RESUMEN
BACKGROUND: The receptor for advanced glycation endproducts (RAGE) has been implicated as a critical molecule in the pathogenesis of experimental asthma/allergic airway inflammation (AAI). It has been previously shown that RAGE acts both upstream of interleukin-33 (IL-33) release and downstream of IL-33 release via RAGE-dependent IL-33-induced accumulation of type 2 innate lymphoid cells (ILC2s) in the lungs, which perpetuate type 2 inflammation and mucus metaplasia. However, the mechanism by which RAGE mediates downstream IL-33-induced type 2 inflammatory responses is unknown. OBJECTIVE: This study tested the hypothesis that ILC2s are recruited to the lungs via RAGE-dependent vascular cell adhesion molecule 1 (VCAM-1) expression on lung endothelial cells. METHODS: House dust mite extract, Alternaria alternata extract, or rIL-33 was used to induce AAI/VCAM-1 expression in wild-type (WT) and RAGE-knockout (RAGE-KO) mice. Intravenous (i.v.) anti-VCAM-1 or intraperitoneal (i.p.) ß7 blocking antibody administration was used to determine the role of VCAM-1 in IL-33-induced AAI. RESULTS: Enhanced VCAM-1 expression in the lungs by HDM, AA, or rIL-33 exposure was found to be RAGE-dependent. In addition, stimulation of primary mouse lung endothelial cells with IL-33 induced VCAM-1 expression in WT, but not RAGE-KO cells. Administration of VCAM-1 and ß7-integrin blocking antibodies reduced IL-33-induced eosinophilic inflammation, mucus metaplasia, and type 2 inflammatory responses. CONCLUSION: This study demonstrates that allergen- and cytokine-induced VCAM-1 expression is RAGE-dependent and contributes to lung ILC2 accumulation and downstream eosinophilic inflammation, mucus metaplasia, and type 2 inflammatory responses.
Asunto(s)
Asma/patología , Células Endoteliales/metabolismo , Inflamación/inducido químicamente , Interleucina-33/farmacología , Receptor para Productos Finales de Glicación Avanzada/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Alérgenos/inmunología , Animales , Asma/inducido químicamente , Células Cultivadas , Citocinas/metabolismo , Interleucina-33/inmunología , Pulmón/patología , Linfocitos/inmunología , RatonesRESUMEN
Reactive oxygen species, including superoxide, generally are considered neurotoxic molecules whose effects can be alleviated by antioxidants. Different from this view, we show that scavenging of superoxide with an antioxidant enzyme is associated with deficits in hippocampal long-term potentiation (LTP), a putative neural substrate of memory, and hippocampal-mediated memory function. Using transgenic mice that overexpress extracellular superoxide dismutase (EC-SOD), a superoxide scavenger, we found that LTP was impaired in hippocampal area CA1 despite normal LTP in area CA3. The LTP impairment in area CA1 could be reversed by inhibition of EC-SOD. In addition, we found that EC-SOD transgenic mice exhibited impaired long-term memory of fear conditioning to contextual cues despite exhibiting normal short-term memory of the conditioning experience. These findings strongly suggest that superoxide, rather than being considered exclusively a neurotoxic molecule, should also be considered a signaling molecule necessary for normal neuronal function.
Asunto(s)
Aprendizaje por Asociación , Espacio Extracelular/enzimología , Potenciación a Largo Plazo , Trastornos de la Memoria/genética , Superóxido Dismutasa/biosíntesis , Animales , Reacción de Prevención , Señales (Psicología) , Potenciales Postsinápticos Excitadores/fisiología , Miedo , Heterocigoto , Hipocampo/citología , Hipocampo/metabolismo , Hipocampo/fisiopatología , Humanos , Técnicas In Vitro , Potenciación a Largo Plazo/genética , Masculino , Trastornos de la Memoria/fisiopatología , Ratones , Ratones Transgénicos , Umbral del Dolor , Técnicas de Placa-Clamp , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Conducta Espacial , Superóxido Dismutasa/genética , Transmisión Sináptica/genéticaRESUMEN
Extracellular superoxide dismutase (EC SOD) is generally the least abundant SOD isozyme in tissues, while the intracellular Cu,Zn SOD is usually the most abundant isozyme. The biological significance of EC SOD is unknown. Immunolocalization studies show that EC SOD is in the connective tissue surrounding smooth muscle in vessels and airways within the lung. Endothelium derived relaxing factor, thought to be a nitric oxide (NO) species, is a primary mediator of vascular relaxation. During NO.'s diffusion between the endothelium and smooth muscle, extracellular superoxide would be the most efficient scavenger of NO(.). High levels of extracellular superoxide dismutase in vessels could, therefore, be essential to enable NO. to modulate vascular tone. To evaluate the hypothesis that vessel walls are functionally rich in extracellular superoxide scavenging capacity, this study quantitates the EC SOD levels in pulmonary and systemic vessels and in airways. Both pulmonary and systemic arteries in humans and baboons were found to contain high activities of EC SOD. The level of EC SOD in all human and baboon arteries examined is greater than or equal to the level of intracellular Cu,Zn SOD, and EC SOD accounted for over 70% of the total SOD activity in some vessels examined. Immunolocalization of EC SOD in human and baboon vessels show similar distributions of this enzyme in pulmonary and systemic vessels. EC SOD is located beneath the endothelium, surrounding smooth muscle cells, and throughout the adventitia of vessels. The high level of EC SOD in vessels, and its localization between endothelial and smooth muscle cells, suggest that regulation of superoxide may be particularly important in this region, possibly in regulating vascular tone.
Asunto(s)
Bronquios/enzimología , Pulmón/irrigación sanguínea , Músculo Liso Vascular/enzimología , Papio/metabolismo , Superóxido Dismutasa/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Estudios de Evaluación como Asunto , Femenino , Humanos , Inmunohistoquímica , Masculino , Especificidad de la Especie , Resistencia Vascular/fisiologíaRESUMEN
The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung tissue and is believed to protect the lung from oxidative damage that results in diseases such as pulmonary fibrosis. This study tests the hypothesis that proteolytic removal of the heparin-binding domain of EC-SOD results in clearance of the enzyme from the extracellular matrix of pulmonary tissues and leads to a loss of antioxidant protection. Using a polyclonal antibody to mouse EC-SOD, the immunodistribution of EC-SOD in normal and bleomycin-injured lungs was examined. EC-SOD labeling was strong in the matrix of vessels, airways, and alveolar surfaces and septa in control lungs. At 2 d post-treatment, a slight increase in EC-SOD staining was evident. In contrast, lungs examined 4 or 7 d post-treatment, showed an apparent loss of EC-SOD from the matrix and surface of alveolar septa. Notably, at 7 d post-treatment, the truncated form of EC-SOD was found in the bronchoalveolar lavage fluid of bleomycin-treated mice, suggesting that EC-SOD is being removed from the extracellular matrix through proteolysis. However, loss of EC-SOD through proteolysis did not correlate with a decrease in overall pulmonary EC-SOD activity. The negligible effect on EC-SOD activity may reflect the large influx of intensely staining inflammatory cells at day 7. These results indicate that injuries leading to pulmonary fibrosis have a significant effect on EC-SOD distribution due to proteolytic removal of the heparin-binding domain and may be important in enhancing pulmonary injuries by altering the oxidant/antioxidant balance in alveolar interstitial spaces.
Asunto(s)
Pulmón/enzimología , Fibrosis Pulmonar/enzimología , Superóxido Dismutasa/metabolismo , Animales , Antioxidantes/metabolismo , Bleomicina , Líquido del Lavado Bronquioalveolar/química , Modelos Animales de Enfermedad , Matriz Extracelular/enzimología , Heparina/metabolismo , Hidrólisis , Inmunohistoquímica/métodos , Pulmón/patología , Ratones , Unión Proteica , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patologíaRESUMEN
Transgenic mice, which had been transfected with the human extracellular superoxide dismutase gene, causing an approximate five-fold increase in brain parenchymal extracellular superoxide dismutase activity, were used to investigate the role of extracellular superoxide dismutase in ischemic brain injury. Transgenic (n = 21) and wild-type (n = 19) mice underwent 90 min of intraluminal middle cerebral artery occlusion and 24 h of reperfusion. Severity of resultant hemiparesis and cerebral infarct size were measured. Wild-type mice had larger infarcts (cortex: wild type =37+/-14 mm3, transgenic = 27+/-13 mm3, P=0.03; subcortex: wild type = 33+/-14 mm3, transgenic = 23+/-10 mm3, P = 0.02). Neurological scores, however, were similar (P = 0.29). Other mice underwent autoradiographic determination of intra-ischemic cerebral blood flow. The volume of tissue at risk of infarction (defined as volume of tissue where blood flow was <25 ml/100g/min) was similar between groups (cortex: wild type = 51+/-15 mm3, transgenic = 47+/-9 mm3, P=0.65; subcortex: wild type = 39+/-16 mm3, transgenic= 37+/-17 mm3, P=0.81). These results indicate that antioxidant scavenging of free radicals by extracellular superoxide dismutase plays an important role in the histological response to a focal ischemic brain insult.
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Infarto Cerebral/prevención & control , Hemiplejía/prevención & control , Ataque Isquémico Transitorio/fisiopatología , Superóxido Dismutasa/genética , Animales , Presión Sanguínea , Temperatura Corporal , Infarto Cerebral/patología , Circulación Cerebrovascular , Círculo Arterial Cerebral/anomalías , Círculo Arterial Cerebral/anatomía & histología , Lateralidad Funcional , Hematócrito , Hemiplejía/fisiopatología , Humanos , Inmunidad Innata/genética , Ataque Isquémico Transitorio/genética , Ataque Isquémico Transitorio/patología , Masculino , Ratones , Ratones Transgénicos , Factores de Riesgo , Superóxido Dismutasa/metabolismoRESUMEN
In this study we demonstrate that, in addition to blood, alpha1-microglobulin (alpha1m) is present in most tissues, including liver, heart, eye, kidney, lung, pancreas, and skeletal muscle. Western blotting of perfused and homogenized rat tissue supernatants revealed alpha1m in its free, monomeric form and in high molecular weight forms, corresponding to the complexes fibronectin-alpha1m and alpha1-inhibitor-3-alpha1m, which have previously been identified in plasma. The liver also contained a series of alpha1m isoforms with apparent molecular masses between 40 and 50 kD. These bands did not react with anti-inter-alpha-inhibitor antibodies, indicating that they do not represent the alpha1m-bikunin precursor protein. Similarly, the heart contained a 45-kD alpha1m band and the kidney a 50-kD alpha1m band. None of these alpha1m isoforms was present in plasma. Immunohistochemical analysis of human tissue demonstrated granular intracellular labeling of alpha1m in hepatocytes and in the proximal epithelial cells of the kidney. In addition, alpha1m immunoreactivity was detected in the interstitial connective tissue of heart and lung and in the adventitia of blood vessels as well as on cell surfaces of cardiocytes. alpha1m mRNA was found in the liver and pancreas by polymerase chain reaction, suggesting that the protein found in other tissues is transported via the bloodstream from the production sites in liver and pancreas. The results of this study indicate that in addition to its role in plasma, alpha1m may have important functions in the interstitium of several tissues. (J Histochem Cytochem 46:887-893, 1998)
Asunto(s)
Glicoproteínas/metabolismo , Glicoproteínas de Membrana , Inhibidor de la Tripsina de Soja de Kunitz , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/sangre , Humanos , Inmunohistoquímica , Especificidad de Órganos , Isoformas de Proteínas/sangre , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: To examine the molecular structure and ultrastructural distribution of a novel amine oxidase in human ciliary body. METHODS: Human ciliary bodies were solubilized with a nonionic detergent. The solubilized material was subjected to affinity chromatography with 2B4.14.1, a monoclonal antibody which recognizes a family of ciliary body glycoproteins. Proteins eluted from the affinity column were further separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Peptides produced from a 2B4.14. 1-reactive protein with an approximate molecular weight of 100 kDa were analyzed by Edman degradation. The protein thus identified was further examined by Western blotting and immunoelectron microscopy with anti-peptide antisera. RESULTS: Peptide sequences from the 100 kDa ciliary body protein were identical to the predicted protein sequence of an amine oxidase identified recently in a human placental cDNA library. The identity of the ciliary body protein was confirmed by Western blotting with rabbit antiserum generated against the predicted carboxy-terminal peptide of human placenta amine oxidase. Western blotting under nonreducing conditions and following glycosidase digestion indicated that the native enzyme is a disulfide-linked homodimer with multiple N-linked oligosaccharide side chains. By immunoelectron microscopy, the ciliary body amine oxidase was localized to the plasma membranes of inner epithelial cells. CONCLUSIONS: Human placenta amine oxidase is present on the plasma membranes of ciliary body inner epithelial cells. This finding provides a potential explanation for amine oxidase enzyme activity detected in previous studies of anterior segment tissues. Though the functional role of human placenta amine oxidase in the eye is unclear, it may contribute to the production of H2O2 in aqueous humor.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/química , Amina Oxidasa (conteniendo Cobre)/metabolismo , Cuerpo Ciliar/enzimología , Monoaminooxidasa/química , Monoaminooxidasa/metabolismo , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Western Blotting , Cuerpo Ciliar/química , Cobre , Electroforesis en Gel de Poliacrilamida , Epitelio/enzimología , Humanos , Metaloproteínas/química , Metaloproteínas/metabolismo , Microscopía Inmunoelectrónica , Persona de Mediana EdadRESUMEN
Maintenance of low vascular tone within the placenta is mediated by nitric oxide (NO). The half-life of NO is very short, as superoxide anion (O2-) rapidly inactivates NO to form peroxynitrite. Superoxide dismutases compete with NO for O2-. By scavenging O2-, superoxide dismutase promotes the vasodilatory action of NO. Extracellular superoxide dismutase (EC-SOD) is present in high concentrations within the extracellular matrix of systemic arteries and has been proposed to mediate vascular smooth muscle tone by increasing NO bioavailability. The localization and activity of EC-SOD within the human placenta has not been determined. Placental EC-SOD may be involved in placental vascular tone, and abnormal activity may lead to pre-eclampsia secondary to increased O2--mediated inactivation of NO. To investigate this possibility, the activity and localization of human placental EC-SOD was determined in normal women, and then compared to pre-eclamptic women. Placental EC-SOD localized within the villous extracellular matrix around arterioles, and there were no differences in distribution between normal and pre-eclamptic women. There were no differences in placental EC-SOD activity between normal and pre-eclamptic subjects in either center (33.7+/-4.1 versus 33.1+/-2.5, P=0.6), or peripheral (34.3+/-5.6 versus 34.0+/-3.5, P=0.9) samples. EC-SOD localization around villous vessels suggests that EC-SOD serves potentially to protect the fetal vasculature from O2-, in both normal and pre-eclamptic pregnancies. Placental EC-SOD distribution and activity is not different between pre-eclamptic and normal women, suggesting that EC-SOD is not involved in the vascular changes seen in pre-eclampsia.
Asunto(s)
Espacio Extracelular/enzimología , Placenta/enzimología , Superóxido Dismutasa/metabolismo , Adulto , Arteriolas/enzimología , Western Blotting , Vellosidades Coriónicas/irrigación sanguínea , Vellosidades Coriónicas/enzimología , Matriz Extracelular/enzimología , Femenino , Humanos , Técnicas para Inmunoenzimas , EmbarazoRESUMEN
The distinction of malignant mesothelioma from tumors metastatic to the serosal membranes can often be made based on the results of histochemical or immunohistochemical studies. However, in some cases, these techniques are inadequate to make a firm diagnosis. In these instances, electron microscopic studies with the observation of a constellation of characteristic ultrastructural findings may permit an unequivocal diagnosis of mesothelioma.
Asunto(s)
Mesotelioma/diagnóstico , Mesotelioma/ultraestructura , Microscopía Electrónica , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Diagnóstico Diferencial , Humanos , Ácido Hialurónico/metabolismo , Inmunohistoquímica , Uniones Intercelulares/ultraestructura , Filamentos Intermedios/ultraestructura , Mesotelioma/metabolismo , Microvellosidades/ultraestructura , Sarcoma/diagnóstico , Sarcoma/metabolismo , Sarcoma/ultraestructuraRESUMEN
Extracellular superoxide dismutase (EC-SOD) is one of three mammalian SOD isozymes. Although there is knowledge of the functional role of EC-SOD in arteries, little is known about the function of EC-SOD in other tissues, including the brain. As a first step toward improving our understanding of EC-SOD in the brain, we studied the localization of EC-SOD in the central nervous system of the adult mouse using immunohistochemistry. We detected EC-SOD staining in a subpopulation of neurons throughout the brain as well as in tanycytes in the mediobasal hypothalamus. Particularly prominent EC-SOD staining was observed in neurons of the hilar region of the hippocampus, the lateral habenular nucleus of the thalamus, and the suprachiasmatic nuclei of the hypothalamus. Substantial numbers of neurons were distributed throughout the striatum and cortex; the morphology and distribution of these cells was similar to neurons previously shown to contain the neuronal isoform of nitric oxide synthase. In contrast to other regions with prominent EC-SOD immunoreactivity, EC-SOD localization in tanycytes occurred in a region lacking a blood-brain barrier. The high levels of EC-SOD present in discrete populations of cells in these regions suggest that EC-SOD plays an important, specialized role in the physiology and/or pathology in the brain.
Asunto(s)
Encéfalo/enzimología , Espacio Extracelular/enzimología , Superóxido Dismutasa/metabolismo , Animales , Especificidad de Anticuerpos , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Diencéfalo/enzimología , Hipocampo/enzimología , Técnicas para Inmunoenzimas , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Neostriado/enzimología , Fracciones Subcelulares/enzimologíaRESUMEN
CNS oxygen (O2) toxicity is complex, and the etiology of its most severe manifestation, O2 convulsions, is yet to be determined. A role for depletion of the brain GABA pool has been proposed, although recent data have implicated production of reactive O2 species, e.g. H2O2, in this process. We hypothesized that the production of H2O2 and NH3 produced by monoamine oxidase (MAO) would lead to depletion of GABA and production of nitric oxide (NO.) respectively, and thereby enhance CNS O2 toxicity. In this study, rats treated with an MAO inhibitor (pargyline) or a nitric oxide synthase inhibitor (LNNA) were protected against O2-induced convulsions. Selected cerebral amino acids including arginine were measured in control and O2 treated rats (6 ATA, 20 min) with or without drug pretreatment. After O2 exposure, the cerebral pools of glutamate, aspartate, and GABA decreased significantly while glutamine content increased relative to control (P < 0.05). After treatment with either enzyme inhibitor, glutamine, glutamate and aspartate concentrations were maintained near control levels. Remarkably, GABA depletion by O2 was not prevented despite protection from seizures by both pargyline and LNNA. The NO. precursor, arginine, was increased significantly in the brain by toxic O2 exposure, but both pargyline and LNNA inhibited this effect. Simultaneous norepinephrine measurements indicated that its storage substantially decreased during hyperoxia (P < 0.05), but this effect too was blocked by either pargyline or LNNA. These data indicate that protection against O2 by these inhibitors is not related to preservation of the GABA pool. More importantly, O2 dependent norepinephrine metabolism and NO. synthesis appear to be interactive during CNS O2 toxicity.
Asunto(s)
Aminoácidos/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Óxido Nítrico/metabolismo , Norepinefrina/metabolismo , Oxígeno/efectos adversos , Animales , Arginina/análogos & derivados , Arginina/farmacología , Oxigenoterapia Hiperbárica , Masculino , Inhibidores de la Monoaminooxidasa/farmacología , Nitroarginina , Concentración Osmolar , Pargilina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Peroxynitrite anion is a powerful oxidant which can initiate nitration and hydroxylation of aromatic rings. Peroxynitrite can be formed in several ways, e.g. from the reaction of nitric oxide with superoxide or from hydrogen peroxide and nitrite at acidic pH. We investigated pH dependent nitration and hydroxylation resulting from the reaction of hydrogen peroxide and nitrite to determine if this reaction proceeds at pH values which are known to occur in vivo. Nitration and hydroxylation products of tyrosine and salicyclic acid were separated with an HPLC column and measured using ultraviolet and electrochemical detectors. These studies revealed that this reaction favored hydroxylation between pH 2 and pH 4, while nitration was predominant between pH 5 and pH 6. Peroxynitrite is presumed to be an intermediate in this reaction as the hydroxylation and nitration profiles of authentic peroxynitrite showed similar pH dependence. These findings indicate that hydrogen peroxide and nitrite interact at hydrogen ion concentrations present under some physiologic conditions. This interaction can initiate nitration and hydroxylation of aromatic molecules such as tyrosine residues and may thereby contribute to the biochemical and toxic effects of the molecules.
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Peróxido de Hidrógeno/química , Inflamación/metabolismo , Nitratos/química , Estrés Oxidativo/fisiología , Nitrito de Sodio/química , Tirosina/química , Concentración de Iones de Hidrógeno , Hidroxilación , Hierro/metabolismo , Salicilatos/química , Ácido Salicílico , Tirosina/análogos & derivados , Tirosina/síntesis químicaRESUMEN
AIMS: Recent studies suggest the importance of oxidant stress in the progression of pulmonary fibrosis. The aim of this study was to investigate extracellular superoxide dismutase (ECSOD), the major antioxidant enzyme of the extracellular matrix of human lung, in biopsy-proven idiopathic pulmonary fibrosis (IPF) related to usual interstitial pneumonia (UIP). METHODS AND RESULTS: Fibrotic areas and fibroblastic foci in UIP lungs were notable for absence of ECSOD by immunohistochemistry. Western blotting showed significantly lowered immunoreactivity of ECSOD in fibrotic compared with non-fibrotic areas of the diseased lung. The only cell type that showed intense ECSOD positivity in UIP was the interstitial mast cell. In order to investigate the mechanism for ECSOD depletion in fibrotic areas, alveolar epithelial cells were exposed to tumour necrosis factor-alpha and transforming growth factor (TGF)-beta1; TGF-beta suggested a trend towards decreased synthesis. Patients with UIP were also assessed to determine whether this disease is associated with a naturally occurring mutation in ECSOD (Arg213Gly) which leads to a loss of tissue binding of ECSOD. No significant differences could be found in the allele or genotype frequencies of this polymorphism between 63 UIP patients and 61 control subjects. CONCLUSION: Overall, consistent with several other antioxidant enzymes, ECSOD is very low in fibrotic areas of UIP, which may further increase the oxidant burden in this disease.
Asunto(s)
Enfermedades Pulmonares Intersticiales/enzimología , Enfermedades Pulmonares Intersticiales/patología , Fibrosis Pulmonar/enzimología , Fibrosis Pulmonar/patología , Superóxido Dismutasa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Sustitución de Aminoácidos , Secuencia de Bases , Estudios de Casos y Controles , Línea Celular , ADN/genética , Femenino , Variación Genética , Genotipo , Humanos , Inmunohistoquímica , Pulmón/enzimología , Enfermedades Pulmonares Intersticiales/genética , Masculino , Persona de Mediana Edad , Oxidantes/metabolismo , Fibrosis Pulmonar/genética , Superóxido Dismutasa/genéticaRESUMEN
The role of extracellular superoxide in the pathogenesis of vasogenic edema was studied using transgenic mice expressing a 5-fold increase in extracellular superoxide dismutase (EC-SOD) activity in their brains. Increased EC-SOD expression offered significant protection against edema development after cold-induced injury (44% less edema than nontransgenic littermates, p < 0.05). Since iron may contribute to vasogenic edema by catalyzing the production of hydroxyl radical from superoxide and hydrogen peroxide, the effects of the chelator deferoxamine were studied. Deferoxamine reduced edema formation after cold-induced injury (43% less edema than controls, p < 0.05); however, treatment with iron-saturated deferoxamine also reduced edema development in mice (32-48% less edema, p < 0.05). This suggested that the protection offered by deferoxamine was independent of its ability to chelate iron. An iron-independent mechanism by which superoxide can contribute to vasogenic edema is via reaction with nitric oxide to produce the potentially toxic peroxynitrite anion, which is also scavenged by deferoxamine. Mice treated with an inhibitor of nitric oxide synthase were protected against cold-induced edema (37% less edema, p < 0.05). EC-SOD transgenic mice received no additional protection by inhibition of nitric oxide synthesis, supporting this novel alternative mechanism of edema formation.
Asunto(s)
Edema Encefálico/etiología , Frío , Óxido Nítrico/metabolismo , Superóxido Dismutasa/metabolismo , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Edema Encefálico/enzimología , Edema Encefálico/metabolismo , Edema Encefálico/prevención & control , Deferoxamina/farmacología , Depuradores de Radicales Libres , Hidróxidos , Radical Hidroxilo , Quelantes del Hierro , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico SintasaRESUMEN
About 8% of our cases of mesothelioma occur in women, with a median age of 59 years. Our percentage is lower than other series reported in the literature because of the large number of occupationally exposed men referred to our laboratory. Tumor arose in the pleura in 86% of the women in our study, and the majority were epithelial. Pleural plaques were found in half of the women for which this information was available, and asbestosis was found in only 16%. A history of exposure to asbestos was identified in three quarters of the women, more than half of whom were household contacts of asbestos workers. Occupational exposure to asbestos was identified in only 19% of patients. An elevated tissue asbestos burden was noted in 70% of women from whom lung tissue was available for analysis. The main fiber type identified was amosite, followed by tremolite and chrysotile. These findings and those from other countries suggest a need for reassessment of the background rate of mesothelioma in industrialized nations.
Asunto(s)
Asbestosis/patología , Mesotelioma/patología , Neoplasias Peritoneales/patología , Peritoneo/patología , Pleura/patología , Neoplasias Pleurales/patología , Adulto , Anciano , Anciano de 80 o más Años , Asbesto Amosita/análisis , Asbestos Anfíboles/análisis , Asbestos Serpentinas/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fibras Minerales/análisisRESUMEN
The proteinase "inhibitor" alpha 2-macroglobulin (alpha 2M) is able to entrap and form covalent linkages with diverse proteins during a transient proteinase-activated state. These complexes are rapidly endocytosed after binding to receptors present on macrophages and other cells. We have previously shown that compared to free hen egg lysozyme (HEL), alpha 2M-complexed HEL undergoes enhanced macrophage uptake, processing, and presentation to T hybridoma clones in vitro. Inasmuch as it is not clear whether T hybridoma responses accurately reflect primary immune responses in vivo, we studied antibody production in rabbits using two Ag complexed with either human alpha 2M (H alpha 2M) or a homologous protein purified from rabbit plasma, alpha 1-macroglobulin (R alpha 1M). Pathogen-free NZW rabbits received s.c. injections with adjuvant-free preparations of free HEL or porcine pancreatic elastase (PPE), H alpha 2M-HEL-PPE complexes, R alpha 1M-HEL-PPE complexes, or mixtures of the uncomplexed proteins. Complexing the Ag to alpha 2M resulted in 10 to 500-fold higher IgG titers compared to uncomplexed controls. Injection of Ag complexed to either H alpha 2M or R alpha 1M resulted in levels of anti-HEL IgG comparable to those elicited by emulsification in CFA. Inasmuch as inflammatory proteinases such as neutrophil elastase can initiate covalent complex formation with alpha 2M, we propose that "proteinase-activated" alpha 2M may mediate receptor-enhanced Ag uptake by macrophages, resulting in augmented Ag processing and antibody production.
Asunto(s)
Formación de Anticuerpos , Presentación de Antígeno , Antígenos/administración & dosificación , alfa-Macroglobulinas/administración & dosificación , Animales , Células Presentadoras de Antígenos/fisiología , Antígenos/inmunología , Endocitosis , Inmunoglobulina G/biosíntesis , Muramidasa/inmunología , Conejos , alfa-Macroglobulinas/fisiologíaRESUMEN
Studies examining the biochemical characteristics and pharmacological properties of extracellular superoxide dismutase (EC SOD) have been severely limited because of difficulties in purifying the enzyme. Recently EC SOD was found to exist in high concentrations in the arteries of most mammals examined and it is the predominant form of SOD activity in many arteries. We now describe a three-step, high-yield protocol for the purification of EC SOD from human aorta. In the first step, the high affinity of EC SOD for heparin is utilized to obtain a fraction in which EC SOD constitutes roughly 13% of the total protein compared with only 0.3% of that of the starting material. In addition, over 80% of the original EC SOD activity present in the aortic homogenate was retained after the first step of purification. EC SOD was further purified using a combination of cation- and anion-exchange chromatography. The overall yield of EC SOD from this purification procedure was 46%, with over 4 mg of EC SOD obtained from 230 g of aorta. Purified EC SOD was found to exist predominantly as a homotetramer composed of two disulphide-linked dimers. However, EC SOD was also found to form larger multimers when analysed by native PAGE. It was shown by urea denaturation that the formation of multimers increased the thermodynamic stability of the protein. Limited proteolysis of EC SOD suggested that there is one interchain disulphide bond covalently linking two subunits. This disulphide bond involves cysteine-219 and appears to link the heparin-binding domains of the two subunits.
Asunto(s)
Aorta/enzimología , Superóxido Dismutasa/química , Secuencia de Aminoácidos , Disulfuros/química , Estabilidad de Enzimas , Glicoproteínas/química , Humanos , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
Although reactive O2 species appear to participate in central nervous system (CNS) O2 toxicity, the exact roles of different reactive O2 species are undetermined. To study the contribution of extracellular superoxide anion (O2-) to CNS O2 toxicity we constructed transgenic mice overexpressing human extracellular superoxide dismutase (ECSOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) in the brain. Remarkably, when exposed to 6 atm (1 atm = 101.3 kPA) of hyperbaric oxygen for 25 min, transgenic mice demonstrated higher mortality (83%) than nontransgenic litter-mates (33%; P < 0.017). Pretreatment with diethyldithiocarbamate, which inhibits both ECSOD and Cu/Zn superoxide dismutase (Cu/Zn SOD) activity, increased resistance to CNS O2 toxicity, in terms of both survival (100% in transgenics and 93% in nontransgenics) and resistance to seizures (4-fold increase in seizure latency in both transgenic and nontransgenic mice; P < 0.05). Thus, O2- apparently protects against CNS O2 toxicity. We hypothesized that O2- decreased toxicity by inactivating nitric oxide (NO.). To test this, we inhibited NO. synthase (EC 1.14.23) with N omega-nitro-L-arginine to determine whether NO. contributes to enhanced CNS O2 toxicity in transgenic mice. N omega-nitro-L-arginine protected both transgenic and nontransgenic mice against CNS O2 toxicity (100% survival and a 4-fold delay in time to first seizure; P < 0.05), as well as abolishing the difference in sensitivity to CNS O2 toxicity between transgenic and nontransgenic mice. These results implicate NO. as an important mediator in CNS O2 toxicity and suggest that ECSOD increases CNS O2 toxicity by inhibiting O2(-)-mediated inactivation of NO.
Asunto(s)
Espacio Extracelular/metabolismo , Oxigenoterapia Hiperbárica , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/toxicidad , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Animales , Encéfalo/efectos de los fármacos , Radicales Libres , Ratones , Ratones Transgénicos , Convulsiones/etiologíaRESUMEN
BACKGROUND: Extracellular superoxide dismutase (EC-SOD) is a principal enzymatic scavenger of the superoxide anion in extracellular spaces. It is thought to be important as a defense against superoxide-mediated damage to both cell surfaces and extracellular matrix proteins. EC-SOD may also be important in regulating intercellular signalling by extracellular superoxide. EC-SOD is believed to be mainly located in the extracellular matrix of tissues. However, the specific localization of EC-SOD is unknown. Knowledge of the distribution of EC-SOD is an essential step in defining its functions. EXPERIMENTAL DESIGN: Using light microscopic immunohistochemistry, electron microscopic immunocytochemistry and an EC-SOD affinity-purified polyclonal rabbit antibody to human recombinant EC-SOD, we evaluated the distribution of EC-SOD in human lungs. RESULTS: These studies revealed that in the lung EC-SOD is primarily located in the extracellular matrix. Specifically, EC-SOD is found in areas containing high amounts of type I collagen and other unidentified matrix elements, but was not seen in areas rich in elastin or cartilage. In the lung, EC-SOD is predominantly located around larger vessels and airways, and, to a lesser degree, in the extracellular matrix around alveolar and capillary regions. Some EC-SOD was found in bronchiolar epithelial cell junctions and around the surface of vascular and airway smooth muscle cells. No labeling was seen on endothelial cell surfaces of capillaries, small muscular, or large elastic vessels in the lung. Labeling for EC-SOD was limited to the extracellular spaces, consistent with it being a secreted protein, with the exception of a small amount of intracellular labeling seen in bronchial epithelial cells and type II cells. CONCLUSIONS: The labeling distribution of EC-SOD in human lungs was defined using immunohistochemistry and immunocytochemical techniques. The findings suggest that EC-SOD is not diffusely located throughout the lung, but has a specific distribution in the extracellular matrix. These findings on the distribution of EC-SOD suggest that specific roles for EC-SOD may include the protection of matrix elements such as collagen from oxidative stress and that it may also function in the regulation of intercellular signals that are modulated by reactive oxygen species.