RESUMO
INTRODUCTION: Patients with systemic lupus erythematosus (SLE) possessing anti-phospholipid antibodies (aPLs) are often complicated by thrombotic vascular events. aPLs commonly associated with the complications are anti-cardiolipin/ß2-glycoprotein I antibodies (aCL/ß2GPI) and anti-phosphatidylserine/prothrombin antibodies (aPS/PT). However, the pathological mechanisms leading to thrombosis remain unclear. We explored clinical features of SLE patients with aCL/ß2GPI and aPS/PT and investigated thrombogenic effects of their IgG fractions. MATERIALS AND METHODS: We enrolled 97 SLE patients and 38 healthy control volunteers and performed activated protein C (APC) resistance screening test using their plasma samples. To detect the direct effect of aPLs IgG on APC, we developed an APC sensitivity ratio assay. Effects of aPLs IgG on monocytes were studied by measuring the surface expression of tissue factor (TF) and excretion of TNF-α from peripheral blood mononuclear cell culture. RESULTS AND CONCLUSION: Thrombotic complications among SLE patients were closely associated with aCL/ß2GPI or aPS/PT, with higher prevalence in patients with both antibodies. Addition of aPLs(+)-IgG to the APC sensitivity ratio assay led to significant suppression of the anticoagulant activity of APC. The suppression was more pronounced in double-positive cases. TF expression on monocytes and concentration of TNF-α in culture medium were increased by aPLs, again more pronounced in double-positive cases. These results indicate that the effects of aCL/ß2GPI and aPS/PT are synergic both for APC anticoagulant activity and for production of TF and TNF-α from mononuclear cells. These modes of thrombogenic action of aPLs could be an important target for developing specific measures to prevent complications of SLE.
Assuntos
Anticorpos Antifosfolipídeos/imunologia , Anticoagulantes/uso terapêutico , Genes APC/fisiologia , Leucócitos Mononucleares/metabolismo , Tromboplastina/metabolismo , Trombose/sangue , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticoagulantes/farmacologia , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
: Antiphospholipid syndrome, which often complicates systemic lupus erythematosus (SLE), features high occurrence of arterial and/or venous thrombosis and recurrent fetal loss. However, which antibody subclass contributes to which clinical event remains uncertain. We newly developed an up-to-date enzyme immunoassay system using the AcuStar automated analyzer (Instrumentation Laboratory, Bedford, Massachusetts, USA) for parallel detection of six subclasses of antiphospholipid antibodies (aPLs): anticardiolipin antibodies (aCL) of IgG, IgM, and IgA and anti-ß2-glycoprotein I antibodies (aß2GPI) of IgG, IgM, and IgA. They were measured in 276 healthy volunteers and 138 patients with SLE: 45 with thromboembolic complications (29 arterial; 16 venous) and 93 without. Lupus anticoagulant activity in their plasma was measured according to the guidelines recommended by the Subcommittee on Lupus Anticoagulant/Phospholipid-Dependent Antibodies. aCL/ß2GPI was measured with a standard ELISA kit commonly used in Japan. The positive results of IgG aCL, IgA aCL, and IgG aß2GPI were closely associated with thromboembolic complications, whereas IgM aCL and IgM aß2GPI were not. receiver operating characteristic analysis revealed that the accuracy of predicting thromboembolic complications based on the composite test results of the former three antibodies were obviously higher than by each alone. Regarding agreement with the test results of lupus anticoagulant activity, IgG aß2GPI showed the closest match. Patients with SLE frequently possess various combinations of the six aPL subclasses, and this antibody spectrum is closely associated with thromboembolic events in these patients. This new automated enzyme immunoassay system allows simultaneous analysis of the profile of aPL subclasses for the differential diagnosis of antiphospholipid antibody syndrome in its early stage.
Assuntos
Anticorpos Antifosfolipídeos/análise , Síndrome Antifosfolipídica/diagnóstico , Técnicas Imunoenzimáticas/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Anticardiolipina/análise , Anticorpos Anticardiolipina/classificação , Anticorpos Antifosfolipídeos/classificação , Síndrome Antifosfolipídica/complicações , Autoanticorpos/análise , Autoanticorpos/classificação , Criança , Diagnóstico Diferencial , Feminino , Humanos , Imunoglobulina A/análise , Imunoglobulina G/análise , Imunoglobulina M/análise , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-Idade , Tromboembolia/etiologia , Adulto Jovem , beta 2-Glicoproteína I/imunologiaRESUMO
We sought to determine whether oxidative stress and anti-oxidative activity could act as biomarkers that discriminate patients with chronic fatigue syndrome (CFS) from healthy volunteers at acute and sub-acute fatigue and resting conditions. We calculated the oxidative stress index (OSI) from reactive oxygen metabolites-derived compounds (d-ROMs) and the biological antioxidant potential (BAP). We determined changes in d-ROMs, BAP, and OSI in acute and sub-acute fatigue in two healthy groups, and compared their values at rest between patients with CFS (diagnosed by Fukuda 1994 criteria) and another group of healthy controls. Following acute fatigue in healthy controls, d-ROMs and OSI increased, and BAP decreased. Although d-ROMs and OSI were significantly higher after sub-acute fatigue, BAP did not decrease. Resting condition yielded higher d-ROMs, higher OSI, and lower BAP in patients with CFS than in healthy volunteers, but lower d-ROMs and OSI when compared with sub-acute controls. BAP values did not significantly differ between patients with CFS and controls in the sub-acute condition. However, values were significantly higher than in the resting condition for controls. Thus, measured of oxidative stress (d-ROMS) and anti-oxidative activity (BAP) might be useful for discriminating acute, sub-acute, and resting fatigue in healthy people from patients with CFS, or for evaluating fatigue levels in healthy people.
Assuntos
Antioxidantes/metabolismo , Síndrome de Fadiga Crônica/diagnóstico , Estresse Oxidativo , Espécies Reativas de Oxigênio/sangue , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Síndrome de Fadiga Crônica/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Therapeutic hypothermia protects neurons after injury to the central nervous system (CNS). Microglia express toll-like receptors (TLRs) that play significant roles in the pathogenesis of sterile CNS injury. To elucidate the possible mechanisms involved in the neuroprotective effect of therapeutic hypothermia, we examined the effects of hypothermic culture on TLR3-activated microglial release of interferon (IFN)- ß and nitric oxide (NO), which are known to be associated with neuronal cell death. When rat or mouse microglia were cultured under conditions of hypothermia (33°C) and normothermia (37°C) with a TLR3 agonist, polyinosinic-polycytidylic acid, the production of IFN- ß and NO in TLR3-activated microglia at 48 h was decreased by hypothermia compared with that by normothermia. In addition, exposure to recombinant IFN- ß and sodium nitroprusside, an NO donor, caused death of rat neuronal pheochromocytoma PC12 cells in a concentration-dependent manner after 24 h. Taken together, these results suggest that the attenuation of microglial production of IFN- ß and NO by therapeutic hypothermia leads to the inhibition of neuronal cell death.
Assuntos
Hipotermia/metabolismo , Interferon beta/metabolismo , Microglia/metabolismo , Óxido Nítrico/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Células Cultivadas , Camundongos , Microglia/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Células PC12 , Poli I-C/farmacologia , Ratos , Ratos Wistar , Temperatura , Receptor 3 Toll-Like/antagonistas & inibidoresRESUMO
PURPOSE: Therapeutic hypothermia protects neurons following injury to the central nervous system (CNS). Microglia express toll-like receptors (TLRs) that play significant roles in pathological processes in sterile CNS injury. We have examined the effects of culture temperature on the TLR2-activated microglial production of cytokines and nitric oxide (NO), which are known to be associated with CNS damage, and the possible involvement of nuclear factor-κB (NF-κB) activation underlying such effects. METHODS: Rat microglia were cultured with a selective TLR2 agonist, Pam(3)CSK(4), under hypothermic, normothermic, and hyperthermic conditions, and with Pam(3)CSK(4) in the presence of a NF-κB activation inhibitor at 37 °C. Cytokine and NO levels and NF-κB p65 activation were measured. RESULTS: The production of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), and NO and the activation of NF-κB p65 were reduced by hypothermia, but augmented by hyperthermia at 3-6, 24-48, 48, and 0.5 h, post-treatment initiation, respectively. Pharmacological inhibition of NF-κB activation impaired the Pam(3)CSK(4)-induced TNF-α, IL-10, and NO production. CONCLUSIONS: In TLR2-activated microglia, hypothermia reduced, while hyperthermia increased, the early activation of NF-κB and the subsequent NF-κB-mediated production of TNF-α, IL-10, and NO in a time-dependent manner, suggesting that attenuation of these factors via suppression of NF-κB in microglia is one possible neuroprotective mechanism of therapeutic hypothermia. Moreover, temperature-dependent changes in microglial TNF-α production during the early phase and IL-10 and NO production during the late phase indicate that these factors might be useful as clinical markers to monitor hypothermia-related neuronal protection and hyperthermia-related neuronal injury.
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Técnicas de Cultura de Células , Microglia/metabolismo , Temperatura , Fator de Transcrição RelA/metabolismo , Animais , Interleucina-10/metabolismo , Lipopeptídeos/farmacologia , Óxido Nítrico/metabolismo , Ratos , Ratos Wistar , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Therapeutic hypothermia protects neurons after severe brain injury. Activated microglia produce several neurotoxic factors, such as pro-inflammatory cytokines and nitric oxide (NO), during neuron destruction. Hence, suppression of microglial release of these factors is thought to contribute partly to the neuroprotective effects of hypothermia. After brain insults, adenosine triphosphate (ATP) is released from injured cells and activates microglia. Here, we examined the acute effects of temperature on ATP-activated microglial production of inflammatory factors, and the possible involvement of p38 mitogen-activated protein kinase (p38) underlying such effects. METHODS: Microglia were cultured with ATP at 33, 37, and 39°C, or with ATP in the presence of a p38 inhibitor, SB203580, at 37°C. Cytokine and NO levels, and p38 activation were measured. RESULTS: Compared to 37°C, TNF-α was reduced at 33°C and augmented at 39°C for 1.5 h. IL-6 was reduced at 33°C for 6 h. NO was reduced at 33°C, but augmented at 39°C for 6 h. p38 was reduced at 33°C for 1 min. SB203580 inhibited ATP-induced TNF-α, IL-6, and NO production. CONCLUSION: Lowering temperature rapidly reduced p38 activation and the subsequent p38-regulated production of pro-inflammatory cytokines and NO in ATP-activated microglia, suggesting that attenuation of early phase inflammatory responses via suppression of p38 in microglia is one possible neuroprotective mechanism of therapeutic hypothermia. Temperature elevation increased TNF-α and NO production in these cells. These temperature-dependent changes imply that monitoring of TNF-α and NO in the cerebrospinal fluid during the early phase might be useful as biomarkers for responses to therapeutic hypothermia and hyperthermia.
Assuntos
Citocinas/metabolismo , Microglia/metabolismo , Óxido Nítrico/metabolismo , Temperatura , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Lesões Encefálicas/metabolismo , Células Cultivadas , Imidazóis , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Fosforilação , Piridinas , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
INTRODUCTION: In systemic lupus erythematosus (SLE) patients, the prevalence of arteriosclerosis obliterans (ASO) is high despite a lack of common risk factors for ASO. The main objective of this study was to investigate a possible direct role of anti-phospholipid antibodies (aPLs), which are frequently detected in SLE patients, in the pathogenesis of ASO. MATERIALS AND METHODS: We examined tissue factor (TF) expression on the monocyte surface by flow cytometric analysis in 89 SLE patients with or without ASO and/or aPLs and studied the in vitro effect of purified IgG fractions from plasma of SLE patients or normal healthy volunteers (aPLs(+) IgG, n=8; aPLs(-) IgG, n=6; Normal IgG, n=6) on the expression of TF and production of TNF-α and IL-1ß in healthy peripheral blood mononuclear cells (PBMCs) or isolated monocytes. RESULTS: We confirmed that high expression of monocyte TF was strongly associated with the prevalence of ASO and the presence of aPLs. Treatments of PBMCs with aPLs(-) IgG or normal IgG did not significantly increase expression of TF, TNF-α, and IL-1ß messenger RNA (mRNA) and the production of TNF-α and IL-1ß. However, stimulation of PBMCs with aPLs(+) IgG caused significant increase in expression of TF, TNF-α, and IL-1ß mRNA. Moreover, aPLs(+) IgG stimulated PBMCs and significantly enhanced the production of TNF-α and IL-1ß. CONCLUSION: These results suggest that IgG-aPLs cause persistently high TF expression and inflammatory cytokine production by interacting with peripheral blood monocytes and lymphocytes, which may be an important mechanism in the pathogenesis of ASO peculiar to SLE patients.