RESUMO
The proximate cause of autoantibodies characteristic of systemic autoimmune diseases has been controversial. One hypothesis is that autoantibodies are the result of polyclonal nonspecific B cell activation. Alternatively, autoantibodies could be the result of antigen-driven B cell activation, as observed in secondary immune responses. We have approached this question by studying monoclonal anti-DNA autoantibodies derived from unmanipulated spleen cells of the autoimmune MRL/lpr mouse strain. This analysis shows that anti-DNAs, like rheumatoid factors (19), are the result of specific antigen-driven stimulation. In addition, correlation of sequences with fine specificity shows that: (a) somatic mutations can cause specificity for dsDNA and that such mutations are selected for; (b) arginine residues play an important role in determining specificity; and (c) anti-idiotypes that recognize the majority of anti-DNA are probably not specific for any one family of V regions.
Assuntos
Autoanticorpos/genética , Doenças Autoimunes/genética , DNA/imunologia , Mutação , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Doenças Autoimunes/imunologia , Sequência de Bases , Células Clonais , DNA de Cadeia Simples/imunologia , Ensaio de Imunoadsorção Enzimática , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , RNA Mensageiro/genética , Especificidade da EspécieRESUMO
BACKGROUND: Secretory leucocyte protease inhibitor (SLPI), which is present in many physiological fluids including saliva, sputum and nasal discharge, is the most effective inhibitor of chymase. Previously, we demonstrated that chymase is able to cleave SLPI and that the cleaved portion, cSLPI, is a biomarker of chymase activity. OBJECTIVE: We investigated the potential of cSLPI as a biomarker of chymase activity in subjects with allergic rhinitis (AR) and asthmatic airway disease. METHODS: Baseline sputum samples were collected from atopic asthmatics and healthy controls (HC). Nasal lavages (NAL) were performed in subjects with AR both at baseline and following a nasal challenge with allergen or placebo. Levels of cSLPI and chymase were determined by Western analysis, and tryptase and alpha-2 macroglobulin were measured by immunoassay. RESULTS: As compared with HC, asthmatics showed a significant increase in baseline cSLPI/total SLPI ratios and an increase in chymase levels. There was a high correlation of cSLPI/SLPI ratios to chymase levels in normal individuals and untreated asthmatics. In the NAL of patients with AR, as compared with placebo, allergen challenge increased inflammatory biomarkers, including cSLPI/SLPI ratios, chymase levels, tryptase levels and alpha2-macroglobulin levels. Correlations were observed between cSLPI/SLPI ratios and chymase levels and cSLPI/SLPI ratios and alpha2-macroglobulin levels; no correlation was seen between cSLPI/SLPI ratios and tryptase levels. CONCLUSION: Our data indicate that cSLPI reflects chymase activity in AR and asthma. Hence, cSLPI may serve as a biomarker for disease activity and for monitoring the efficacy of novel anti-inflammatory treatments in chymase-mediated diseases.
Assuntos
Quimases/metabolismo , Hipersensibilidade Respiratória/metabolismo , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Adolescente , Adulto , Idoso , Biomarcadores/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Líquido da Lavagem Nasal/química , Líquido da Lavagem Nasal/imunologia , Hipersensibilidade Respiratória/enzimologia , Hipersensibilidade Respiratória/imunologia , Estudos Retrospectivos , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/metabolismo , Inibidor Secretado de Peptidases Leucocitárias/imunologia , Escarro/enzimologia , Escarro/imunologia , Escarro/metabolismo , Triptases/metabolismo , alfa-Macroglobulinas/metabolismoRESUMO
BACKGROUND: We aimed to study the reproducibility of several biomarkers of allergic rhinitis to investigate their potential as outcome measures in clinical intervention trials. Furthermore, we investigated the kinetics of the biomarkers studied in nasal lavage and brush material following a placebo-controlled nasal allergen challenge. METHODS: We performed a skin prick test and measured serum specific immunoglobulin (Ig) E levels and inflammatory biomarkers in nasal lavage and brush material in 20 patients with allergic rhinitis on 2 separate days (washout, 14-21 days). The patients were then randomly assigned to undergo an intranasal challenge with a relevant allergen (n=10) or diluent (n=10) in order to assess the kinetics of several biomarkers of allergic airway inflammation in nasal lavage and brush samples. RESULTS: Baseline serum IgE levels and skin wheal sizes were highly reproducible measurements, with a coefficient of variation (CV) of 13.4% and 18.2%, respectively. This was not the case with the majority of inflammatory biomarkers, whose CV varied considerably (range, 6.1%-224.1%). The nasal allergen challenge induced an increase in composite symptom scores in all patients. Compared to placebo, tryptase (P=.004), eosinophilic cationic protein (ECP) (P=.03) and alpha2-macroglobulin (P=.002) were increased in nasal lavage at 20 minutes post allergen. Nasal lavage ECP levels and nasal brush eosinophils were still significantly increased at 7 hours (P=.03 and P=.04), but all statistical significance had been lost at 24 hours post challenge. CONCLUSION: Serum specific IgE assays and skin prick tests exhibited good reproducibility in patients with clinically stable allergic rhinitis. We were also able to investigate the kinetics of allergen-induced upper airway inflammatory markers in nasal lavage and brush material. Hence, nasal allergen challenge, when used in combination with nasal lavage and brush sampling, is a suitable research tool for early drug development.
Assuntos
Anti-Inflamatórios/uso terapêutico , Rinite Alérgica Perene/tratamento farmacológico , Rinite Alérgica Sazonal/tratamento farmacológico , Adulto , Alérgenos/imunologia , Biomarcadores/sangue , Feminino , Humanos , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Líquido da Lavagem Nasal/imunologia , Testes de Provocação Nasal , Nariz/imunologia , Reprodutibilidade dos Testes , Rinite Alérgica Perene/sangue , Rinite Alérgica Sazonal/sangue , Testes CutâneosRESUMO
The protection of drinking water is a key issue in a Public Health Department's activity. A substantial amount of planning and monitoring work is involved in the development and implementation of a water safety plan, aimed not only at the enforcement of public health regulations, but also at the improvement of the quality water. We provide an overview of the quality monitoring program of the municipality of Prato, a highly populated and industrialized area, where ground water is contaminated by anthropogenic pollutants such as trichloroethylene, tetrachloroethylene and nitrate. We show how, in spite of the intrinsically poor quality of the basic water resource, the careful application of an appropriate prevention plan, with the cooperation of the local water authority, allows the delivery of drinking water of increasing safety and quality.
Assuntos
Indústrias , Saúde Pública , Purificação da Água/normas , Abastecimento de Água/normas , Monitoramento Ambiental/métodos , Humanos , Indústrias/legislação & jurisprudência , Itália , Nitratos/análise , Política Pública , Rios , Solventes/análise , Tetracloroetileno/análise , Tricloroetileno/análise , Poluição Química da Água/análise , Poluição Química da Água/prevenção & controle , Abastecimento de Água/legislação & jurisprudênciaRESUMO
BACKGROUND: Previous investigators have shown that systemic markers of inflammation may be increased in patients with acute ischemic syndromes or after percutaneous coronary revascularization and that persistent elevation in these markers is predictive of excess risk of subsequent adverse cardiac events. By virtue of its cross-reactivity with the glycoprotein IIb/IIIa, avbeta3, and alphaMbeta2 receptors, abciximab may reduce inflammatory processes. Methods and Results-- Assays for the inflammatory markers C-reactive protein, interleukin-6, and tumor necrosis factor-alpha were performed on serum samples obtained from 160 patients in a placebo-controlled, randomized trial of abciximab during angioplasty. Eighty patients each had received a placebo or abciximab bolus plus a 12-hour infusion. Serum samples were drawn at baseline (before revascularization), 24 to 48 hours after study drug administration, and 4 weeks after study drug administration. Between baseline and 24 to 48 hours, the increase in C-reactive protein was 32% less in patients receiving abciximab than placebo (P=0.025); the rise in interleukin-6 levels was 76% less in the abciximab group (P<0.001); and the rise in tumor necrosis factor-alpha levels was 100% less with abciximab therapy (P=0.112). By 4 weeks, most marker levels had returned to baseline, with no significant differences between placebo and abciximab groups. CONCLUSIONS: Systemic markers of inflammation increase in the first 24 to 48 hours after angioplasty, but the magnitude of that rise is diminished by periprocedural abciximab. Some of the long-term clinical benefit derived from this agent may be related to an anti-inflammatory effect.
Assuntos
Angioplastia Coronária com Balão , Anticorpos Monoclonais/administração & dosagem , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Inflamação/prevenção & controle , Inibidores da Agregação Plaquetária/administração & dosagem , Abciximab , Angioplastia Coronária com Balão/efeitos adversos , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Feminino , Humanos , Inflamação/sangue , Inflamação/etiologia , Infusões Intravenosas , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismo , Estados UnidosRESUMO
BACKGROUND: Abciximab and ticlopidine reduce adverse cardiovascular events after percutaneous transluminal coronary angioplasty (PTCA). The goal of the current study was to determine if combined abciximab/ticlopidine therapy inhibits arterial thrombosis more effectively than either treatment alone. The effect of each therapy on platelet-leukocyte interactions was also investigated. METHODS AND RESULTS: Whole blood samples from 14 patients undergoing PTCA who received abciximab therapy, ticlopidine therapy, or both treatments were evaluated using dynamic experimental systems. Mural thrombus formation under arterial shear conditions (1500 s(-1)) was determined in a parallel plate flow chamber. Shear-induced platelet aggregation was evaluated using a cone-and-plate viscometer at a shear rate of 3000 s(-1). Of the 3 treatments, combined abciximab/ticlopidine therapy produced the most consistent reduction in shear-induced platelet aggregation and the most prolonged inhibition of mural thrombosis. Three days after PTCA, abciximab/ticlopidine treatment decreased mural thrombus formation to approximately 50% of baseline values. Abciximab treatment alone inhibited mural thrombosis for only 1 day after PTCA, whereas ticlopidine treatment alone had no significant effect. Two hours after PTCA, abciximab therapy significantly decreased the number of circulating platelet-neutrophil aggregates but significantly enhanced P-selectin-mediated leukocyte adhesion on the collagen/von Willebrand factor-platelet surface. CONCLUSIONS: Combined therapy with abciximab and ticlopidine has a prolonged inhibitory effect on mural thrombosis formation relative to either treatment alone. Further, we demonstrated an unexpected effect of abciximab in enhancing P-selectin-mediated leukocyte adhesion.
Assuntos
Angioplastia Coronária com Balão , Anticorpos Monoclonais/uso terapêutico , Anticoagulantes/uso terapêutico , Plaquetas/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Leucócitos/efeitos dos fármacos , Trombose/prevenção & controle , Ticlopidina/uso terapêutico , Abciximab , Anticorpos Monoclonais/farmacologia , Anticoagulantes/farmacologia , Plaquetas/fisiologia , Quimioterapia Combinada , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Leucócitos/fisiologia , Glicoproteínas de Membrana/fisiologia , Selectina-P/fisiologia , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Trombose/etiologia , Ticlopidina/farmacologiaRESUMO
BACKGROUND: Both inherited predisposition and platelet hyperreactivity have been associated with ischemic coronary events, but mechanisms that support genetic differences among platelets from different subjects are generally lacking. Associations between the platelet Pl(A2) polymorphism of GP IIIa and coronary syndromes raise the question as to whether this inherited variation may contribute to platelet hyperreactivity. METHODS AND RESULTS: In this study, we characterized functional parameters in platelets from healthy donors with the Pl(A) (HPA-1) polymorphism, a Leu (Pl(A1)) to Pro (Pl(A2)) substitution at position 33 of the GP IIIa subunit of the platelet GP IIb/IIIa receptor (integrin alpha(IIb)beta(3)). We studied 56 normal donors (20 Pl(A1,A1), 20 Pl(A1,A2), and 16 Pl(A2,A2)). Compared with Pl(A1,A1) platelets, Pl(A2)-positive platelets showed a gene dosage effect for significantly greater surface-expressed P-selectin, GP IIb/IIIa-bound fibrinogen, and activated GP IIb/IIIa in response to low-dose ADP. Surface expression of GP IIb/IIIa was similar in resting platelets of all 3 genotypes but was significantly greater on Pl(A2,A2) platelets after ADP stimulation (P=0.003 versus Pl(A1,A1); P=0.03 versus Pl(A1,A2)). Pl(A1,A2) platelets were more sensitive to inhibition of aggregation by pharmacologically relevant concentrations of aspirin and abciximab. CONCLUSIONS: Pl(A2)-positive platelets displayed a lower threshold for activation, and platelets heterozygous for Pl(A) alleles showed increased sensitivity to 2 antiplatelet drugs. These in vitro platelet studies may have relevance for in vivo thrombotic conditions.
Assuntos
Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Polimorfismo Genético/fisiologia , Abciximab , Difosfato de Adenosina/farmacologia , Adulto , Substituição de Aminoácidos/genética , Anticorpos Monoclonais/farmacologia , Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Feminino , Fibrinogênio/metabolismo , Dosagem de Genes , Genótipo , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Masculino , Selectina-P/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Polimorfismo Genético/genética , Valores de ReferênciaRESUMO
BACKGROUND: Platelet glycoprotein IIb/IIIa blockade with abciximab (ReoPro) improves the clinical outcomes of percutaneous coronary intervention. This registry was conducted to characterize the effects of repeated administration of abciximab during intervention. METHODS AND RESULTS: We recruited 500 consecutive patients at 22 centers in the United States who were receiving abciximab for at least a second time during percutaneous coronary intervention. Safety was measured as the incidence of hypersensitivity reactions, major bleeding, and thrombocytopenia. Efficacy was assessed as event-free clinical success. Human antichimeric antibody (HACA) responses were also characterized. There were no cases of hypersensitivity (95% upper confidence bound, 0.3%), major bleeding, or death. Clinical success was 94.4%. Thrombocytopenia occurred in 23 patients (4.6%; 95% CI, 2.8% to 6.4%), including 12 (2.4%; 95% CI, 1.1% to 3.7%) who developed profound thrombocytopenia (<20x10(9) cells/L). In 2 patients (0.4%), profound thrombocytopenia did not develop until after hospital discharge; in 4 (0.8%), profound thrombocytopenia recurred despite platelet transfusion. Before a first readministration, a positive HACA titer was present in 22 of 454 patients (4.8%); after a first readministration, an additional 82 of 432 (19.0%) became HACA-positive. HACA did not neutralize the in vitro inhibition of platelet aggregation by abciximab or correlate with clinical events. CONCLUSIONS: The results, including overall rates of thrombocytopenia, were consistent with randomized clinical trials of first abciximab treatment. However, there was a shift from mild to profound thrombocytopenia, and cases of delayed presentation and of recurrent thrombocytopenia were seen. These findings suggest that indications and guidelines for first-time use apply to retreatment, particularly the systematic monitoring for thrombocytopenia.
Assuntos
Angioplastia Coronária com Balão , Anticorpos Monoclonais/administração & dosagem , Doença das Coronárias/terapia , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Sistema de Registros/estatística & dados numéricos , Trombocitopenia/diagnóstico , Grau de Desobstrução Vascular/efeitos dos fármacos , Abciximab , Angioplastia Coronária com Balão/efeitos adversos , Anticorpos/sangue , Anticorpos/farmacologia , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Aspirina/administração & dosagem , Doença das Coronárias/sangue , Intervalo Livre de Doença , Esquema de Medicação , Hemorragia/etiologia , Heparina/administração & dosagem , Humanos , Fragmentos Fab das Imunoglobulinas/efeitos adversos , Fragmentos Fab das Imunoglobulinas/imunologia , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/efeitos adversos , Inibidores da Agregação Plaquetária/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complicações Pós-Operatórias/prevenção & controle , Trombocitopenia/etiologia , Resultado do Tratamento , Estados UnidosRESUMO
BACKGROUND: We evaluated platelet activation and aggregation in patients with acute myocardial infarction (AMI) treated with thrombolytic therapy alone or with reduced-dose thrombolysis and concomitant abciximab. METHODS AND RESULTS: The study was performed in 20 control subjects and 51 patients with AMI before and after reperfusion with either alteplase or reteplase or reduced doses of these agents with concomitant abciximab. Platelet activation was assayed by platelet surface expression of P-selectin. Turbidometric platelet aggregation in response to ADP was measured in patients before thrombolytic therapy and 90 minutes and 24 hours after the beginning of thrombolytic therapy. P-selectin expression was greater at baseline in patients than normal control subjects (30.4% versus 9. 8%, P<0.0001) but was identical between the 2 groups after stimulation with ADP (64.4% versus 69.3%, P=0.37). However, at 24 hours, basal P-selectin expression declined in patients (P=0.0025 versus baseline), whereas ADP-stimulated P-selectin expression was lower in patients than in control subjects (48% versus 69%, P=0. 0004). When combined with reduced doses of either alteplase or reteplase, abciximab achieved 91% and 83% inhibition of 5 and 20 micromol/L ADP-induced platelet aggregation, which decreased to 46% and 40%, respectively, at 24 hours. No appreciable difference in the platelet inhibition profile of abciximab was observed between the 2 thrombolytics. CONCLUSIONS: Platelet activation and aggregation are heightened in the setting of thrombolysis for AMI. Despite this enhanced level of platelet activation, abciximab, combined with a reduced-dose thrombolytic, inhibited platelet aggregation similarly to the level reported in elective settings.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Infarto do Miocárdio/tratamento farmacológico , Ativadores de Plasminogênio/administração & dosagem , Inibidores da Agregação Plaquetária/administração & dosagem , Agregação Plaquetária/efeitos dos fármacos , Terapia Trombolítica , Ativador de Plasminogênio Tecidual/administração & dosagem , Abciximab , Idoso , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Angiografia Coronária , Feminino , Fibrinolíticos/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Selectina-P/biossíntese , Proteínas Recombinantes/administração & dosagem , Resultado do TratamentoRESUMO
OBJECTIVES: This study determined the incidence of pseudothrombocytopenia during abciximab therapy administered for percutaneous coronary interventions and compared the clinical course of patients with pseudothrombocytopenia with the clinical courses of patients with thrombocytopenia and patients with normal platelet counts. BACKGROUND: Although pseudothrombocytopenia has been previously reported during therapy with abciximab, the incidence and significance of this occurrence are unknown. The failure to differentiate pseudothrombocytopenia from thrombocytopenia could lead to unnecessary interruption of abciximab infusions or to platelet transfusions. METHODS: The incidences of pseudothrombocytopenia and thrombocytopenia were determined in four large placebo-controlled abciximab trials: c7E3 Fab Antiplatelet Therapy in Unstable Refractory Angina (CAPTURE), Evaluation of 7E3 for the Prevention of Ischemic Complications (EPIC), Evaluation of Percutaneous Transluminal Coronary Angioplasty to Improve Long-term Outcome of c7E3 GpIIb/IIIa Receptor Blockade (EPILOG) and Evaluation of Platelet IIb/IIIa Inhibitor for Stenting (EPISTENT). The clinical features, bleeding complications and major clinical outcomes of patients with pseudothrombocytopenia and those with thrombocytopenia were compared with each other and with those of patients with normal platelet count. RESULTS: Pseudothrombocytopenia occurred in 2.1% (95% confidence intervals [CI]: 1.7%, 2.5%) of abciximab-treated patients and in 0.6% of placebo-treated patients (p < 0.001). Thrombocytopenia occurred in 3.7% (95% CI: 3.2%, 4.2%) of abciximab-treated patients and in 1.8% (95% CI: 1.3%, 2.3%) of placebo-treated patients (p < 0.001). Patients with thrombocytopenia had significantly higher rates of major bleeding, major decreases in hemoglobin and increased transfusion requirements of both blood and platelets compared with those without thrombocytopenia. By contrast, pseudothrombocytopenic patients did not differ from patients with normal platelet counts in any of the measures of blood loss or transfusion requirements. Thrombocytopenic patients, but not those with pseudothrombocytopenia, had increased rates of revascularization at 30 days and six months. As previously reported, there was also a higher rate of death and myocardial infarction in the thrombocytopenic patients. CONCLUSIONS: Pseudothrombocytopenia is the cause of more than one third (36.3%) of low platelet counts in patients undergoing coronary interventions who are treated with abciximab. This study demonstrates that pseudothrombocytopenia is a benign laboratory condition that does not increase bleeding, stroke, transfusion requirements or the need for repeat revascularization. It is important to recognize pseudothrombocytopenia so that the beneficial effects of abciximab are not lost by premature termination of therapy.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Fragmentos Fab das Imunoglobulinas/efeitos adversos , Inibidores da Agregação Plaquetária/efeitos adversos , Trombocitopenia/induzido quimicamente , Abciximab , Idoso , Angina Instável/sangue , Angina Instável/tratamento farmacológico , Angina Instável/cirurgia , Anticorpos Monoclonais/administração & dosagem , Intervalos de Confiança , Ponte de Artéria Coronária , Diagnóstico Diferencial , Método Duplo-Cego , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Incidência , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/administração & dosagem , Contagem de Plaquetas/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Estudos Prospectivos , Trombocitopenia/sangue , Trombocitopenia/diagnósticoRESUMO
OBJECTIVE: Enhanced GPIIb/IIIa binding and inhibition of platelet aggregation of eptifibatide by the reduction of ionized plasma calcium concentrations have been reported. The present study compared the importance of Ca2+ chelation on the in vitro platelet inhibitory profiles of the GPIIb/IIIa antagonists abciximab, eptifibatide and tirofiban. METHODS AND RESULTS: Turbidimetric platelet aggregation dose response curves of the various GPIIb/IIIa antagonists were performed using platelet rich plasma (PRP) anticoagulated with either trisodium citrate, or the non-chelating anticoagulant, PPACK. The concentrations of antagonist that resulted in 50% inhibition of TRAP-induced (10 microM) platelet aggregation (IC50) were measured in the presence of either citrate or PPACK. In addition, the influence of Ca2+ chelation on the binding properties (relative affinity, on- and off-rates) of abciximab for the GPIIb/IIIa receptor on platelets was measured. For all three agonists, the IC50 concentrations were lower for platelets treated with citrate than PPACK, but the degree of difference varied among the agents. The mean TRAP IC50 values for citrate and PPACK were 88.2 +/- 12.2 nM and 126.1 +/- 28.4 nM for abciximab (1.4 fold enhancement; p = 0.0007), 75.9 +/- 13.3 nM and 142.6 +/- 32.6 nM for tirofiban (1.9-fold enhancement; p = 0.001), and 260.2 +/- 62.5 nM and 810.3 +/- 182.5 nM for eptifibatide (3.1-fold enhancement; p = 0.001). A similar shift in effective inhibitor concentrations for abciximab was observed with ADP (10 microM). The relative affinities (EC50), on- and off-rates of abciximab for the platelet GPIIb/IIIa receptor in the presence of trisodium citrate and PPACK were equivalent. CONCLUSIONS: These data confirm previous observations that Ca2+ chelation afforded by citrate decreases the effective inhibitor concentrations of GPIIb/IIIa antagonists, as assessed by turbidimetric platelet aggregation. However, the extent of decrease was less for abciximab and tirofiban, compared to eptifibatide.
Assuntos
Cálcio/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Abciximab , Anticorpos Monoclonais/farmacologia , Anticoagulantes/farmacologia , Cálcio/metabolismo , Quelantes/farmacologia , Ácido Cítrico/farmacologia , Relação Dose-Resposta a Droga , Eptifibatida , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Cinética , Peptídeos/farmacologia , Tirofibana , Tirosina/análogos & derivados , Tirosina/farmacologiaRESUMO
In addition to inhibition of platelet aggregation, GPIIb-IIIa antagonists may reduce thrombotic events via other mechanisms. In a novel whole blood flow cytometric system, we investigated the effects of GPIIb-IIIa antagonists, in the presence or absence of thrombin inhibitors, on platelet surface-bound factor V/Va and platelet surface phospholipids. Diluted venous blood was incubated with either buffer or a GPIIb-IIIa antagonist (abciximab, tirofiban, or eptifibatide). Some samples were pre-incubated with clinically relevant concentrations of unfractionated heparin (UFH), a low molecular weight heparin, a direct thrombin inhibitor, or buffer only. Platelets were then activated and labeled with mAb V237 (factor V/Va-specific) or annexin V (binds phosphatidylserine), fixed, and analyzed by flow cytometry. In the absence of thrombin inhibitors, GPIIb-IIIa antagonists (especially abciximab) significantly reduced agonist-induced platelet procoagulant activity, as determined by reduced binding of V237 and annexin V. At high pharmacologic concentrations, unfractionated heparin and enoxaparin, but not hirudin, further reduced factor V/Va binding to the surface of activated platelets in the presence of GPIIb-IIa antagonists. Agonist-induced platelet procoagulant activity was reduced in a patient with Glanzmann's thrombasthenia. We conclude that GPIIb-IIIa antagonists reduce platelet procoagulant activity in whole blood and heparin and enoxaparin augment this reduction. Fibrinogen binding to GPIIb-IIIa is important in the generation of platelet procoagulant activity.
Assuntos
Fator V/metabolismo , Fosfatidilserinas/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Tirosina/análogos & derivados , Abciximab , Anticorpos Monoclonais/farmacologia , Plaquetas/metabolismo , Colágeno/farmacologia , Relação Dose-Resposta a Droga , Eptifibatida , Citometria de Fluxo , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Recém-Nascido , Proteínas de Membrana/metabolismo , Peptídeos/farmacologia , Ligação Proteica , Trombastenia/sangue , Trombina/antagonistas & inibidores , Trombina/farmacologia , Tirofibana , Tirosina/farmacologiaAssuntos
Asma/imunologia , Rinite Alérgica Perene/imunologia , Rinite Alérgica Sazonal/imunologia , Adulto , Alérgenos/imunologia , Asma/diagnóstico , Biomarcadores/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Rinite Alérgica Perene/diagnóstico , Rinite Alérgica Sazonal/diagnóstico , Adulto JovemRESUMO
AIMS: The aim of this study was to assess the inter- and intra-laboratory variation of the concentration-response to the GPIIb/IIIa-antagonists abciximab and eptifibatide on platelet aggregometry and to compare results with flow cytometric tests as well as the rapid platelet function analyser (RPFA). METHODS: In five different laboratory sites, blood from three to five healthy donors was spiked with abciximab or eptifibatide, followed by the assessment of: (1) aggregometry (anticoagulant: sodium citrate 3.18% or hirudin 5 microg/ml); (2) flow cytometry (fibrinogen binding or PAC1-expression), or (3) RPFA. Dose-response curves were established on the basis of a sigmoidal Imax)-model [I=(Imax)*Cg)/(IC50g + Cg)]. RESULTS: For citrated blood, aggregation induced by 20 microM ADP was blocked up to 100% by both GPIIb/IIIa-antagonists, IC50 values varied between 0.11-0.22 microg/ml for eptifibatide and 1.25-2.3 microg/ml for abciximab. I(max) of the response to 5 microg/ml collagen ranged from 46% to 100%, and IC50 values varied between 0.28-0.34 microg/ml for eptifibatide and 2.3-3.8 microg/ml for abciximab. In hirudinized blood, IC50 values for eptifibatide were 1.5- to 3-fold higher than those obtained with citrated plasma. Inhibition of PAC1-expression by abciximab (IC50) 0.84 microg/ml) showed results similar those of the RPFA (approx. 1.0 microg/ml); larger differences between PAC1 and RPFA results were observed for eptifibatide. Based on aggregometry, eptifibatide concentrations for 80% inhibition varied from 0.27 to 0.55 microg/ml, and were considerably less when the RPFA was taken as basis (0.15 or 0.22 microg/ml). A similar pattern was observed for abciximab. CONCLUSIONS: We found quite a low inter- and intra-laboratory variation in the in vitro pharmacodynamic characterization of GPIIb/IIIa-antagonists by aggregometry, making results of these tests obtained from different laboratories during clinical trials at least comparable. The RPFA exhibits a higher sensitivity to inhibitory GPIIb/IIIa-effects, in keeping with the "real" inhibition of the activated receptor (PAC1) as assessed with more elaborate flow cytometry.
Assuntos
Técnicas de Laboratório Clínico/normas , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Abciximab , Anticorpos Monoclonais/farmacologia , Técnicas de Laboratório Clínico/estatística & dados numéricos , Relação Dose-Resposta a Droga , Eptifibatida , Fibrinogênio , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Microesferas , Variações Dependentes do Observador , Peptídeos/farmacologia , Testes de Função Plaquetária/instrumentação , Testes de Função Plaquetária/normas , Testes de Função Plaquetária/estatística & dados numéricos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologiaRESUMO
Abciximab (ReoPro) is a mouse-human chimeric monoclonal antibody Fab fragment of the parent murine monoclonal antibody 7E3, and was the first of these agents approved for use as adjunct therapy for the prevention of cardiac ischemic complications in patients undergoing percutaneous coronary intervention (PCI). Abciximab binds with high avidity to both the non-activated and activated form of the GPIIb/IIIa receptor of platelets, the major adhesion receptor involved in aggregation. Additional cardiovascular indications for abciximab are unstable angina, carotid stenting, ischemic stroke and peripheral vascular diseases. Abciximab also interacts with two other integrin receptors; the a av b b3 receptor, which is present in low numbers on platelets but in high density on activated endothelial and smooth muscle cells, and a aMb b2 integrin which is present on activated leukocytes. Cell types that express integrins GPIIb/IIIa and a av b b3 such as platelets, endothelial and tumor cells have been implicated in angiogenesis, tumor growth and metastasis. Since abciximab interacts with high avidity to integrins GPIIb/IIIa and a av b b3, it is reasonable to assume that it may possess anti-angiogenic properties in angiogenesis-related diseases, as well as anti-metastastatic properties in case of disseminating tumors expressing the target integrin receptors.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Isquemia/terapia , Metástase Neoplásica/tratamento farmacológico , Inibidores da Agregação Plaquetária/uso terapêutico , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Trombose/terapia , Abciximab , Ensaios Clínicos como Assunto , Humanos , Isquemia/fisiopatologia , Metástase Neoplásica/fisiopatologia , Trombose/fisiopatologiaRESUMO
The clinical consequences of immune antibodies generated to abciximab (ReoPro) and infliximab (Remicade) are described. Abciximab, a chimaeric Fab fragment that binds to the beta3 integrin of the GPIIb/IIIa and alphavbeta3 receptors on human platelets, is approved in the US and Europe for use in percutaneous coronary intervention (PCI) to prevent cardiac ischaemic complications. The effects of induced antibodies upon the safety and efficacy of repeat administration of abciximab have been evaluated in the ReoPro Re-administration Registry Study, in which 5.7% of patients were HACA positive before re-treatment. An interim evaluation of 1000 patients has indicated that re-administration of abciximab can be accomplished in the setting of PCI with an acceptable safety and efficacy profile. Infliximab is a chimaeric IgG1 antibody specific for human TNFalpha, and is approved in the US and Europe for the acute treatment of the signs and symptoms of Crohn's disease and for the chronic treatment of rheumatoid arthritis (RA). The incidence of antibodies to infliximab is reported to be approximately 10%; however, an inverse dose-immunogenicity relationship was observed, indicating that higher doses of infliximab (> or = 3 to 10 mg/kg) could reduce the incidence of immune antibodies. The induction of immune antibodies could also be reduced by concomitant administration of low-dose methotrexate and other immunosuppressant agents. Although antibodies to infliximab appeared to be associated with lower serum infliximab concentrations and a slightly higher incidence of infusion reactions, these immune antibodies were generally not associated with a reduction in clinical efficacy. In addition, the antibodies induced to infliximab are specific for infliximab, and do not cross-react with other currently available therapeutic antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Antirreumáticos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Abciximab , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/efeitos adversos , Antirreumáticos/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Fragmentos Fab das Imunoglobulinas/efeitos adversos , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Infliximab , Contagem de PlaquetasAssuntos
Isquemia Encefálica/tratamento farmacológico , Inibidores da Agregação Plaquetária/uso terapêutico , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Humanos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/administração & dosagem , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Terapia TrombolíticaRESUMO
BACKGROUND: Exhaled nitric oxide (eNO) is an established, noninvasive biomarker of active airway inflammation in (atopic) asthma. Treatment with anti-inflammatory therapy, such as inhaled corticosteroids, effectively decreases eNO levels. The NIOX MINO (MINO) is a hand-held, relatively inexpensive, electrochemical device that has been shown to yield comparable eNO measurements to the NIOX stationary unit. AIM: To compare measurements of MINO with another widely used and validated stationary chemiluminescence analyzer, the Ecomedics (ECO). METHODS: We performed subsequent eNO measurements on ECO and MINO in 50 subjects (19 healthy volunteers, 18 healthy smokers and 13 non-smoking, atopic asthmatics, not on controller therapy) on two visits 4-10 days apart. The mean of three acceptable measurements by ECO and the first acceptable measurement with the MINO were used for analysis. RESULTS: Both devices yielded reproducible eNO values for all subjects on both visits, with an overall CV of 22.7% (ECO) and 18.3% (MINO). A significant correlation was found between both devices (r=0.97, p<0.0001). Bland-Altman plots showed a high degree of agreement for the entire study population (mean difference MINO vs ECO=-10%; 95% limit of agreement were -36% and +28%) and in the three individual subgroups. CONCLUSIONS: Exhaled NO values measured with the MINO are reproducible and in agreement with the ECO. Our results add further evidence to the reliability of the MINO and warrant its applicability in research and clinical practice.
Assuntos
Asma/metabolismo , Medições Luminescentes/instrumentação , Óxido Nítrico/análise , Adulto , Asma/fisiopatologia , Biomarcadores/análise , Testes Respiratórios/instrumentação , Eletroquímica , Desenho de Equipamento , Expiração/fisiologia , Feminino , Humanos , Medições Luminescentes/métodos , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Guias de Prática Clínica como Assunto , Adulto JovemRESUMO
BACKGROUND: Exhaled nitric oxide (eNO) is a validated noninvasive marker of airway inflammation in asthma. In patients with allergic rhinitis (AR), increased levels of nasal nitric oxide (nNO) have also been measured. However, the applicability of nNO as a marker of upper airway inflammation awaits validation. AIM: To test the longitudinal reproducibility of standardized nNO measurements in patients with AR and the effects of nasal allergen challenge. METHODS: Twenty patients with clinically stable, untreated AR participated in a combined study design. First, reproducibility of nNO was tested over 1, 7, and 14-21 days. Subsequently, the effect of nasal allergen challenge on nNO was studied in a placebo-controlled, parallel design. Nasal NO was measured with a chemoluminescence analyzer. Ten subjects randomly underwent a standardized nasal allergen challenge; 10 subjects received placebo. Response to nasal challenge was monitored by composite symptom scores. RESULTS: There was a good reproducibility of nNO up to 7 days [coefficient of variation (CV) over 1 (16.45%) and 7 days (21.5%)], decreasing over time [CV (14-21 days): 38.3%]. As compared with placebo, allergen challenge caused a significant increase in symptom scores (P < 0.001), accompanied by a decrease in nNO at 20 min postchallenge (P = 0.001). Furthermore, there was a gradual increase in nNO at 7 h, reaching significance at 24-h postallergen (P = 0.04). CONCLUSIONS: Similar to eNO in asthma, nNO is a noninvasive marker, potentially suitable to monitor upper airway inflammation following allergen-induced late response. Present data show a good reproducibility of nNO measurements, decreasing over time, probably because of subclinical seasonal influences.
Assuntos
Mucosa Nasal/metabolismo , Óxido Nítrico/biossíntese , Rinite Alérgica Perene/diagnóstico , Rinite Alérgica Sazonal/diagnóstico , Adulto , Alérgenos/administração & dosagem , Animais , Antígenos de Dermatophagoides/administração & dosagem , Antígenos de Plantas/administração & dosagem , Biomarcadores/metabolismo , Gatos/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Provocação Nasal , Poaceae/imunologia , Pólen/imunologia , Reprodutibilidade dos Testes , Rinite Alérgica Perene/metabolismo , Rinite Alérgica Sazonal/metabolismoRESUMO
Bovine von Willebrand factor (vWF) was digested with protease I (P-I), a metalloprotease isolated from rattlesnake venom. Digestion of vWF for 24 h with P-I yielded a terminal digest consisting of an equimolar mixture of two major fragments (apparent Mr 250K and 200K). The 250-kilodalton (kDa) fragment consists of a 125-kDa chain from one subunit and a 45- and 78-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment consists of a 97-kDa chain from one subunit and a 35- and 61-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment binds to heparin, and the heparin binding domain is located on the 97-kDa polypeptide chain. This fragment also competes with labeled, native vWF for binding to formalin-fixed human platelets, with an IC50 of 12.5 micrograms/mL (65 nM). However, native vWF has an IC50 of 2.5 micrograms/mL, indicating that the affinity of the 200-kDa fragment for platelets is approximately one-fifth that of vWF. The 200-kDa fragment agglutinates platelets, but its agglutinating ability is only 5% that of the native molecule. Only the 200-kDa fragment is recognized by monoclonal antibodies 2 and H-9, which are directed against vWF and inhibit vWF binding to platelet glycoprotein Ib (GPIb). Immunological studies, using nine monoclonal antibodies directed against vWF, and the demonstration that the heparin and GPIb binding domains are located on only one fragment suggest that the two fragments are composed of different regions of the vWF subunit. Analysis of the P-I cleavage pattern suggests that all vWF subunits are not cleaved in the same fashion. The first cleavage on half of the subunits generates the 45-kDa terminal and 175-kDa intermediate digest products. The 175-kDa chain is again cleaved, producing the 97- and 78-kDa terminal polypeptide chains. However, the first cleavage of the other subunits generates the 35-kDa terminal and the 186-kDa intermediate digest product, which upon cleavage produces the 125- and 61-kDa terminal polypeptide chains. Immunological data support the asymmetric cleavage pattern. An epitope for a monoclonal antibody is present on both the 186- and 175-kDa intermediate digest products but is only found on one terminal digest fragment, the 78-kDa polypeptide chain, suggesting that the 186- and 175-kDa polypeptides are cleaved at different sites.(ABSTRACT TRUNCATED AT 400 WORDS)