RESUMEN
Systemic lupus erythematosus is a chronic, relapsing autoimmune disease that can affect multiple organ systems. An increased prevalence of drug allergy has been reported in lupus patients compared with the general population. Using a cohort of 417 lupus patients, we found a history of sulpha allergy in 27.3% of patients. European-American lupus patients with sulpha allergy are about two times more likely to suffer from lymphopenia, two times more likely to have anti-Ro autoantibody, and four times less likely to have anti-nRNP antibodies compared with lupus patients without a reported sulpha allergy (P = 0.0075, 0.025, and 0.032, respectively). In African-American lupus patients, a history of sulpha allergy was associated with over three times increased odds of developing pericarditis (P = 0.005).
Asunto(s)
Hipersensibilidad a las Drogas/epidemiología , Lupus Eritematoso Sistémico/inmunología , Sulfonamidas/efectos adversos , Adulto , Anticuerpos Antinucleares/sangre , Autoanticuerpos/sangre , Hipersensibilidad a las Drogas/etnología , Femenino , Humanos , Masculino , Ribonucleoproteínas/inmunologíaRESUMEN
OBJECTIVE: To test the hypothesis that thrombomodulin (TM) may be a target for lupus anticoagulant (LAC) antibodies. METHODS: A recombinant soluble form of TM was produced and used as an antigen for an ELISA to detect antibodies to TM (TMAB). Sixty-one samples from 58 patients identified by the coagulation laboratory as having a LAC and 200 patients with unexplained thrombosis were evaluated along with 201 healthy controls. RESULTS: Eighteen (30%) of the 58 patients with a LAC and 20 (10%) of 200 patients with unexplained thrombosis had antibodies to TM. Similar antibodies were found in only 4 (2%) of 201 normal controls. TMAB show selectivity for TM lacking chondroitin sulfate, but do not otherwise have an immunodominant region. The IgG from 6 patients with TMAB was purified, and it bound TM in our ELISA. Three of the 6 IgG fractions inhibited protein C activation 40% to 70% compared to no inhibition in 7 healthy controls. CONCLUSION: Some patients with LAC and unexplained thrombosis have antibodies to TM that may arise in response to TM that has been altered and lost its chondroitin sulfate attachment. Antibodies to TM may be an important risk factor for inflammation and thrombosis in these patients.
Asunto(s)
Anticuerpos/inmunología , Inhibidor de Coagulación del Lupus/inmunología , Trombomodulina/inmunología , Trombosis/inmunología , Adulto , Anciano , Anticuerpos/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inhibidor de Coagulación del Lupus/sangre , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/inmunología , Trombosis/sangreRESUMEN
Cultured human umbilical vein endothelial cells inhibited tumor necrosis factor-alpha release from whole blood or isolated mononuclear cells exposed to endotoxin. In contrast, the endothelial cells augmented neutrophil elastase release in the same blood. A protein with these functional properties was isolated from endothelial cell-conditioned media and, surprisingly, was identified as calmodulin. Authentic calmodulin mimicked the effect of endothelium. 125I-Calmodulin bound to a high affinity site on monocytic cell lines (Kd approximately 30 nM, in agreement with its functional activity). Cross-linking of 125I-calmodulin to monocytic cells identified a candidate calmodulin receptor. We conclude that calmodulin possesses an extracellular signaling role in addition to its intracellular regulatory functions. Calmodulin released at sites of tissue injury or possibly by specific mechanisms in the endothelium can bind to receptors, modulating the activities of inflammatory cells.
Asunto(s)
Proteínas de Unión a Calmodulina/fisiología , Calmodulina/farmacología , Endotelio Vascular/fisiología , Elastasa de Leucocito/sangre , Monocitos/fisiología , Neutrófilos/enzimología , Secuencia de Aminoácidos , Animales , Calmodulina/sangre , Calmodulina/química , Proteínas de Unión a Calmodulina/efectos de los fármacos , Bovinos , Línea Celular , Células Cultivadas , Reactivos de Enlaces Cruzados , Células HL-60 , Humanos , Cinética , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Transducción de Señal , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/biosíntesis , Venas UmbilicalesRESUMEN
BACKGROUND: The thrombomodulin-dependent protein C anticoagulant pathway plays a major physiologic role in the down-regulation of the coagulation process. In cell culture, inflammatory cytokines or endotoxin can down-regulate endothelial thrombomodulin (TM) suggesting that suppressed TM expression may contribute to thrombotic complications noted in Gram-negative sepsis. EXPERIMENTAL DESIGN: In the present study, we have examined TM expression in the kidneys of septic rats utilizing indirect immunofluorescence and have quantified TM antigen and TM activity in extracts of the same kidneys by enzyme-linked immunosorbent assays and protein C activation assays, respectively. Conscious Sprague-Dawley rats were injected intravenously with LD95 doses of live E. coli (N = 30), or endotoxin (N = 30). Control animals (N = 30) were injected with equivalent volumes of saline. The rats were killed 30, 90, 180, 360, and 720 minutes after the initiation of sepsis. RESULTS: Glomerular capillary thrombosis developed by 180 minutes in approximately half of the animals after the initiation of sepsis. We failed to demonstrate suppressed TM expression in the kidneys of septic animals using immunofluorescence. Neither enzyme-linked immunosorbent assays, nor protein C activation assays showed decreased levels in TM antigen expression or activity at different time points during the sepsis. CONCLUSIONS: These results indicate that suppressed TM expression does not contribute to the development of the glomerular capillary thrombosis in this septic rat model.
Asunto(s)
Arteriolas/metabolismo , Infecciones por Escherichia coli/metabolismo , Corteza Renal/metabolismo , Glomérulos Renales/metabolismo , Choque Séptico/metabolismo , Trombomodulina/biosíntesis , Animales , Arteriolas/patología , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Endotoxinas , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Infecciones por Escherichia coli/patología , Técnica del Anticuerpo Fluorescente , Corteza Renal/irrigación sanguínea , Corteza Renal/patología , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/patología , Masculino , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Choque Séptico/patología , Trombomodulina/análisis , Trombosis/metabolismo , Trombosis/patologíaRESUMEN
OBJECTIVE: To investigate the hypothesis that soluble E-selectin (sE-selectin) may be detected in synovial fluid (SF) and play a role in inflammatory arthritis. METHODS: We used a sandwich ELISA to measure sE-selectin in the SF of 58 patients with rheumatoid arthritis (RA), 9 with psoriatic arthritis (PsA), 30 with osteoarthritis (OA), 13 with gout, and 9 with calcium pyrophosphate dihydrate crystal deposition disease (CPPD). RESULTS: SF sE-selectin values in RA (mean 1.49 ng/ml, 0.18-3.90) and PsA (mean 1.36 ng/ml, 0.88-2.31) were significantly higher than those with OA (mean 0.83 ng/ml, 0.00-1.83), gout (mean 1.04 ng/ml, 0.11-3.42), or CPPD (mean 0.80 ng/ml, 0.20-1.47). Elevated SF sE-selectin was associated with elevated serum sE-selectin, erythrocyte sedimentation rate, and SF white blood cell count. CONCLUSION: Our findings suggest that endothelial cell activation and E-selectin may contribute to the development of inflammatory processes.
Asunto(s)
Artritis/metabolismo , Moléculas de Adhesión Celular/metabolismo , Líquido Sinovial/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Artritis/etiología , Artritis Gotosa/metabolismo , Artritis Psoriásica/metabolismo , Artritis Reumatoide/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/sangre , Condrocalcinosis/metabolismo , Selectina E , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoartritis/metabolismo , SolubilidadRESUMEN
Thrombomodulin (TM) binds thrombin to form a complex that activates the plasma anticoagulant zymogen protein C. TM is an integral membrane glycoprotein that contains a chondroitin sulfate moiety. Interaction with thrombin involves both the protein component of TM, specifically the growth factor-like repeats 4-6 (TM 4-6), and chondroitin sulfate. Removal of chondroitin sulfate decreases the affinity for thrombin approximately 10-fold and shifts the Ca2+ dependence of protein C activation from simple saturation at > or = 500 microM Ca2+ to a distinct optimum at approximately 100 microM Ca2+. Thrombin possesses two regions of high positive charge, anion binding exosites 1 and 2. Anion binding exosite 1 interacts with the growth factor region of TM while exosite 2 is involved in binding prothrombin activation fragment 2 or heparin. We demonstrate that recombinant TM, truncated at the membrane-spanning domain, or TM 4-6 can bind thrombin when fragment 2 is present either covalently attached (meizothrombin des-fragment 1) or in reversible association. With meizothrombin des-fragment 1, the Ca2+ dependence of protein C activation is independent of the presence of the chondroitin sulfate on TM. At 0.27 mM Ca2+, TM containing chondroitin sulfate binds thrombin (Kd(app) = 0.3 nM) approximately 45 times tighter than meizothrombin des-fragment 1 (Kd(app) = 14 nM). However, the chondroitin-free form binds thrombin (Kd(app) = 2.4 nM) only approximately 4 times tighter than meizothrombin des-fragment 1 (Kd(app) = 9.4 nM). These studies suggest that occupancy of anion binding exosite 2 by either chondroitin sulfate or fragment 2 alters thrombin conformation resulting in the altered Ca2+ dependence of protein C activation.
Asunto(s)
Calcio/metabolismo , Proteína C/metabolismo , Trombina/química , Trombina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcio/farmacología , Bovinos , Sulfatos de Condroitina/farmacología , Precursores Enzimáticos/química , Humanos , Cinética , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Protrombina/metabolismo , Especificidad por Sustrato , Trombomodulina/metabolismoRESUMEN
OBJECTIVE: To investigate the state of endothelial cell activation in vasculitis, scleroderma, and systemic lupus erythematosus (SLE). METHODS: We used a sandwich ELISA to quantitate a soluble form of endothelial leukocyte adhesion molecule-1 (sELAM) in serum. RESULTS: sELAM was detected in serum from healthy individuals (mean 0.92 ng/ml). Levels were significantly higher in patients with giant cell arteritis (mean 2.04 ng/ml), polyarteritis nodosa (mean 2.08 ng/ml), scleroderma (mean 2.27 ng/ml), and SLE (mean 3.93 ng/ml). Elevated values were present in patients with both active and inactive disease. sELAM levels of > 3 ng/ml identified most patients with recent onset or active disease. CONCLUSION: Our findings may reflect a low degree of endothelial cell activation in healthy persons that is increased in inflammatory diseases involving blood vessels. Elevated serum sELAM levels may reflect ongoing inflammatory processes in these diseases.
Asunto(s)
Moléculas de Adhesión Celular/sangre , Lupus Eritematoso Sistémico/sangre , Esclerodermia Sistémica/sangre , Vasculitis/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Selectina E , Ensayo de Inmunoadsorción Enzimática , Femenino , Arteritis de Células Gigantes/sangre , Humanos , Masculino , Persona de Mediana Edad , Poliarteritis Nudosa/sangre , Poliarteritis Nudosa/tratamiento farmacológico , Prednisona/uso terapéutico , Valores de ReferenciaRESUMEN
We tested sera from 56 patients to determine the seroprevalence of antibodies to hepatitis C virus (HCV) in polyarteritis nodosa (PAN), to assess the specificity of these antibodies for hepatitis C virus encoded antigens, and to evaluate the clinical features in patients with HCV infection and PAN. Eleven (20%) were positive for anti-HCV by an enzyme immunoassay. Three (5%) had specific antibodies to HCV encoded antigens detected by recombinant immunoblot assay (RIBA II) and had HCV RNA detected by polymerase chain reaction. Patients with HCV infection were more likely to have liver and skin involvement and a diminished serum complement.
Asunto(s)
Hepacivirus/inmunología , Anticuerpos Antihepatitis/sangre , Hepatitis C/complicaciones , Poliarteritis Nudosa/etiología , Adulto , Secuencia de Bases , Femenino , Hepatitis C/inmunología , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Poliarteritis Nudosa/inmunología , Radioinmunoensayo , Pruebas SerológicasRESUMEN
A quantitative sandwich ELISA for E-selectin in the fluid phase (soluble E-selectin, sEs) has been developed that is sensitive to 100 pg/ml. The assay shows no reactivity with either L- or P-selectins. We have used this to determine the fate of E-selectin after cell-surface expression and to test whether levels measured in vivo may represent the state of endothelial activation. E-selectin was first detectable in supernatants of IL-1-stimulated endothelial cells at 24 h, and increased slowly up until 72 h. However, over this time period the total E-selectin detectable in the system (cells plus supernatants) declined dramatically. 125I-surface-labeled endothelial cells cultured for 24 h show an E-selectin of reduced m.w. in the supernatant, indicating that the molecule is shed from the surface. The shed form also appears to be slightly smaller than the intact membrane form as determined from immunoprecipitation and molecular sieving studies. In addition, the cytoplasmic domain of the molecule found in supernatants of activated endothelial cells and in serum is not intact as determined by loss of reactivity with an antipeptide antibody specific for the cytoplasmic domain. We have examined the sera of 71 normal individuals. Without exception, sEs was found in serum in the range of 0.13 to 2.8 ng/ml, suggesting that even in the absence of overt inflammatory processes E-selectin is being synthesized and released into the bloodstream. In addition, bacteremic patients with hypotension, but not those without, showed markedly elevated sEs values. As determined by cell-binding studies, the blood-derived form of E-selectin is biologically active.
Asunto(s)
Moléculas de Adhesión Celular/análisis , Endotelio Vascular/metabolismo , Choque Séptico/sangre , Adulto , Secuencia de Bases , Moléculas de Adhesión Celular/sangre , Moléculas de Adhesión Celular/aislamiento & purificación , Cromatografía en Gel , Selectina E , Endotelio Vascular/química , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interleucina-1/farmacología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Sepsis/sangreRESUMEN
The authors investigated the release of an endothelial cell-specific protein (E92) by cultured porcine aortic endothelial cell cultures. Under normal culture conditions, endothelial cells released little or no E92 into the culture supernatant. Treatment with thrombin (0.01 to 10 units/ml), endotoxin (0.01 to 10 micrograms/ml), or interleukin-1 (0.01 to 3.0 units/ml), however, caused significant, dose-dependent increases in E92 detectable in the culture supernatants. Time-course experiments showed that maximum release of E92 into cellular supernatants occurred 24 hours after stimulation with all mediators. Parallel experiments used 51Cr-loaded endothelial cells as a measure of lethal cellular injury. None of the mediators caused significant injury at the doses observed to induce release of E92. These results suggest that the release of E92 into the supernatants of cultured endothelial cells is an inducible event. The data also support the hypothesis that detection of E92 antigen in sera from patients with rheumatic disease represents a marker of in vivo vascular endothelial cell activity.
Asunto(s)
Endotelio Vascular/metabolismo , Endotoxinas/farmacología , Escherichia coli , Interleucina-1/farmacología , Proteínas de la Membrana/metabolismo , Trombina/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacosRESUMEN
In order to determine whether endothelial cell antigens are detectable in serum from patients with rheumatic disease characterized by vascular involvement, we developed an ELISA using a monoclonal antibody directed against a 92,000 molecular weight endothelial cell specific antigen designated E92. Sera were assayed from 191 patients and 34 healthy controls. E92 was undetectable or present in very low concentrations in healthy controls and was elevated in most patients with an active rheumatic disease. Our results indicate that circulating endothelial antigens are present in rheumatic vascular syndromes and may provide a measure of endothelial cell function.
Asunto(s)
Antígenos de Superficie/análisis , Endotelio Vascular/inmunología , Glicoproteínas de Membrana/análisis , Enfermedades Reumáticas/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Peso Molecular , Enfermedades Reumáticas/patologíaRESUMEN
Clinical and laboratory data in 124 patients with rheumatoid arthritis treated with methotrexate (MTX) were retrospectively reviewed over the initial 2 years after the start of treatment. Clinical improvement occurred in 103 (83%) patients after 12 weeks of treatment. At 2 years of followup, 60 patients (48%) continued to receive MTX with sustained clinical benefit. It has been discontinued in 64 (52%) patients (adverse drug reactions in 38, lack of clinical benefit in 15, and miscellaneous reasons in 11). Patients with adverse drug reactions had higher initial serum creatinine and blood urea nitrogen values than patients without adverse drug reactions.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Metotrexato/uso terapéutico , Adulto , Anciano , Femenino , Estudios de Seguimiento , Humanos , Masculino , Metotrexato/administración & dosificación , Metotrexato/efectos adversos , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
Methotrexate therapy has been effective in the treatment of RA with short term experience suggesting little serious adverse reactions. Our review of 168 patients receiving methotrexate has identified nine patients with probable or possible methotrexate-induced pulmonary toxicity, giving a prevalence of 5% and an incidence of 3.9 per 100 patients per year. No clinical or laboratory features showed an association that could potentially predict the development of pulmonary disease. All patients experienced complete recovery with supportive care and/or corticosteroid therapy. Clinical monitoring for this complication is warranted in all patients receiving long term methotrexate therapy for RA.