RESUMO
Neutrophil extracellular traps (NETs) are web-like structures consisting of DNA, histones and granule proteins, released from neutrophils in thrombus formation, inflammation, and cancer. We asked if plasma levels of the NET markers myeloperoxidase (MPO)-DNA and citrullinated histone H3 (H3Cit)-DNA, are elevated in liver cirrhosis and hepatocellular carcinoma (HCC) and if the levels correlate with clinical parameters. MPO-DNA, H3Cit-DNA, and thrombin-antithrombin (TAT) complex, as a marker of coagulation activity, were measured using ELISA in plasma from 82 patients with HCC, 95 patients with cirrhosis and 50 healthy controls. Correlations were made to clinical parameters and laboratory data and patients were followed for a median of 22.5 months regarding thrombosis development. H3Cit-DNA was significantly (p < 0.01) elevated in plasma from cirrhosis (66.4 ng/mL) and HCC (63.8 ng/mL) patients compared to healthy controls (31.8 ng/mL). TAT levels showed similar pattern (3.1, 3.7, and 0.0 µg/mL respectively, p < 0.01). MPO-DNA was significantly (p < 0.01) elevated in cirrhosis patients (0.53 O.D.) as compared to controls (0.33 O.D.). Levels of MPO-DNA and H3Cit-DNA correlated positively with Child-Pugh and MELD score. TAT was increased in all Child-Pugh and MELD groups. In multivariable logistic regression, Child B and C liver cirrhosis were independent predictors of elevated H3Cit-DNA in plasma. Levels of MPO-DNA and H3Cit-DNA were similar in patients with or without history of thrombosis, or thrombus formation during follow-up. In conclusion, plasma markers of NET formation are elevated in liver cirrhosis and correlate to the degree of liver dysfunction in patients with liver cirrhosis and/or HCC. The presence of HCC did not further increase the plasma levels of NET markers as compared to patients with cirrhosis only.
Assuntos
Carcinoma Hepatocelular/imunologia , Cirrose Hepática/imunologia , Neoplasias Hepáticas/imunologia , Fígado/imunologia , Neutrófilos/imunologia , Trombose/imunologia , Idoso , Antitrombina III/imunologia , Biomarcadores/sangue , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Citrulinação , DNA/sangue , Armadilhas Extracelulares/imunologia , Feminino , Histonas/sangue , Humanos , Inflamação , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/sangue , Cirrose Hepática/diagnóstico , Cirrose Hepática/patologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/patologia , Peptídeo Hidrolases/sangue , Peptídeo Hidrolases/imunologia , Peroxidase/sangue , Trombose/sangue , Trombose/diagnóstico , Trombose/patologiaRESUMO
The mechanisms underlying host HIV control hold much promise in the search for a functional HIV cure. We investigated the host genomic signatures in elite controllers or rapid progressors following recent infection and the correlates of immune reconstitution during combination antiretroviral therapy. We characterized the HIV-specific longitudinal host transcriptional response of peripheral blood mononuclear cells from elite controllers, rapid progressors, immune responders and non-responders using a RT-qPCR array in a cohort of recently HIV-infected Brazilian individuals. The elite controllers expressed unique transcripts early in infection that were closely associated with specialized cross-presentation between XCR1+ DCs and antigen-specific CD8+ T cells (XCL1). The natural suppression of HIV was also associated with the highly functional co-expression of cytokines and chemokines (CCL2, TNF and IL-10) concomitant with the maintenance of important anti-inflammatory and anticoagulant properties (Antithrombin III). Immune responders exhibited exclusively upregulated mRNAs possibly related to stem cell mobilization before combination antiretroviral therapy (neutrophil elastase). Our longitudinal approach to gene expression permitted us to discover previously unrecognized determinants that contribute to natural or antiretroviral-mediated HIV-1 immune control.
Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Leucócitos Mononucleares/imunologia , Transcriptoma/imunologia , Terapia Antirretroviral de Alta Atividade , Antitrombina III/genética , Antitrombina III/imunologia , Antivirais/uso terapêutico , Brasil , Contagem de Linfócito CD4 , Quimiocinas/genética , Quimiocinas/imunologia , Estudos de Coortes , Apresentação Cruzada/imunologia , Citocinas/genética , Citocinas/imunologia , Perfilação da Expressão Gênica/métodos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Sobreviventes de Longo Prazo ao HIV , HIV-1/efeitos dos fármacos , Antígenos HLA-B/imunologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/efeitos dos fármacosRESUMO
Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin-antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution.
Assuntos
Artérias/imunologia , Complemento C5a/imunologia , Fibrinolisina/imunologia , Trombose Venosa/imunologia , Animais , Antitrombina III/efeitos dos fármacos , Antitrombina III/imunologia , Artérias/efeitos dos fármacos , Artérias/patologia , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Complemento C3a/biossíntese , Complemento C3a/imunologia , Complemento C5a/biossíntese , Complexo de Ataque à Membrana do Sistema Complemento/efeitos dos fármacos , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Fator Xa/imunologia , Fator Xa/metabolismo , Fibrinolisina/metabolismo , Humanos , Camundongos , Peptídeo Hidrolases/efeitos dos fármacos , Peptídeo Hidrolases/imunologia , Ativadores de Plasminogênio/administração & dosagem , Trombina/imunologia , Trombina/metabolismo , Trombose Venosa/tratamento farmacológico , Trombose Venosa/patologiaRESUMO
The instant blood-mediated inflammatory reaction (IBMIR) has been studied in whole blood models of human allo-islet transplantation for short periods (<6 h). Beyond this time frame the innate response to intraportally transplanted islets is less well described. A novel whole blood model was applied to study blood-islet-graft interactions up to 48 h. Heparinized polyvinyl chloride tubing was sealed into small bags containing venous blood together with allogeneic human islets and exocrine tissue, respectively. The bags were attached to a rotating wheel (37°C). Concentrated glucose and sodium hydrogen carbonate were added every 12 h to maintain physiological limits for sustained immune cell functions. Plasma was collected at repeated time points for analyses of coagulation/complement activation and chemokine/cytokine production. Immune cell infiltration was analyzed using immunohistochemistry. Coagulation and platelet activation markers, thrombin-antithrombin complex (TAT) and soluble CD40 ligand (sCD40L) showed early high concentrations (at 6-12 h). sC5b-9 steadily increased over 48 h. At 6 h neutrophils and monocytes surrounded the clotted cellular grafts with a following massive infiltration of neutrophils. High and increasing concentrations of CXCR1/2 ligands [IL-8 and growth-regulated oncogene α/ß/γ (Gro-α/ß/γ)] and IL-6 were produced in response to human islets and exocrine tissue. The CCR2 ligand monocyte chemoattractant protein 1 (MCP-1) exhibited increasing concentrations in response to exocrine tissue. The CXCR3 ligand interferon-inducible T cell α chemoattractant (I-TAC) was produced in response to both human islets and exocrine tissue from 6 h. Monokine induced by γ interferon (Mig) and interferon γ-induced protein 10 (IP-10) showed a later response, preferentially to exocrine tissue and with larger variations among preparations. An extended blood model of clinical islet transplantation allowed characterization of early immune activation in response to human islets and exocrine tissue. Increased production of chemokines targeting CXCR1/2, CCR2, and CXCR3 was observed, accompanied by massive intraislet neutrophil infiltration over 48 h. The model proved to be useful in exploring early blood-mediated reactions to cellular transplants and has relevance for evaluation of pharmacological interventions to prevent graft loss.
Assuntos
Imunidade Inata , Inflamação/sangue , Transplante das Ilhotas Pancreáticas/imunologia , Antitrombina III/imunologia , Coagulação Sanguínea , Antígenos CD40/sangue , Antígenos CD40/imunologia , Células Cultivadas , Quimiocinas/sangue , Quimiocinas/imunologia , Ativação do Complemento , Humanos , Imunidade Celular , Inflamação/imunologia , Interleucinas/sangue , Interleucinas/imunologia , Peptídeo Hidrolases/sangue , Peptídeo Hidrolases/imunologia , Ativação Plaquetária , Transplante HomólogoRESUMO
UNLABELLED: ESSENTIALS: Antithrombin III (AT)ß binds heparin with higher affinity than ATα. A conformation-specific antibody against ATß, TPP2009, was made to investigate ATß in hemostasis. TPP2009 bound specifically to heparin-ATß and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATß in hemostasis. BACKGROUND: Antithrombin III (AT)ß is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site. OBJECTIVES: To characterize a conformation-specific antibody against ATß and begin to investigate the role of ATß in maintaining hemostasis. METHODS: Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound ATß (ATß*H) by the use of phage display. RESULTS: SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATß*H and glycosaminoglycan-associated ATß on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50 ) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added ATß and heparin. In purified systems with ATß and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50 , 25.7 nm) and cynomolgus monkey (EC50 , 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies. CONCLUSIONS: TPP2009 specifically targets a unique conformational epitope on ATß*H and blocks ATß-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATß in hemostasis.
Assuntos
Anticorpos/farmacologia , Antitrombina III/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Coagulantes/farmacologia , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Especificidade de Anticorpos , Antitrombina III/química , Antitrombina III/imunologia , Sítios de Ligação de Anticorpos , Testes de Coagulação Sanguínea , Linhagem Celular , Técnicas de Visualização da Superfície Celular , Coagulantes/imunologia , Coagulantes/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Mapeamento de Epitopos , Humanos , Ligação Proteica , Estrutura Secundária de Proteína , Coelhos , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
INTRODUCTION: Given its central role in mediating heparin-induced anti-coagulation, antithrombin (AT) gene mutations may result in heparin resistance. This study investigates the relationship between familial AT gene mutations and tolerance to heparin. METHODS: The medical history of a male patient with heparin resistance who received heart surgery and six of his family members was reviewed. Activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen (Fib), D-dimer (D=D), and platelet count were determined to assess coagulation function. AT activity and the AT gene were also analyzed. For the newly identified gene mutations, polymorphisms were excluded in 120 healthy Kazak controls. RESULTS: Two mutations were identified in exon 7 of the AT gene, SERPINC1: g.1267G>A (p.A391T) found in five participants, including the index patient, and g.1334G>A, a silent mutation, in two family members. The g.1267G>A mutation may alter focal AT protein conformation. Neither of these mutations was observed in the healthy Kazak controls. Although all coagulation parameters and AT activity were within the normal ranges for the index patient and his family members, the platelet levels were significantly lower than that observed for the healthy Kazak controls (p=0.001). There was no significant difference in AT antigen levels between the groups; however, participants with the g.1267G>A mutation had a 44.25% reduction in heparin binding compared to the control group (p<0.001). CONCLUSION: We identified a novel hereditary mutation, g.1267G>A (p.A391T), in the AT gene, which reduces its heparin binding capacity and might be associated with resistance to heparin.
Assuntos
Antitrombina III/genética , Antitrombina III/imunologia , Antitrombinas/imunologia , Predisposição Genética para Doença/genética , Heparina/imunologia , Adulto , Idoso , Anticoagulantes/imunologia , Sequência de Bases , Feminino , Humanos , Cazaquistão , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Ligação Proteica , Tempo de Protrombina , Adulto JovemRESUMO
INTRODUCTION: Dengue virus (DENV) is transmitted by the mosquito vector, and causes a wide range of symptoms that lead to dengue fever (DF) or life-threatening dengue hemorrhagic fever (DHF). The host and viral correlates that contribute to DF and DHF are complex and poorly understood, but appear to be linked to inflammation and impaired coagulation. Full-length osteopontin (FL-OPN), a glycoprotein, and its activated thrombin-cleaved product, trOPN, integrate multiple immunological signals through the induction of pro-inflammatory cytokines. MATERIALS AND METHOD: To understand the role of OPN in DENV-infection, we assessed circulating levels of FL-OPN, trOPN, and several coagulation markers (D-dimer, thrombin-antithrombin complex [TAT], thrombomodulin [TM], and ferritin in blood obtained from 65 DENV infected patients in the critical and recovery phases of DF and DHF during a dengue virus epidemic in the Philippines in 2010. RESULTS: Levels of FL-OPN, trOPN, D-dimer, TAT, and TM were significantly elevated in the critical phase in both the DF and DHF groups, as compared with healthy controls. During the recovery phase, FL-OPN levels declined while trOPN levels increased dramatically in both the DF and DHF groups. FL-OPN levels were directly correlated with D-dimer and ferritin levels, while the generation of trOPN was associated with TAT levels, platelet counts, and viral RNA load. CONCLUSION: Our study demonstrated the marked elevation of plasma levels of FL-OPN and thrombin-cleaved OPN product, trOPN, in DENV-infection for the first time. Further studies on the biological functions of these matricellular proteins in DENV-infection would clarify its pathogenesis.
Assuntos
Coagulação Sanguínea , Vírus da Dengue/fisiologia , Dengue/complicações , Dengue/imunologia , Osteopontina/imunologia , Trombina/imunologia , Antitrombina III/análise , Antitrombina III/imunologia , Dengue/sangue , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Produtos de Degradação da Fibrina e do Fibrinogênio/imunologia , Interações Hospedeiro-Patógeno , Humanos , Inflamação/sangue , Inflamação/complicações , Inflamação/imunologia , Inflamação/virologia , Osteopontina/sangue , Peptídeo Hidrolases/análise , Peptídeo Hidrolases/imunologia , Trombina/análise , Trombomodulina/análise , Trombomodulina/imunologiaRESUMO
The exacerbated disease due to immune- and coagulative-mediated pulmonary injury during acute respiratory viruses infection results in severe morbidity and mortality. Identifying novel approaches to modulate virus-induced inflammation-coagulation interactions could be important alternatives for treating acute respiratory viruses infections. In this study we investigated the effect of the probiotic strain Lactobacillus rhamnosus CRL1505 on lung TLR3-mediated inflammation, and its ability to modulate inflammation-coagulation interaction during respiratory viral infection. Our findings reveal for the first time that a probiotic bacterium is able to influence lung immune-coagulative reaction triggered by TLR3 activation, by modulating the production of proinflammatory and anti-inflammatory cytokines as well as expression of tissue factor and thrombomodulin in the lung. We also demonstrated that the preventive treatment with the probiotic bacteria beneficially modulates the fine tune balance between clearing respiratory viruses (respiratory syncytial virus and influenza virus) and controlling immune-coagulative responses in the lung, allowing normal lung function to be maintained in the face of a viral attack. Our data also pinpoint a crucial role for IL-10 in the immune protection induced by L. rhamnosus CRL1505 during respiratory viral infections. These observations might be helpful to propose new preventive or therapeutic approaches to better control virus-inflammatory lung damage using probiotic functional foods.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Lacticaseibacillus rhamnosus , Probióticos/farmacologia , Animais , Antitrombina III/imunologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Chlorocebus aethiops , Citocinas/imunologia , Cães , Alphainfluenzavirus , Contagem de Leucócitos , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Peptídeo Hidrolases/imunologia , Pneumonia/sangue , Pneumonia/induzido quimicamente , Pneumonia/imunologia , Poli I-C , Infecções por Vírus Respiratório Sincicial/sangue , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano , Células VeroRESUMO
OBJECTIVE: To express recombinant antithrombin-III (AT-III) in an E.coli expression system, prepare the antiserum of AT-III and determine its titer. METHODS: Gene segment of AT-III was acquired by cloning technology. Then prokaryotic expression plasmid pET32a(+)-AT-III was constructed and transformed into E.coli competent cells BL21. The cells were induced by IPTG to express AT-III. After purified, the product was used to immunize New Zealand rabbits. Then antiserum was detected using indirect ELISA and Western blotting. RESULTS: Specific bands appeared at about M(r); 77 000, indicating prokaryotic expression protein detected by SDS-PAGE and Western blotting. From the rabbits immunized by the purified fusion protein, we aquired AT-III antiserum, of which the highest titer was 1:12 800 as shown by indirect ELISA. Western blotting showed the antiserum had the ability of specific binding to AT-III protein expressed by 293T and CHO or purified AT-III protein. CONCLUSION: Antiserum of human AT-III has been prepared successfully.
Assuntos
Antitrombina III/genética , Soros Imunes/isolamento & purificação , Especificidade de Anticorpos , Antitrombina III/imunologia , Clonagem Molecular , Escherichia coli/genética , Humanos , Soros Imunes/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
BACKGROUND: Although there have been dramatic strides made recently in the treatment of chronic hepatitis C virus infection, interferon-α based therapy remains challenging for certain populations, including those with unfavorable IL28B genotypes, psychiatric co-morbidity, HIV co-infection, and decompensated liver disease. We have recently shown that ATIII, a serine protease inhibitor (serpin), has broad antiviral properties. RESULTS: We now show that ATIII is capable of inhibiting HCV in the OR6 replicon model at micromolar concentrations. At a mechanistic level using gene-expression arrays, we found that ATIII treatment down-regulated multiple host cell signal transduction factors involved in the pathogenesis of cirrhosis and hepatocellular carcinoma, including Jun, Myc and BMP2. Using a protein interactive network analysis we found that changes in gene-expression caused by ATIII were dependent on three nodes previously implicated in HCV disease progression or HCV replication: NFκB, P38 MAPK, and ERK1/2. CONCLUSIONS: Our findings suggest that ATIII stimulates a novel innate antiviral host cell defense different from current treatment options.
Assuntos
Antitrombina III/metabolismo , Antivirais/metabolismo , Hepacivirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antitrombina III/imunologia , Antivirais/imunologia , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hepacivirus/imunologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacos , Replicação Viral/imunologiaRESUMO
The enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. Activated thrombin activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis and is an attractive target to develop profibrinolytic drugs. TAFI can be activated by thrombin, thrombin/thrombomodulin, or plasmin, but the in vivo physiologic TAFI activator(s) are unknown. Here, we generated and characterized MA-TCK26D6, a monoclonal antibody raised against human TAFI, and examined its profibrinolytic properties in vitro and in vivo. In vitro, MA-TCK26D6 showed a strong profibrinolytic effect caused by inhibition of the plasmin-mediated TAFI activation. In vivo, MA-TCK26D6 significantly decreased fibrin deposition in the lungs of thromboembolism-induced mice. Moreover, in the presence of MA-TCK26D6, plasmin-α(2)-antiplasmin complexes in plasma of thromboembolism-induced mice were significantly increased compared with a control antibody, indicative of an acceleration of fibrinolysis through MA-TCK26D6. In this study, we show that plasmin is an important TAFI activator that hampers in vitro clot lysis. Furthermore, this is the first report on an anti-TAFI monoclonal antibody that demonstrates a strong profibrinolytic effect in a mouse thromboembolism model.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Carboxipeptidase B2/imunologia , Fibrinólise/imunologia , Tromboembolia/terapia , Animais , Afinidade de Anticorpos/imunologia , Antitrombina III/imunologia , Carboxipeptidase B2/metabolismo , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Humanos , Técnicas In Vitro , Pulmão/irrigação sanguínea , Pulmão/imunologia , Camundongos , Camundongos Mutantes , Peptídeo Hidrolases/imunologia , Especificidade da Espécie , Tromboembolia/imunologia , Tromboembolia/metabolismoRESUMO
A new sensitive, selective and simple immune resonance scattering spectral assay was proposed for the determination of trace amounts of Antithrombin-III (AT-III). It was based on the immune reaction of AT-III with the goat-anti-human AT-III antibody and the resonance scattering effect of the immunocomplex particles in pH 7.2 Tris-HCl buffer solution and in the presence of polyethelene glycol (PEG)6000. The results showed that the resonance scattering signal of AT-III and goat-anti-human AT-III antibody was very weak. However, AT-III was combined with goat-anti-human AT-III antibody specifically, and aggregated to form immunocomplex particles, which enhanced the resonance scattering intensity greatly and produced three resonance scattering peaks at 368 nm, 491 nm and 536 nm respectively. The strongest resonance scattering peak was at 491 nm. In the present paper, the influences of pH, goat-anti-human AT-III antibody and PEG-6000 concentration, incubation temperature, reactive time, and foreign substances were investigated. The result showed that the resonance scattering intensity at 491 nm (I(RS)) is linear to the AT-III concentration in the range of 62.5 to 850 ng x mL(-1), under the optimum conditions of 0.30 mL goat-anti-human AT-III antibody, 30 mg x mL(-1) polyethelene glycol-6000, being incubated at 37 degrees C for 15 min, the voltage at 400 V, and the excitation and emission slit width both at 5.0 nm. Its regress equation is deltaI(RS) 0.062 5c + 1.36, and a relative coefficient of 0.996, with a detection limit of 29.4 ng x mL(-1). The results of co-existing substance tolerance test showed that 1.40 x 10(5) ng x mL(-1) glycine, 9.0 x 10(3) ng x mL(-1) L-glutamic acid, 5.0 x 10(5) ng x mL(-1) glucose, 5.0 x 10(4) ng x mL(-1) urea, 1.5 x 10(4) ng x mL(-1) IgG, 3.0 x 10(4) ng x mL(-1) human serum albumin, and 1.5 x 10(4) ng x mL(-1) bovine serum albumin did not interfere with the resonance scattering determination of 2.50 x 10(2) ng x mL(-1) AT-III, when the relative error was within +/- 10%. Four polyethelene glycols, i. e. polyethelene glycol-4000, polyethelene glycol-6000, polyethelene glycol-10000 and polyethelene glycol-20000, on the immune resonance scattering spectral system were examined in details. The results show that polyethelene glycol-6000 has low blank and high deltaI(RS) value, and was chosen for use. The new resonance scattering spectral method features high sensitivity, good selectivity, simplicity and rapidity, and was applied to the quantitative analysis of AT-III in plasma and serum samples with satisfactory results. Its recovery is in the range of 90.2%-108.9%.
Assuntos
Antitrombina III/análise , Antitrombina III/imunologia , Humanos , Concentração de Íons de Hidrogênio , Espalhamento de Radiação , TemperaturaRESUMO
Complement is an essential part of the innate immune system, which clears pathogens without requirement for previous exposure, although it also greatly enhances the efficacy and response of the cellular and humoral immune systems. Kaposi's sarcoma-associated herpesvirus (KSHV) is the most recently identified human herpesvirus and the likely aetiological agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. We previously reported that the KSHV complement control protein (KCP) was expressed on infected cells and virions, and could inhibit complement through decay-accelerating activity (DAA) of the classical C3 convertase and cofactor activity (CFA) for factor I (FI)-mediated degradation of C4b and C3b, as well as acting as an attachment factor for binding to heparan sulphate on permissive cells. Here, we determined the ability of a panel of monoclonal anti-KCP antibodies to block KCP functions relative to their recognized epitopes, as determined through binding to recombinant KCP containing large (entire domain) or small (2-3 amino acid residue) alterations. One antibody recognizing complement control protein (CCP) domain 1 blocked heparin binding, DAA and C4b CFA, but was poor at blocking C3b CFA, while a second antibody recognizing CCP4 blocked C3b CFA and 80% DAA, but not C4b CFA or heparan sulphate binding. Two antibodies recognizing CCP2 and CCP3 were capable of blocking C3b and C4b CFA and heparan sulphate binding, but only one could inhibit DAA. These results show that, while KCP is a multifunctional protein, these activities do not completely overlap and can be isolated through incubation with monoclonal antibodies.
Assuntos
Antígenos CD55/imunologia , Herpesvirus Humano 8/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antitrombina III/imunologia , Ligação Competitiva/imunologia , Células CHO , C3 Convertase da Via Clássica do Complemento/imunologia , Complemento C3b/imunologia , Complemento C4b/imunologia , Cricetinae , Cricetulus , Mapeamento de Epitopos/métodos , Heparitina Sulfato/metabolismo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Upregulation of the activated Factor VII (FVIIa)/Tissue Factor complex, downregulation of natural anticoagulation pathways, and inhibition of fibrinolysis, are major contributors to coagulopathies associated with acute inflammation. Provision of FVIIa, and consequent downstream coagulation-related proteases, also stimulates further inflammatory changes, which can result in disseminated intravascular coagulation. Thus, the potential protective effects in vivo of a genetic-based reduction in FVII levels have been investigated in a murine model of acute inflammation, namely lipopolysaccharide (LPS)-induced lethal endotoxaemia. Mice with a total FVII deficiency do not survive the neonatal period. Therefore mice expressing low levels of FVII (FVII(tTA/tTA)), producing sufficient amounts of FVII for survival (approximately 5% of wild-type (WT) FVII), were employed to investigate in vivo pathways involved in the crosstalk between coagulation, inflammation, and survival, consequent to administration of a lethal dose of LPS. The FVII(tTA/tTA) mice presented with reduced mortality, coagulation, and inflammatory responses in comparison with similarly treated WT mice after administration of LPS. The attenuated inflammatory responses in FVII(tTA/tTA) mice were associated with downregulation of Egr-1 signalling. Administration, in vivo, of specific inhibitors of FXa and thrombin demonstrated that the inflammatory responses were unaltered in WT mice, but further reduced in FVII(tTA/tTA) mice. Therefore, a FVII deficiency enhances survival from lethal endotoxaemia both through attenuation of inflammatory responses that result directly from reduced FVIIa levels, and, indirectly, from downregulation of coagulation proteases downstream of the FVII-dependent cascade.
Assuntos
Endotoxemia/imunologia , Deficiência do Fator VII/imunologia , Ancrod/farmacologia , Animais , Anticoagulantes/farmacologia , Antitrombina III/imunologia , Biomarcadores/análise , Coagulação Sanguínea/imunologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Proteína 1 de Resposta de Crescimento Precoce/imunologia , Fator Xa/imunologia , Fibrinogênio/imunologia , Fondaparinux , Hirudinas/farmacologia , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Peptídeo Hidrolases/imunologia , Polissacarídeos/farmacologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais/imunologia , Trombina/imunologiaRESUMO
Antithrombin (AT), a member of the serine protease inhibitor family, is the key regulator of thrombin activity in vivo. Thrombin inhibition is accomplished by the formation of covalent thrombin-AT (TAT) complex. The rate of inhibition is accelerated by heparin, which also leads to the formation of a substantial amount of cleaved AT. We produced a murine monoclonal antibody (mAb) (M9) that is specific for the two forms of AT, in which the reactive center loop is inserted into beta-sheet A, i.e. cleaved and latent AT. The antibody has no measurable affinity for native AT. Using M9 as a catcher antibody in conjunction with a mAb (M27) that does not bind latent AT, we developed a sandwich assay that measures cleaved AT without interference from latent and native AT. The concentration in healthy subjects was determined to be 1.3 mg L(-1) (range: 1.0-1.9), which was about 100-fold lower than the plasma concentration of native AT and 1000-fold higher than the concentration of the TAT complex. The cleaved AT concentration is higher than what would be expected from the rate of formation of cleaved AT in vitro in conjunction with TAT complex formation in the presence of heparin. The concentration of cleaved AT did not correlate with the TAT concentration in plasma from patients with venous thrombosis.
Assuntos
Anticorpos Monoclonais , Antitrombina III/química , Ensaio de Imunoadsorção Enzimática , Fragmentos de Peptídeos/sangue , Animais , Afinidade de Anticorpos , Antitrombina III/imunologia , Heparina/farmacologia , Humanos , Camundongos , Fragmentos de Peptídeos/imunologia , Peptídeo Hidrolases/imunologia , Conformação Proteica , Trombose Venosa/sangueRESUMO
Increased thrombin generation occurs in the airways of asthmatic patients. Thrombin activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme which after activation by a thrombin-thrombomodulin complex inhibits fibrinolysis. The aim of this study was to evaluate TAFI concentration and activity in plasma of bronchial asthma patients challenged with Dermatophagoides pteronyssinus (Dp) extracts. The study was performed on 23 asthma patients mean age 28.7 +/- 9.8 years with a typical history and positive skin prick test with Dp allergens. Seventeen patients developed both early asthmatic reaction (EAR) and late asthmatic reaction (LAR), 6 patients developed only EAR. Blood was collected before allergen challenge (AO), 1 (A1), 8 (A2) and 24 hours (A3) after allergen challenge. Five healthy persons, mean age 24.5 +/- 3.6 years with negative skin prick tests to common aeroallergens served as negative controls. Healthy controls underwent sham bronchial provocation with 0.9% solution of NaCl. TAFI antigen and activity, levels of thrombin-antithrombin III complexes (TAT) and prothrombin fragments F 1+2 were measured in plasma by ELISA method. At A0 in dual responders mean TAFI concentration (101.4 +/- 23.82%) and activity (19.6 +/- 5.92 microg/ml) were higher than in healthy controls (69.35 +/- 21.49%; p < 0.05 and 10.49 +/- 3.53 microg/ml; p = 0.01; respectively). Significant fall in TAFI plasma concentration was detected at A1 and the decreased concentration of TAFI persisted until A3 (85.15 +/- 26.36%; p < 0.05). No significant change in plasma TAFI concentration was observed in healthy controls or in patients who responded with an EAR only. Allergen induced inflammation is associated with significant changes in plasma TAFI concentration.
Assuntos
Asma/sangue , Carboxipeptidase B2/sangue , Trombina/metabolismo , Adulto , Alérgenos/imunologia , Animais , Antitrombina III/imunologia , Asma/imunologia , Testes de Provocação Brônquica , Carboxipeptidase B2/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fibrinólise , Humanos , Masculino , Peptídeo Hidrolases/sangue , Peptídeo Hidrolases/imunologia , Pyroglyphidae/imunologia , Trombomodulina/metabolismoRESUMO
Soluble inhibitory factors produced by CD8+ T-cells have been shown to inhibit HIV-1 replication and may play a critical role in vivo in anti-viral host defense. CD8+ T-cell-modified antithrombin III (ATIII) accounts for some of the described CD8+ T-cell anti-viral activity. We demonstrate that CD4+ T-cells, CD8+ T-cells, and natural killer cells react to an ATIII gradient by cell migration. Furthermore, exogenously added ATIII induced a G-protein-coupled signal transduction process in CD4+ T-cells and inhibited TNF-alpha-induced NF-kappaB activation. Heat and/or heparin treatment prior to the anti-viral inhibition test increased the anti-HIV activity up to 1000-fold. Our data indicate that anti-viral inactive ATIII can be activated having promising anti-viral properties as complementary candidate for the treatment of HIV infection.
Assuntos
Antitrombina III/farmacologia , Antivirais/farmacologia , Animais , Fármacos Anti-HIV/farmacologia , Anticorpos Monoclonais/farmacologia , Antitrombina III/química , Antitrombina III/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Bovinos , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Heparina/química , Temperatura Alta , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ritonavir/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transfecção , Replicação Viral/efeitos dos fármacos , Zidovudina/farmacologiaRESUMO
BACKGROUND: As an ideal candidate for xenotransplantation, the compatibility of physiological porcine organs with those of humans is an essential premise. In this study, we analyzed hepatic coagulant, fibrinolytic, and anticoagulant functions between Banna Minipig Inbreds (BMIs) and humans to evaluate such hepatic compatibility. METHODS: BMI factors II, V, VII, X, and XII were added to the corresponding factor-deficient human plasma to determine prothrombin times (PT) and activated partial thromboplastin times (APTT). Human tissue plasminogen activator (t-PA) was added to both BMI and human plasma to determine plasmin activity. The antithrombin-III (AT-III) activity of plasma was analyzed with the STA-Stago autoanalyzer using an AT-III assay kit. RESULTS: Both PT and APTT were reduced but within normal parameters when BMI factors II, V, VII, X, and XII were added to the corresponding factor-deficient human plasma. The activities of BMI coagulation factors II, V, VII, X, and XII were 3.2, 3.7, 4.7, 2.9, and 4.5 times those of humans, respectively. The activity of plasmin was significantly higher in BMI plasma than in humans when human t-PA was added to both. The normal range of human AT-III activity was 90-108% while BMI AT-III was 124.50 +/- 2.38%. CONCLUSIONS: The activities of coagulation factors and AT-III were higher in BMIs than in humans. BMI coagulation factors XII, VII, and X trigger human intrinsic, extrinsic, and common pathways, respectively, which functioned normally. In addition, BMI plasminogen could be activated by human t-PA.
Assuntos
Anticoagulantes/imunologia , Coagulantes/imunologia , Fibrinólise/imunologia , Fígado/imunologia , Adulto , Animais , Antitrombina III/imunologia , Humanos , Masculino , Tempo de Protrombina , Especificidade da Espécie , Suínos , Porco Miniatura , TriazinasRESUMO
A high-affinity monoclonal antibody (M27), raised against the human thrombin-antithrombin complex, has been identified and characterized. The epitope recognized by M27 was located to the linear sequence FIREVP (residues 411-416), located in the C-terminal cleavage peptide of antithrombin. This region overlaps, by two residues, the putative binding site of antithrombin for the serpin-enzyme complex receptor. Studies in rats and with HepG2 cells in culture indicated that the Fab fragment of M27 does not block binding and uptake of the thrombin-antithrombin complex, suggesting that this region does not play a major role in the recognition and clearance of the thrombin-antithrombin complex. M27 blocked the ability of antithrombin to inhibit thrombin as well as antithrombin cleavage, both in the presence and absence of heparin.