RESUMEN
This investigation was to elucidate the basis for augmentation of nitric-oxide synthesis in neutrophils exposed to hyperbaric oxygen. Hyperoxia increases synthesis of reactive species leading to S-nitrosylation of ß-actin, which causes temporary inhibition of ß(2) integrin adherence. Impaired ß(2) integrin function and actin S-nitrosylation do not occur in neutrophils from mice lacking type-2 nitric-oxide synthase (iNOS) or when incubated with 1400W, an iNOS inhibitor. Similarly, effects of hyperoxia were abrogated in cells depleted of focal adhesion kinase (FAK) by treatment with small inhibitory RNA and those exposed to a specific FAK inhibitor concurrent with hyperoxia. Nitric oxide production doubles within 10 min exposure to hyperoxia but declines to approximately half-maximum production over an additional 10 min. Elevated nitric oxide production did not occur after FAK depletion or inhibition, or when filamentous actin formation was inhibited by cytochalasin D. Intracellular content of iNOS triples over the course of a 45-min exposure to hyperoxia and iNOS dimers increase in a commensurate fashion. Confocal microscopy and immunoprecipitation demonstrated that co-localization/linkage of FAK, iNOS, and filamentous actin increased within 15 min exposure to hyperoxia but then decreased below the control level. Using isolated enzymes in ex vivo preparations an association between iNOS and filamentous actin mediated by FAK could be demonstrated and complex formation was impeded when actin was S-nitrosylated. We conclude that iNOS activity is increased by an FAK-mediated association with actin filaments but peak nitric oxide production is transient due to actin S-nitrosylation during exposure to hyperoxia.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Antígenos CD18/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Regulación de la Expresión Génica , Neutrófilos/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Animales , Citoesqueleto/metabolismo , Dimerización , Fibrinógeno/metabolismo , Radicales Libres , Glutatión Transferasa/metabolismo , Ratones , Ratones Noqueados , Neutrófilos/metabolismo , Óxido Nítrico Sintasa de Tipo II/química , Oxígeno/metabolismo , Conejos , Especies de Nitrógeno Reactivo , SolubilidadRESUMEN
The investigation goal was to identify mechanisms for reversal of actin S-nitrosylation in neutrophils after exposure to high oxygen partial pressures. Prior work has shown that hyperoxia causes S-nitrosylated actin (SNO-actin) formation, which mediates ß(2) integrin dysfunction, and these changes can be reversed by formylmethionylleucylphenylalanine or 8-bromo-cyclic GMP. Herein we show that thioredoxin reductase (TrxR) is responsible for actin denitrosylation. Approximately 80% of cellular TrxR is localized to the cytosol, divided between the G-actin and short filamentous actin (sF-actin) fractions based on Triton solubility of cell lysates. TrxR linkage to sF-actin requires focal adhesion kinase (FAK) based on immunoprecipitation studies. S-Nitrosylation accelerates actin filament turnover (by mechanisms described previously (Thom, S. R., Bhopale, V. M., Yang, M., Bogush, M., Huang, S., and Milovanova, T. (2011) Neutrophil ß(2) integrin inhibition by enhanced interactions of vasodilator stimulated phosphoprotein with S-nitrosylated actin. J. Biol. Chem. 286, 32854-32865), which causes FAK to disassociate from sF-actin. TrxR subsequently dissociates from FAK, and the physical separation from actin impedes denitrosylation. If SNO-actin is photochemically reduced with UV light or if actin filament turnover is impeded by incubations with cytochalasin D, latrunculin B, 8-bromo-cGMP, or formylmethionylleucylphenylalanine, FAK and TrxR reassociate with sF-actin and cause SNO-actin removal. FAK-TrxR association can also be demonstrated using isolated enzymes in ex vivo preparations. Uniquely, the FAK kinase domain is the site of TrxR linkage. We conclude that through its scaffold function, FAK influences TrxR activity and actin S-nitrosylation.
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Antígenos CD18/metabolismo , Citoesqueleto/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Neutrófilos/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Antígenos CD18/genética , Línea Celular , Citoesqueleto/genética , Quinasa 1 de Adhesión Focal/genética , Humanos , Neutrófilos/citología , Transporte de Proteínas/fisiología , Ratas , Reductasa de Tiorredoxina-Disulfuro/genéticaRESUMEN
We hypothesized that circulating microparticles (MPs) play a role in pro-inflammatory effects associated with carbon monoxide (CO) inhalation. Mice exposed for 1h to 100 ppm CO or more exhibit increases in circulating MPs derived from a variety of vascular cells as well as neutrophil activation. Tissue injury was quantified as 2000 kDa dextran leakage from vessels and as neutrophil sequestration in the brain and skeletal muscle; and central nervous system nerve dysfunction was documented as broadening of the neurohypophysial action potential (AP). Indices of injury occurred following exposures to 1000 ppm for 1h or to 1000 ppm for 40 min followed by 3000 ppm for 20 min. MPs were implicated in causing injuries because infusing the surfactant MP lytic agent, polyethylene glycol telomere B (PEGtB) abrogated elevations in MPs, vascular leak, neutrophil sequestration and AP prolongation. These manifestations of tissue injury also did not occur in mice lacking myeloperoxidase. Vascular leakage and AP prolongation were produced in naïve mice infused with MPs that had been obtained from CO poisoned mice, but this did not occur with MPs obtained from control mice. We conclude that CO poisoning triggers elevations of MPs that activate neutrophils which subsequently cause tissue injuries.
Asunto(s)
Monóxido de Carbono/toxicidad , Enfermedades del Sistema Nervioso Central/inducido químicamente , Exposición por Inhalación/efectos adversos , Material Particulado/toxicidad , Enfermedades Vasculares/inducido químicamente , Animales , Monóxido de Carbono/administración & dosificación , Enfermedades del Sistema Nervioso Central/metabolismo , Enfermedades del Sistema Nervioso Central/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Material Particulado/administración & dosificación , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patologíaRESUMEN
Production of reactive species in neutrophils exposed to hyperoxia causes S-nitrosylation of ß-actin, which increases formation of short actin filaments, leading to alterations in the cytoskeletal network that inhibit ß(2) integrin-dependent adherence (Thom, S. R., Bhopale, V. M., Mancini, D. J., and Milovanova, T. N. (2008) J. Biol. Chem. 283, 10822-10834). In this study, we found that vasodilator-stimulated protein (VASP) exhibits high affinity for S-nitrosylated short filamentous actin, which increases actin polymerization. VASP bundles Rac1, Rac2, cyclic AMP-dependent, and cyclic GMP-dependent protein kinases in close proximity to short actin filaments, and subsequent Rac activation increases actin free barbed end formation. Using specific chemical inhibitors or reducing cell concentrations of any of these proteins with small inhibitory RNA abrogates enhanced free barbed end formation, increased actin polymerization, and ß(2) integrin inhibition by hyperoxia. Alternatively, incubating neutrophils with formylmethionylleucylphenylalanine or 8-bromo-cyclic GMP activates either cyclic AMP-dependent or cyclic GMP-dependent protein kinase, respectively, outside of the short F-actin pool and phosphorylates VASP on serine 153. Phosphorylated VASP abrogates the augmented polymerization normally observed with S-nitrosylated actin, VASP binding to actin, elevated Rac activity, and elevated formation of actin free barbed ends, thus restoring normal ß(2) integrin function.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Antígenos CD18/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Citoesqueleto de Actina/genética , Actinas/genética , Animales , Antígenos CD18/genética , Moléculas de Adhesión Celular/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Activación Enzimática/efectos de los fármacos , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , N-Formilmetionina Leucil-Fenilalanina/farmacología , Fosfoproteínas/genética , Unión Proteica/efectos de los fármacos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Diabetic patients undergoing hyperbaric oxygen therapies (HBO(2)T) for refractory lower extremity neuropathic ulcers exhibit more than a twofold elevation (p=0.004) in circulating stem cells after treatments and the post-HBO(2)T CD34(+) cell population contains two- to threefold higher levels of hypoxia inducible factors-1, -2, and -3, as well as thioredoxin-1 (p<0.003), than cells present in blood before HBO(2)T. Skin margins obtained from 2-day-old abdominal wounds exhibit higher expression of CD133, CD34, hypoxia inducible factor-1, and Trx-1 vs. margins from refractory lower extremity wounds and expression of these proteins in all wounds is increased due to HBO(2)T (p<0.003). HBO(2)T is known to mobilize bone marrow stem cells by stimulating nitric oxide synthase. We found that nitric oxide synthase activity is acutely increased in patients' platelets following HBO(2)T and remains elevated for at least 20 hours. We conclude that HBO(2) T stimulates vasculogenic stem cell mobilization from bone marrow of diabetics and more cells are recruited to skin wounds.
Asunto(s)
Pie Diabético/terapia , Oxigenoterapia Hiperbárica , Células Madre/fisiología , Cicatrización de Heridas/fisiología , Biopsia con Aguja , Plaquetas/enzimología , Movimiento Celular , Pie Diabético/patología , Pie Diabético/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo III/sangreRESUMEN
Fracture healing is a multistage process characterized by inflammation, cartilage formation, bone deposition, and remodeling. Chondrocytes are important in producing cartilage that forms the initial anlagen for the hard callus needed to stabilize the fracture site. We examined the role of FOXO1 by selective ablation of FOXO1 in chondrocytes mediated by Col2α1 driven Cre recombinase. Experimental mice with lineage-specific FOXO1 deletion (Col2α1Cre+FOXO1L/L) and negative control littermates (Col2α1Cre-FOXO1L/L) were used for in vivo, closed fracture studies. Unexpectedly, we found that in the early phases of fracture healing, FOXO1 deletion significantly increased the amount of cartilage formed, whereas, in later periods, FOXO1 deletion led to a greater loss of cartilage. FOXO1 was functionally important as its deletion in chondrocytes led to diminished bone formation on day 22. Mechanistically, the early effects of FOXO1 deletion were linked to increased proliferation of chondrocytes through enhanced expression of cell cycle genes that promote proliferation and reduced expression of those that inhibit it and increased expression of cartilage matrix genes. At later time points experimental mice with FOXO1 deletion had greater loss of cartilage, enhanced formation of osteoclasts, increased IL-6 and reduced numbers of M2 macrophages. These results identify FOXO1 as a transcription factor that regulates chondrocyte behavior by limiting the early expansion of cartilage and preventing rapid cartilage loss at later phases.
Asunto(s)
Condrocitos , Curación de Fractura , Animales , Callo Óseo , Cartílago , Proteína Forkhead Box O1/genética , Ratones , OsteoclastosRESUMEN
Peroxiredoxin 6 (Prdx6), an enzyme with glutathione peroxidase and PLA2 (aiPLA2) activities, is highly expressed in respiratory epithelium, where it participates in phospholipid turnover and antioxidant defense. Prdx6 has been localized by immunocytochemistry and subcellular fractionation to acidic organelles (lung lamellar bodies and lysosomes) and cytosol. On the basis of their pH optima, we have postulated that protein subcellular localization determines the balance between the two activities of Prdx6. Using green fluorescent protein-labeled protein expression in alveolar epithelial cell lines, we showed Prdx6 localization to organellar structures resembling lamellar bodies in mouse lung epithelial (MLE-12) cells and lysosomes in A549 cells. Localization within lamellar bodies/lysosomes was in the luminal compartment. Targeting to lysosome-like organelles was abolished by the deletion of amino acids 31-40 from the Prdx6 NH2-terminal region; deletion of the COOH-terminal region had no effect. A green fluorescent protein-labeled peptide containing only amino acids 31-40 showed lysosomal targeting that was abolished by mutation of S32 or G34 within the peptide. Studies with mutated protein indicated that lipid binding was not necessary for Prdx6 targeting. This peptide sequence has no homology to known organellar targeting motifs. These studies indicate that the localization of Prdx6 in acidic organelles and consequent PLA2 activity depend on a novel 10-aa peptide located at positions 31-40 of the protein.
Asunto(s)
Células Epiteliales/metabolismo , Pulmón/citología , Lisosomas/metabolismo , Peroxiredoxina VI/química , Peroxiredoxina VI/metabolismo , Señales de Clasificación de Proteína , Secuencia de Aminoácidos , Animales , Línea Celular , Células Epiteliales/citología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Liposomas/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Proteínas Mutantes , Mutación/genética , Péptidos/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-ActividadRESUMEN
We hypothesized that oxidative stress from hyperbaric oxygen (HBO(2), 2.8 ATA for 90 min daily) exerts a trophic effect on vasculogenic stem cells. In a mouse model, circulating stem/progenitor cell (SPC) recruitment and differentiation in subcutaneous Matrigel were stimulated by HBO(2) and by a physiological oxidative stressor, lactate. In combination, HBO(2) and lactate had additive effects. Vascular channels lined by CD34(+) SPCs were identified. HBO(2) and lactate accelerated channel development, cell differentiation based on surface marker expression, and cell cycle entry. CD34(+) SPCs exhibited increases in thioredoxin-1 (Trx1), Trx reductase, hypoxia-inducible factors (HIF)-1, -2, and -3, phosphorylated mitogen-activated protein kinases, vascular endothelial growth factor, and stromal cell-derived factor-1. Cell recruitment to Matrigel and protein synthesis responses were abrogated by N-acetyl cysteine, dithioerythritol, oxamate, apocynin, U-0126, neutralizing anti-vascular endothelial growth factor, or anti-stromal cell-derived factor-1 antibodies, and small inhibitory RNA to Trx reductase, lactate dehydrogenase, gp91(phox), HIF-1 or -2, and in mice conditionally null for HIF-1 in myeloid cells. By causing an oxidative stress, HBO(2) activates a physiological redox-active autocrine loop in SPCs that stimulates vasculogenesis. Thioredoxin system activation leads to elevations in HIF-1 and -2, followed by synthesis of HIF-dependent growth factors. HIF-3 has a negative impact on SPCs.
Asunto(s)
Células de la Médula Ósea/metabolismo , Diferenciación Celular , Proliferación Celular , Oxigenoterapia Hiperbárica , Neovascularización Fisiológica , Estrés Oxidativo , Células Madre/metabolismo , Tejido Subcutáneo/irrigación sanguínea , Moduladores de la Angiogénesis/farmacología , Proteínas Angiogénicas/metabolismo , Animales , Antioxidantes/metabolismo , Comunicación Autocrina , Biomarcadores/metabolismo , Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Ciclo Celular , Diferenciación Celular/efectos de los fármacos , Movimiento Celular , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Combinación de Medicamentos , Glutatión/metabolismo , Factor 1 Inducible por Hipoxia/deficiencia , Factor 1 Inducible por Hipoxia/genética , Ácido Láctico/metabolismo , Laminina/metabolismo , Ratones , Ratones Noqueados , Neovascularización Fisiológica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Proteoglicanos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Células Madre/efectos de los fármacos , Tiorredoxinas/metabolismo , Factores de TiempoRESUMEN
FOXO1 transcription factors affect a number of cell types that are important in the host response. Cell types whose functions are modulated by FOXO1 include keratinocytes in the skin and mucosal dermis, neutrophils and macrophages, dendritic cells, Tregs and B-cells. FOXO1 is activated by bacterial or cytokine stimulation. Its translocation to the nucleus and binding to promoter regions of genes that have FOXO response elements is stimulated by the MAP kinase pathway and inhibited by the PI3 kinase/AKT pathway. Downstream gene targets of FOXO1 include pro-inflammatory signaling molecules (TLR2, TLR4, IL-1ß, and TNF-α), wound healing factors (TGF-ß, VEGF, and CTGF) adhesion molecules (integrins-ß1, -ß3, -ß6, αvß3, CD11b, CD18, and ICAM-1), chemokine receptors (CCR7 and CXCR2), B cell regulators (APRIL and BLYS), T-regulatory modulators (Foxp3 and CTLA-4), antioxidants (GPX-2 and cytoglobin), and DNA repair enzymes (GADD45α). Each of the above cell types are found in oral mucosa and modulated by bacteria or an inflammatory microenvironment. FOXO1 contributes to the regulation of these cells, which collectively maintain and repair the epithelial barrier, formation and activation of Tregs that are needed to resolve inflammation, mobilization, infiltration, and activation of anti-bacterial defenses in neutrophils, and the homing of dendritic cells to lymph nodes to induce T-cell and B-cell responses. The goal of the manuscript is to review how the transcription factor, FOXO1, contributes to the activation and regulation of key leukocytes needed to maintain homeostasis and respond to bacterial challenge in oral mucosal tissues. Examples are given with an emphasis on lineage specific deletion of Foxo1 to explore the impact of FOXO1 on cell behavior, inflammation and susceptibility to infection.
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Proteína Forkhead Box O1/metabolismo , Inmunidad Mucosa , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , Animales , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Susceptibilidad a Enfermedades , Proteína Forkhead Box O1/genética , Regulación de la Expresión Génica , Humanos , Linfocitos/inmunología , Linfocitos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Enfermedades Periodontales/etiología , Enfermedades Periodontales/metabolismo , Enfermedades Periodontales/patología , Transducción de SeñalAsunto(s)
Células Endoteliales/metabolismo , Mediadores de Inflamación/metabolismo , Células Madre/metabolismo , Cicatrización de Heridas , Heridas y Lesiones/patología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Heridas y Lesiones/fisiopatologíaRESUMEN
Type 1 diabetes impairs fracture healing. We tested the hypothesis that diabetes affects chondrocytes to impair fracture healing through a mechanism that involves the transcription factor FOXO1. Type 1 diabetes was induced by streptozotocin in mice with FOXO1 deletion in chondrocytes (Col2α1Cre+FOXO1L/L) or littermate controls (Col2α1Cre-FOXO1L/L) and closed femoral fractures induced. Diabetic mice had 77% less cartilage and 30% less bone than normoglycemics evaluated histologically and by micro-computed tomography. Both were reversed with lineage-specific FOXO1 ablation. Diabetic mice had a threefold increase in osteoclasts and a two- to threefold increase in RANKL mRNA or RANKL-expressing chondrocytes compared with normoglycemics. Both parameters were rescued by FOXO1 ablation in chondrocytes. Conditions present in diabetes, high glucose (HG), and increased advanced glycation end products (AGEs) stimulated FOXO1 association with the RANKL promoter in vitro, and overexpression of FOXO1 increased RANKL promoter activity in luciferase reporter assays. HG and AGE stimulated FOXO1 nuclear localization, which was reversed by insulin and inhibitors of TLR4, histone deacetylase, nitric oxide, and reactive oxygen species. The results indicate that chondrocytes play a prominent role in diabetes-impaired fracture healing and that high levels of glucose, AGEs, and tumor necrosis factor-α, which are elevated by diabetes, alter RANKL expression in chondrocytes via FOXO1.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Fracturas del Fémur/metabolismo , Proteína Forkhead Box O1/metabolismo , Curación de Fractura/genética , Animales , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Diabetes Mellitus Experimental/genética , Fracturas del Fémur/genética , Proteína Forkhead Box O1/genética , Curación de Fractura/efectos de los fármacos , Regulación de la Expresión Génica , Glucosa/farmacología , Productos Finales de Glicación Avanzada/farmacología , Ratones , Ratones Noqueados , Ligando RANK/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Microtomografía por Rayos XRESUMEN
BACKGROUND: In this study, we evaluated alternative possibility for CFSE beryllium flow cytometric test against beryllium blood lymphocyte proliferation test (BeLPT) as a standard radioactive clinical screening method to identify sensitization to beryllium. METHODS: Delta PD (the ratio of divided cell population to the total number of cells with subtracted counts of unstimulated cells) of specific beryllium-induced pathogenic CD3+ CD4+ T-lymphocytes and stimulation index (SI) in CFSE proliferation test was compared with delta counts per minute (mean test CPM minus mean control CPM) and SI in radioactive blood BeLPT. RESULTS: Comparison analysis of CFSE and BeLPT demonstrated excellent agreement between delta PD and delta CPM (kappa = 0.845, P << 0.0001). We determined 6.8% positive subjects in the beryllium-exposed, Be-LPT-negative group. The decreased mean difference of these indexes to percentage of average and the long tail in the plot reflects increased sensitivity. CFSE/CD4+ T-cell proliferation assay has 100% specificity, significantly higher sensitivity and efficiency than BeLPT. CONCLUSIONS: Both delta PD, measured by the precursor frequencies method in CFSE assay and delta CPM, defined by tritiated thymidine in BeLPT, can be used for the enumeration of beryllium specific CD4+ T-cell proliferation and may substantially improve the quality of the early diagnosis of beryllium hypersensitivity.
Asunto(s)
Beriliosis/diagnóstico , Berilio/efectos adversos , Bioensayo/métodos , Citometría de Flujo/métodos , Hipersensibilidad/diagnóstico , Timidina , Tritio , Beriliosis/inmunología , Beriliosis/fisiopatología , Bioensayo/normas , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Diagnóstico Precoz , Citometría de Flujo/normas , Humanos , Hipersensibilidad/inmunología , Hipersensibilidad/fisiopatología , Valor Predictivo de las Pruebas , Coloración y Etiquetado/métodos , Células Madre/efectos de los fármacos , Células Madre/inmunologíaRESUMEN
Endothelin-1 (ET-1), tissue plasminogen activator (tPA), and extracellular signal-regulated kinases-mitogen activated protein kinase (ERK-MAPK) are mediators of impaired cerebral hemodynamics after fluid percussion brain injury (FPI) in piglets. Microparticles (MPs) are released into the circulation from a variety of cells during stress, are pro-thrombotic and pro-inflammatory, and may be lysed with polyethylene glycol telomere B (PEG-TB). We hypothesized that MPs released after traumatic brain injury impair hypotensive cerebrovasodilation and that PEG-TB protects the vascular response via MP lysis, and we investigated the relationship between MPs, tPA, ET-1, and ERK-MAPK in that process. FPI was induced in piglets equipped with a closed cranial window. Animals received PEG-TB or saline (vehicle) 30-minutes post-injury. Serum and cerebrospinal fluid (CSF) were sampled and pial arteries were measured pre- and post-injury. MPs were quantified by flow cytometry. CSF samples were analyzed with enzyme-linked immunosorbent assay. MP levels, vasodilatory responses, and CSF signaling assays were similar in all animals prior to injury and treatment. After injury, MP levels were elevated in the serum of vehicle but not in PEG-TB-treated animals. Pial artery dilation in response to hypotension was impaired after injury but protected in PEG-TB-treated animals. After injury, CSF levels of tPA, ET-1, and ERK-MAPK were all elevated, but not in PEG-TB-treated animals. PEG-TB-treated animals also showed reduction in neuronal injury in CA1 and CA3 hippocampus, compared with control animals. These results show that serum MP levels are elevated after FPI and lead to impaired hypotensive cerebrovasodilation via over-expression of tPA, ET-1, and ERK-MAPK. Treatment with PEG-TB after injury reduces MP levels and protects hypotensive cerebrovasodilation and limits hippocampal neuronal cell injury.
Asunto(s)
Lesiones Encefálicas , Región CA1 Hipocampal/patología , Región CA3 Hipocampal/patología , Micropartículas Derivadas de Células/metabolismo , Endotelina-1/líquido cefalorraquídeo , Quinasas MAP Reguladas por Señal Extracelular/líquido cefalorraquídeo , Hipotensión , Activador de Tejido Plasminógeno/líquido cefalorraquídeo , Vasodilatación/fisiología , Animales , Animales Recién Nacidos , Lesiones Encefálicas/sangre , Lesiones Encefálicas/líquido cefalorraquídeo , Lesiones Encefálicas/patología , Región CA1 Hipocampal/metabolismo , Región CA3 Hipocampal/metabolismo , Modelos Animales de Enfermedad , Femenino , Hipotensión/sangre , Hipotensión/líquido cefalorraquídeo , Hipotensión/patología , Masculino , PorcinosRESUMEN
BACKGROUND: Chronic beryllium disease (CBD) is an occupational granulomatous disorder characterized by hypersensitivity to beryllium, mediated by CD4+ T lymphocytes, and predominantly affects the lungs. In this disorder, lymphocyte proliferative responses to beryllium, measured by 3H thymidine incorporation, are used for diagnosis of CBD, for screening asymptomatic workers or former workers to detect unrecognized disease, and for surveillance as a bioassay to detect abnormal exposures. Problems with test variability and the use of radioactivity have recently led to the search for alternative methods. METHODS: We applied a 5,6-carboxyfluorescein diacetate succinimidyl ester flow cytometric technique for measurement of mitogen- and antigen-induced T-lymphocyte proliferation to a group of beryllium-exposed sensitized individuals and beryllium-unexposed controls. RESULTS: We detected mitogen and antigen proliferative responses in CD3+, CD4+, and CD8+ subpopulations. Phytohemagglutinin and Candida stimulated CD4+ and CD8+ T-cell responses, but beryllium appeared to stimulate only CD3+/CD4+ responses. CONCLUSIONS: This technique may provide a sensitive, nonradioactive alternative to the traditional proliferation tests that measure beryllium sensitivity. It offers the added specificity of enabling phenotypic description of the responding cell type and may prove to be easier to standardize for clinical use.
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Contaminantes Ocupacionales del Aire/toxicidad , Beriliosis/patología , Berilio/toxicidad , Linfocitos T/patología , Beriliosis/inmunología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/patología , Proliferación Celular/efectos de los fármacos , Enfermedad Crónica , Citometría de Flujo , Fluoresceínas , Colorantes Fluorescentes , Humanos , Succinimidas , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Timidina/metabolismoRESUMEN
INTRODUCTION: The goals of this study were to investigate the difference in responses between a scuba dive preceded by aerobic exercise (EX) and a nonexercise control dive (CON) and to further evaluate the potential relation between venous gas emboli (VGE) and microparticles (MP). We hypothesized that exercise would alter the quantity and subtype of annexin V-positive MP and VGE. METHODS: Nineteen divers performed two dives to 18 m seawater for 41 min separated by at least 3 d, one of which was preceded by 60 min of treadmill interval exercise. Blood was obtained before exercise, before diving, and 15 min, 2 h, 4 h, and 24 h after surfacing. Intravascular bubbles were quantified by transthoracic echocardiography at 15, 40, 80, and 120 min. RESULTS: The median VGE remained unchanged between the two dives; however, there was a significant increase in VGE in the exercise dive at 40 and 80 min at rest. MP were significantly elevated by approximately 2 times at all time points after CON compared with those after EX. Markers of neutrophil and platelet activation were elevated by both dives, and these elevations were attenuated in the EX dive. CONCLUSIONS: We conclude that some of the differences observed between the EX and CON related to MP and platelet and neutrophil activation provide additional insight into the potential protective benefits of exercise; however, further study is needed to understand the mechanism and true potential of these benefits.
Asunto(s)
Enfermedad de Descompresión/prevención & control , Buceo/fisiología , Ejercicio Físico/fisiología , Activación Neutrófila , Adulto , Micropartículas Derivadas de Células , Enfermedad de Descompresión/inmunología , Embolia Aérea/diagnóstico por imagen , Femenino , Humanos , Masculino , Activación Plaquetaria , UltrasonografíaRESUMEN
Because hyperbaric oxygen treatment mobilizes bone marrow derived-stem/progenitor cells by a free radical mediated mechanism, we hypothesized that there may be differences in mobilization efficiency based on exposure to different oxygen partial pressures. Blood from twenty consecutive patients was obtained before and after the 1st, 10th and 20th treatment at two clinical centers using protocols involving exposures to oxygen at either 2.0 or 2.5 atmospheres absolute (ATA). Post-treatment values of CD34+, CD45-dim leukocytes were always 2-fold greater than the pre-treatment values for both protocols. Values for those treated at 2.5 ATA were significantly greater than those treated at 2.0 ATA by factors of 1.9 to 3-fold after the 10th and before and after the 20th treatments. Intracellular content of hypoxia inducible factors -1, -2, and -3, thioredoxin-1 and poly-ADP-ribose polymerase assessed in permeabilized CD34+ cells with fluorophore-conjugated antibodies were twice as high in all post- versus pre-treatment samples with no significant differences between 2.0 and 2.5 ATA protocols. We conclude that putative progenitor cell mobilization is higher with 2.5 versus 2.0 ATA treatments, and all newly mobilized cells exhibit higher concentrations of an array of regulatory proteins.
Asunto(s)
Antígenos CD34/metabolismo , Oxigenoterapia Hiperbárica , Antígenos Comunes de Leucocito/metabolismo , Neoplasias/terapia , Oxígeno/metabolismo , Células Madre/citología , Anciano , Anciano de 80 o más Años , Células Cultivadas , Femenino , Movilización de Célula Madre Hematopoyética , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/metabolismo , Neoplasias/fisiopatología , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Células Madre/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
BACKGROUND: Spaceflight missions may require crewmembers to conduct Extravehicular Activities (EVA) for repair, maintenance or scientific purposes. Pre-breathe protocols in preparation for an EVA entail 100% hyperoxia exposure that may last for a few hours (5-8 hours), and may be repeated 2-3 times weekly. Each EVA is associated with additional challenges such as low levels of total body cosmic/galactic radiation exposure that may present a threat to crewmember health and therefore, pose a threat to the success of the mission. We have developed a murine model of combined, hyperoxia and radiation exposure (double-hit) in the context of evaluating countermeasures to oxidative lung damage associated with space flight. In the current study, our objective was to characterize the early and chronic effects of repeated single and double-hit challenge on lung tissue using a novel murine model of repeated exposure to low-level total body radiation and hyperoxia. This is the first study of its kind evaluating lung damage relevant to space exploration in a rodent model. METHODS: Mouse cohorts (n=5-15/group) were exposed to repeated: a) normoxia; b) >95% O2 (O2); c) 0.25Gy single fraction gamma radiation (IR); or d) a combination of O2 and IR (O2+IR) given 3 times per week for 4 weeks. Lungs were evaluated for oxidative damage, active TGFß1 levels, cell apoptosis, inflammation, injury, and fibrosis at 1, 2, 4, 8, 12, 16, and 20 weeks post-initiation of exposure. RESULTS: Mouse cohorts exposed to all challenge conditions displayed decreased bodyweight compared to untreated controls at 4 and 8 weeks post-challenge initiation. Chronic oxidative lung damage to lipids (malondialdehyde levels), DNA (TUNEL, cleaved Caspase 3, cleaved PARP positivity) leading to apoptotic cell death and to proteins (nitrotyrosine levels) was elevated all treatment groups. Importantly, significant systemic oxidative stress was also noted at the late phase in mouse plasma, BAL fluid, and urine. Importantly, however, late oxidative damage across all parameters that we measured was significantly higher than controls in all cohorts but was exacerbated by the combined exposure to O2 and IR. Additionally, impaired levels of arterial blood oxygenation were noted in all exposure cohorts. Significant but transient elevation of lung tissue fibrosis (p<0.05), determined by lung hydroxyproline content, was detected as early as 2 week in mice exposed to challenge conditions and persisted for 4-8 weeks only. Interestingly, active TGFß1 levels in +BAL fluid was also transiently elevated during the exposure time only (1-4 weeks). Inflammation and lung edema/lung injury was also significantly elevated in all groups at both early and late time points, especially the double-hit group. CONCLUSION: We have characterized significant, early and chronic lung changes consistent with oxidative tissue damage in our murine model of repeated radiation and hyperoxia exposure relevant to space travel. Lung tissue changes, detectable several months after the original exposure, include significant oxidative lung damage (lipid peroxidation, DNA damage and protein nitrosative stress) and increased pulmonary fibrosis. These findings, along with increased oxidative stress in diverse body fluids and the observed decreases in blood oxygenation levels in all challenge conditions (whether single or in combination), lead us to conclude that in our model of repeated exposure to oxidative stressors, chronic tissue changes are detected that persist even months after the exposure to the stressor has ended. This data will provide useful information in the design of countermeasures to tissue oxidative damage associated with space exploration.
RESUMEN
Inert gases diffuse into tissues in proportion to ambient pressure, and when pressure is reduced, gas efflux forms bubbles due to the presence of gas cavitation nuclei that are predicted based on theory but have never been characterized. Decompression stress triggers elevations in number and diameter of circulating annexin V-coated microparticles (MPs) derived from vascular cells. Here we show that â¼10% MPs from wild-type (WT) but not inflammatory nitric oxide synthase-2 (iNOS) knockout (KO) mice increase in size when exposed to elevated air pressure ex vivo. This response is abrogated by a preceding exposure to hydrostatic pressure, demonstrating the presence of a preformed gas phase. These MPs have lower density than most particles, 10-fold enrichment in iNOS, and generate commensurately more reactive nitrogen species (RNS). Surprisingly, RNS only slowly diffuse from within MPs unless particles are subjected to osmotic stress or membrane cholesterol is removed. WT mice treated with iNOS inhibitor and KO mice exhibit less decompression-induced neutrophil activation and vascular leak. Contrary to injecting naïve mice with MPs from wild-type decompressed mice, injecting KO MPs triggers fewer proinflammatory events. We conclude that nitrogen dioxide is a nascent gas nucleation site synthesized in some MPs and is responsible for initiating postdecompression inflammatory injuries.
Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Enfermedad de Descompresión/metabolismo , Activación Neutrófila/fisiología , Dióxido de Nitrógeno/metabolismo , Lesiones del Sistema Vascular/metabolismo , Animales , Anexina A5/genética , Anexina A5/metabolismo , Permeabilidad de la Membrana Celular/genética , Permeabilidad de la Membrana Celular/fisiología , Micropartículas Derivadas de Células/genética , Colesterol/genética , Colesterol/metabolismo , Inflamación/genética , Inflamación/metabolismo , Inflamación/fisiopatología , Ratones , Ratones Noqueados , Activación Neutrófila/genética , Neutrófilos/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Presión Osmótica/fisiología , Especies de Nitrógeno Reactivo/genética , Especies de Nitrógeno Reactivo/metabolismo , Lesiones del Sistema Vascular/genéticaRESUMEN
The study goal was to use membrane voltage changes during neurohypophysial action potential (AP) propagation as an index of nerve function to evaluate the role that circulating microparticles (MPs) play in causing central nervous system injury in response to decompression stress in a murine model. Mice studied 1 h following decompression from 790 kPa air pressure for 2 h exhibit a 45% broadening of the neurohypophysial AP. Broadening did not occur if mice were injected with the MP lytic agent polyethylene glycol telomere B immediately after decompression, were rendered thrombocytopenic, or were treated with an inhibitor of nitric oxide synthase-2 (iNOS) prior to decompression, or in knockout (KO) mice lacking myeloperoxidase or iNOS. If MPs were harvested from control (no decompression) mice and injected into naive mice, no AP broadening occurred, but AP broadening was observed with injections of equal numbers of MPs from either wild-type or iNOS KO mice subjected to decompression stress. Although not required for AP broadening, MPs from decompressed mice, but not control mice, exhibit NADPH oxidase activation. We conclude that inherent differences in MPs from decompressed mice, rather than elevated MPs numbers, mediate neurological injury and that a component of the perivascular response to MPs involves iNOS. Additional study is needed to determine the mechanism of AP broadening and also mechanisms for MP generation associated with exposure to elevated gas pressure.
Asunto(s)
Potenciales de Acción , Micropartículas Derivadas de Células/metabolismo , Enfermedad de Descompresión/etiología , Descompresión/efectos adversos , Enfermedades de la Hipófisis/etiología , Neurohipófisis/lesiones , Animales , Micropartículas Derivadas de Células/efectos de los fármacos , Enfermedad de Descompresión/metabolismo , Enfermedad de Descompresión/fisiopatología , Modelos Animales de Enfermedad , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Cinética , Ratones , Ratones Noqueados , NADPH Oxidasas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/genética , Peroxidasa/deficiencia , Peroxidasa/genética , Enfermedades de la Hipófisis/metabolismo , Enfermedades de la Hipófisis/fisiopatología , Neurohipófisis/metabolismo , Neurohipófisis/fisiopatología , Polietilenglicoles/farmacología , Trombocitopenia/metabolismo , Trombocitopenia/fisiopatologíaRESUMEN
The study goal was to evaluate responses in humans following decompression from open-water SCUBA diving with the hypothesis that exertion underwater and use of a breathing mixture containing more oxygen and less nitrogen (enriched air nitrox) would alter annexin V-positive microparticle (MP) production and size changes and neutrophil activation, as well as their relationships to intravascular bubble formation. Twenty-four divers followed a uniform dive profile to 18 m of sea water breathing air or 22.5 m breathing 32% oxygen/68% nitrogen for 47 min, either swimming with moderately heavy exertion underwater or remaining stationary at depth. Blood was obtained pre- and at 15 and 120 min postdive. Intravascular bubbles were quantified by transthoracic echocardiography postdive at 20-min intervals for 2 h. There were no significant differences in maximum bubble scores among the dives. MP number increased 2.7-fold, on average, within 15 min after each dive; only the air-exertion dive resulted in a significant further increase to 5-fold over baseline at 2 h postdive. Neutrophil activation occurred after all dives. For the enriched air nitrox stationary at depth dive, but not for other conditions, the numbers of postdive annexin V-positive particles above 1 µm in diameter were correlated with intravascular bubble scores (correlation coefficients â¼0.9, P < 0.05). We conclude that postdecompression relationships among bubbles, MPs, platelet-neutrophil interactions, and neutrophil activation appear to exist, but more study is required to improve confidence in the associations.