Assuntos
Inflamação/enzimologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Desenhos Animados como Assunto , Inibidores de Ciclo-Oxigenase 2/história , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , História do Século XX , História do Século XXI , Humanos , Inflamação/tratamento farmacológico , Inflamação/história , Lactonas/história , Lactonas/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Sulfonas/história , Sulfonas/uso terapêuticoRESUMO
OBJECTIVE: To determine the localization of 3-nitrotyrosine (3-NT), a footprint marker of peroxynitrite (ONOO-) and other reactive nitrogen species, to the inflamed human synovium and to compare this with normal synovial and nonsynovial tissue of human and animal origin. METHODS: Monoclonal and polyclonal antibodies were used to investigate for 3-NT, inducible nitric oxide synthase (iNOS), macrophage marker CD68, and the vascular smooth muscle marker alpha-actin by avidin-biotin immunocytochemistry. RESULTS: In the inflamed synovium, 3-NT was found in the vascular smooth muscle and macrophages. In normal human synovium, 3-NT was present in the vascular smooth muscle and some lining cells and was not associated with immunoreactivity for iNOS. Similarly, 3-NT could be demonstrated in the vascular smooth muscle cells of normal rats and iNOS knockout mice. It was not present in the vascular smooth muscle of healthy, nonsynovial tissue. CONCLUSION: The synovial vasculature in histologically normal human and naive rodent synovium was alone among the normal tissues studied in exhibiting iNOS-independent immunoreactivity for 3-NT. These findings suggest a physiologic role for ONOO- in normal synovial vascular function.
Assuntos
Artrite Reumatoide/patologia , Membrana Sinovial/química , Membrana Sinovial/patologia , Tirosina/análogos & derivados , Tirosina/análise , Actinas/análise , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Feminino , Humanos , Imuno-Histoquímica , Macrófagos/química , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Músculo Liso Vascular/química , Músculo Liso Vascular/patologia , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , RatosRESUMO
Formula-fed babies contract gastroenteritis more than breast-fed babies, which is of concern to mothers who cannot breastfeed or, as with HIV-infected mothers, are discouraged from breastfeeding. The ability of endogenous breastmilk xanthine oxidase to generate the antimicrobial radical nitric oxide has been measured and its influence on the growth of Escherichia coli and Salmonella enteritides examined. Breastmilk, but not formula feed, generated nitric oxide. Xanthine oxidase activity substantially inhibited the growth of both bacteria. An important natural antibiotic system is missing in formula feeds; the addition of xanthine oxidase may improve formula for use when breastfeeding is not a safe option.
Assuntos
Antibacterianos/farmacologia , Leite Humano/enzimologia , Xantina Oxidase/farmacologia , Antibacterianos/isolamento & purificação , Interações Medicamentosas , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Hipoxantina/farmacologia , Leite Humano/metabolismo , Óxido Nítrico/biossíntese , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/crescimento & desenvolvimento , Xantina Oxidase/isolamento & purificação , Xantina Oxidase/metabolismoRESUMO
Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nitric oxide (NO), under anaerobic conditions, in the presence of either NADH or xanthine as reducing substrate. NO production was directly demonstrated by ozone chemiluminescence and showed stoichiometry of approximately 2:1 versus NADH depletion. With xanthine as reducing substrate, the kinetics of NO production were complicated by enzyme inactivation, resulting from NO-induced conversion of XO to its relatively inactive desulfo-form. Steady-state kinetic parameters were determined spectrophotometrically for urate production and NADH oxidation catalyzed by XO and xanthine dehydrogenase in the presence of nitrite under anaerobic conditions. pH optima for anaerobic NO production catalyzed by XO in the presence of nitrite were 7.0 for NADH and =6.0 for xanthine. Involvement of the molybdenum site of XO in nitrite reduction was shown by the fact that alloxanthine inhibits xanthine oxidation competitively with nitrite. Strong preference for Mo=S over Mo=O was shown by the relatively very low NADH-nitrite reductase activity shown by desulfo-enzyme. The FAD site of XO was shown not to influence nitrite reduction in the presence of xanthine, although it was clearly involved when NADH was the reducing substrate. Apparent production of NO decreased with increasing oxygen tensions, consistent with reaction of NO with XO-generated superoxide. It is proposed that XO-derived NO fulfills a bactericidal role in the digestive tract.
Assuntos
Óxido Nítrico/metabolismo , Nitritos/metabolismo , Xantina Desidrogenase/metabolismo , Xantina Oxidase/metabolismo , Anaerobiose , Animais , Compostos de Bifenilo/farmacologia , Domínio Catalítico , Bovinos , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/metabolismo , Cinética , Metaloproteínas/metabolismo , Leite/enzimologia , Molibdênio/metabolismo , NAD/metabolismo , Oniocompostos/farmacologia , Oxirredução , Xantina/metabolismo , Xantina Desidrogenase/antagonistas & inibidores , Xantina Oxidase/antagonistas & inibidoresRESUMO
Calcium release from mouse calvarial organ cultures was used to analyse the well-described biological effects of constant direct current (20 microA) in combination with Faradic products generated at a titanium wire cathode. Constant 20-microA direct current stimulation alone, delivered by agar salt bridges, consistently lowered the media calcium levels. Direct exposure of calvariae to a titanium cathode and its faradic products resulted in further lowering of media calcium levels and also a significant increase in the media pH. Hydrogen peroxide is a faradic product of the titanium cathode, micromolar amounts being generated by our system over 24 hr. H(2)O(2) is pro-resorptive whereas elevated pH stimulates osteoblast activity. We propose that where bone tissue is in direct contact with metal wire cathodes, the faradic products, hydrogen peroxide and hydroxyl ion, are significant factors which, in their own right, further contribute to accelerated remodelling and improved clinical outcome.
Assuntos
Cálcio/fisiologia , Crânio/fisiologia , Animais , Eletrofisiologia , Transporte de Íons/fisiologia , Camundongos , TitânioRESUMO
OBJECTIVE: To investigate endothelial cell adhesion molecule expression and leucocyte adhesion to endothelial cells isolated from the microvasculature of rheumatoid arthritic synovial tissue (SMEC) in comparison with similar cells isolated from healthy subcutaneous adipose tissue (ADMEC) or from umbilical veins (HUVEC). METHODS: Cultured endothelial cells were treated with tumour necrosis factor alpha (TNFalpha) for 2-24 hours before the assessment of cell surface E-selectin, vascular (VCAM-1) or intercellular cell adhesion molecule-I (ICAM-1) expression. Neutrophil and T lymphocyte adhesion to TNFalpha treated endothelial cells was assessed using static and shear dependent assay systems. RESULTS: VCAM-1 expression by SMEC was significantly less sensitive to TNFalpha stimulation than HUVEC or ADMEC. E-selectin expression by SMEC appeared to be more sensitive to TNFalpha stimulation and maximal expression was about 30% greater in comparison with HUVEC or ADMEC. Sensitivity to TNFalpha induction and maximal ICAM-1 expression was similar in all three endothelial cell types. Static neutrophil adhesion to TNFalpha stimulated SMEC was significantly increased in comparison with HUVEC, however this phenomenon was dependent on the presence of neutralising antibodies to ICAM-1. At shear rates in excess of 2.4 dynes/cm(2) significantly more neutrophils and, predominantly CD45RO+, T lymphocytes adhered to TNFalpha stimulated SMEC than HUVEC. CONCLUSION: Rheumatoid synovial endothelial cells differentially regulate E-selectin and VCAM-1. The increased ability of TNFalpha stimulated synovial endothelial cells to support leucocyte adhesion may help to explain the leucocyte, in particular CD45RO+ T-lymphocyte, recruitment observed in the rheumatoid synovium.
Assuntos
Artrite Reumatoide/metabolismo , Moléculas de Adesão Celular/metabolismo , Leucócitos/fisiologia , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Artrite Reumatoide/patologia , Adesão Celular/fisiologia , Técnicas de Cultura de Células , Selectina E/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Ensaio de Imunoadsorção Enzimática , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Membrana Sinovial/patologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
OBJECTIVES/HYPOTHESIS: The objective of this study was to determine the vertical height, horizontal incision width, and extent of undermining that correlated with the lowest closure tension. STUDY DESIGN: Prospective, cadaver study. METHODS: Forty "A-to-T" flaps were made on the torso and lower extremities of fresh cadavers. Ten flaps each were designed at heights of three, four, and five defect radii. Closing tensions were measured for each of these flaps initially, followed by serial base extensions. Based on the information from these first flaps, 10 additional flaps were made at the optimal height and base extensions. These flaps were then serially undermined and tension measurements taken. RESULTS: Our results suggest that making the vertical height of the "A" twice the height of the defect yields a significant decrease in tension of closure when compared with a vertical height one and a half times the defect (P < .01), while increasing the height to two and a half times the defect height provides only a minimal further reduction in closure tension. Extending the base (horizontal) incision one defect diameter in each direction offers the greatest reduction in closing tension. Undermining up to three times the diameter of the defect offers progressive improvement in the tension of closure, while further undermining confers little additional benefit. CONCLUSIONS: Our findings indicate that the ideal A-to-T flap is designed to be twice the height of the original defect, with base extensions one defect diameter in each direction, and undermined to three times the diameter of the defect.
Assuntos
Retalhos Cirúrgicos/fisiologia , Fenômenos Biomecânicos , Humanos , Estudos ProspectivosRESUMO
Pulsed electromagnetic fields (PEMF) are successfully employed in the treatment of a variety of orthopaedic conditions, particularly delayed and nonunion fractures. In this study, we examined PEMF effects on in vitro osteogenesis by bone nodule formation and on mRNA expression of bone morphogenetic proteins 2 and 4 by reverse-transcriptase polymerase chain reaction (RT-PCR) in cultured rat calvarial osteoblasts. PEMF exposure induced a significant increase in both the number (39% over unexposed controls) and size (70% larger compared to unexposed controls) of bone-like nodules formed. PEMF also induced an increase in the levels of BMP-2 and BMP-4 mRNA in comparison to controls. This effect was directly related to the duration of PEMF exposure. This study shows that clinically applied PEMF have a reproducible osteogenic effect in vitro and simultaneously induce BMP-2 and -4 mRNA transcription. This supports the concept that the two effects are related.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Campos Eletromagnéticos , Osteoblastos , Ativação Transcricional/efeitos da radiação , Fator de Crescimento Transformador beta , Animais , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/biossíntese , Diferenciação Celular/efeitos da radiação , Morfogênese/efeitos da radiação , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteoblastos/efeitos da radiação , Ratos , CrânioRESUMO
Xanthine oxidoreductase (XOR) in human mammary epithelial cells was shown to have low true specific activity, similar to that in breast milk. Enzymic activity was increased in response to inflammatory cytokines; increases of 2-2.5-fold being seen with TNF-alpha and IL-1beta and of approximately 8-fold with IFN-gamma. No significant increase was seen with IL-6. A combination of IFN-gamma and TNF-alpha, or of these two cytokines plus IL-1beta, led to responses representing the sum of those obtained by using the individual cytokines. The 8-fold increase in enzymic activity, stimulated by IFN-gamma, corresponded to only a 2-3-fold increase in specific mRNA, suggesting the possibility of post-translational activation; a possibility strongly supported by the corresponding 2-3-fold rise in XOR protein, as determined by ELISA. In no case was cytokine-induced activation accompanied by changes in the oxidase-dehydrogenase ratio of XOR. These data strongly support a role for XOR in the inflammatory response of the human mammary epithelial cell, and provide further evidence of post-translational activation of a low activity form of human XOR, similar to that previously observed in vivo for the breast milk enzyme.
Assuntos
Mama/enzimologia , Citocinas/farmacologia , Xantina Desidrogenase/metabolismo , Xantina Oxidase/metabolismo , Mama/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Feminino , Humanos , Mediadores da Inflamação/farmacologia , Interferon gama/farmacologia , Interleucina-1/farmacologia , Leite Humano/enzimologia , Molibdênio/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Fator de Necrose Tumoral alfa/farmacologia , Xantina Desidrogenase/genética , Xantina Oxidase/genéticaRESUMO
Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophoresis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodonium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopurinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.
Assuntos
Alopurinol/farmacologia , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Hipóxia/complicações , Linfócitos/metabolismo , NAD/metabolismo , Oniocompostos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/enzimologia , Xantina Desidrogenase/fisiologia , Xantina Oxidase/fisiologia , Hipóxia Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Endotélio Vascular/citologia , Humanos , Isquemia/complicações , Isquemia/metabolismo , Linfócitos/patologia , Leite Humano/enzimologia , Oxirredução , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Traumatismo por Reperfusão/etiologia , Espectrofotometria , Especificidade por Substrato , Células Tumorais Cultivadas , Veias UmbilicaisRESUMO
Xanthine oxidoreductase (XOR) catalyses the reduction of the therapeutic organic nitrate, nitroglycerin (glyceryl trinitrate, GTN), as well as inorganic nitrate and nitrite, to nitric oxide (NO) under hypoxic conditions in the presence of NADH. Generation of nitric oxide is not detectable under normoxic conditions and is inhibited by the molybdenum site-specific inhibitors, oxypurinol and (-)BOF 4272. These enzymic reactions provide a mechanism for generation of NO under hypoxic conditions where nitric oxide synthase does not function, suggesting a vasodilatory role in ischaemia.
Assuntos
Nitratos/metabolismo , Óxido Nítrico/biossíntese , Nitrito de Sódio/metabolismo , Xantina Oxidase/metabolismo , Animais , Bovinos , Inibidores Enzimáticos/farmacologia , Cinética , NAD/metabolismo , Nitroglicerina/metabolismo , Oxigênio , Oxipurinol/farmacologia , Triazinas/farmacologia , Xantina Oxidase/antagonistas & inibidoresRESUMO
Subcellular localisation of xanthine oxidoreductase (XOR) was determined by indirect immunofluorescence using confocal microscopy in human endothelial and epithelial cell lines and in primary cultures of human umbilical vein endothelial cells. XOR was diffusely distributed throughout the cytoplasm but with higher intensity in the perinuclear region. In non-permeabilised cells, XOR was clearly seen to be asymmetrically located on the outer surfaces, showing, in many cases, a higher intensity on those faces apposed by closely neighbouring cells. Such specific distribution suggests a functional role for the enzyme in cell-cell interactions, possibly involving signalling via reactive oxygen species.
Assuntos
Endotélio/enzimologia , Células Epiteliais/enzimologia , Xantina Oxidase/metabolismo , Linhagem Celular , Membrana Celular/enzimologia , Citoplasma/enzimologia , Endotélio/citologia , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Espaço Extracelular/enzimologia , Humanos , Veias UmbilicaisAssuntos
Hipóxia/complicações , Membrana Sinovial/irrigação sanguínea , Sinovite/etiologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Expressão Gênica , Glucocorticoides/uso terapêutico , Humanos , Pesquisa , Membrana Sinovial/lesões , Sinovite/tratamento farmacológico , Sinovite/imunologia , Cicatrização/genéticaAssuntos
Complexos Multienzimáticos/sangue , NADH NADPH Oxirredutases/sangue , NAD/metabolismo , Xantina Oxidase/sangue , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Cinética , Complexos Multienzimáticos/química , NADH NADPH Oxirredutases/química , Xantina Oxidase/química , Xantina Oxidase/isolamento & purificaçãoAssuntos
Artrite Reumatoide/fisiopatologia , Reabsorção Óssea/fisiopatologia , Membrana Sinovial/fisiopatologia , Xantina Oxidase/metabolismo , Animais , Artrite Reumatoide/patologia , Reabsorção Óssea/patologia , Humanos , Inflamação , Modelos Químicos , Nitrato Redutase , Nitrato Redutases/metabolismo , Nitrito Redutases/metabolismoRESUMO
Monocyte adhesion to the arterial wall is a key event in the atherosclerotic process. We studied the interactions between human coronary arterial intimal smooth muscle cells (SMCs) and monocytes by examining (i) whether SMCs mediate monocyte adhesion when stimulated by oxidatively modified low density lipoprotein (LDL) or by the cytokines TNF alpha and IL-1, and (ii) the role of the adhesion molecules VCAM-1 and ICAM-1 (vascular cell and intercellular adhesion molecule, respectively) in this process. Preincubation of SMCs with both TNF alpha and IL-1 caused a significant 2-fold increase in VCAM-1 and ICAM-1 expression and a more than 9-fold increase in monocyte adhesion. The latter was significantly inhibited (by 1/3) by neutralising antibodies to VCAM-1 and ICAM-1. Modified LDL also induced a significant 3-fold increase in monocyte adhesion to SMCs, but did not induce VCAM-1 or ICAM-1 expression, nor was this adhesion inhibited by neutralising antibodies to VCAM-1 or ICAM-1. Oxidatively modified LDL, like the proinflammatory cytokines TNF alpha and IL-1, has the ability to enhance monocyte adhesion to human SMCs in vitro. LDL-induced monocyte adhesion to SMCs is distinct from that induced by TNF alpha and IL-1 in its lack of dependence on the classical adhesion pathways involving smooth muscle VCAM-1 and ICAM-1. SMCs are identified as a new cell population which may play an active role in recruiting monocytes to the arterial intima and atherosclerotic plaque.
Assuntos
Adesão Celular , Citocinas/metabolismo , Lipoproteínas LDL/metabolismo , Monócitos/metabolismo , Músculo Liso Vascular/metabolismo , Anticorpos Monoclonais/imunologia , Células Cultivadas , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/metabolismo , Vasos Coronários/citologia , Vasos Coronários/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1/metabolismo , Contagem de Leucócitos , Monócitos/imunologia , Músculo Liso Vascular/citologia , Oxirredução , Valores de Referência , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Antioxidant depletion in a normal adult increases the risk of developing rheumatoid arthritis (1). We discuss how redox processes "control" the inflammatory reaction and suggest that the synovium is a peculiarly "redox-sensitive" microenvironment. This discussion will be illustrated by a zonal model of tissue injury to represent the diseased synovium.