2019-11) Enos, Nathaniel; Takenaka, Hidehito; Scott, Sarah; Salfity, Hai V. N.; Kirk, Maia; Egar, Margaret W.; Sarria, Deborah A.; Slayback-Barry, Denise; Belecky-Adams, Teri; Chernoff, Ellen A. G.; Biology, School of Science
    A previously unreported population of foam cells (foamy macrophages) accumulates in the invasive fibrotic meninges during gap regeneration of transected adult Axolotl spinal cord (salamander Ambystoma mexicanum) and may act beneficially. Multinucleated giant cells (MNGCs) also occurred in the fibrotic meninges. Actin-label localization and transmission electron microscopy showed characteristic foam cell and MNGC podosome and ruffled border-containing sealing ring structures involved in substratum attachment, with characteristic intermediate filament accumulations surrounding nuclei. These cells co-localized with regenerating cord ependymal cell (ependymoglial) outgrowth. Phase contrast-bright droplets labeled with Oil Red O, DiI, and DyRect polar lipid live cell label showed accumulated foamy macrophages to be heavily lipid-laden, while reactive ependymoglia contained smaller lipid droplets. Both cell types contained both neutral and polar lipids in lipid droplets. Foamy macrophages and ependymoglia expressed the lipid scavenger receptor CD36 (fatty acid translocase) and the co-transporter toll-like receptor-4 (TLR4). Competitive inhibitor treatment using the modified fatty acid Sulfo-N-succinimidyl Oleate verified the role of the lipid scavenger receptor CD36 in lipid uptake studies in vitro. Fluoromyelin staining showed both cell types took up myelin fragments in situ during the regeneration process. Foam cells took up DiI-Ox-LDL and DiI-myelin fragments in vitro while ependymoglia took up only DiI-myelin in vitro. Both cell types expressed the cysteine proteinase cathepsin K, with foam cells sequestering cathepsin K within the sealing ring adjacent to the culture substratum. The two cell types act as sinks for Ox-LDL and myelin fragments within the lesion site, with foamy macrophages showing more Ox-LDL uptake activity. Cathepsin K activity and cellular localization suggested that foamy macrophages digest ECM within reactive meninges, while ependymal cells act from within the spinal cord tissue during outgrowth into the lesion site, acting in complementary fashion. Small MNGCs also expressed lipid transporters and showed cathepsin K activity. Comparison of 3H-glucosamine uptake in ependymal cells and foam cells showed that only ependymal cells produce glycosaminoglycan and proteoglycan-containing ECM, while the cathepsin studies showed both cell types remove ECM. Interaction of foam cells and ependymoglia in vitro supported the dispersion of ependymal outgrowth associated with tissue reconstruction in Axolotl spinal cord regeneration.