RESUMEN
A panel of monoclonal antibodies was raised against the low-affinity human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor alpha-chain expressed as recombinant protein on murine FDC-P1 cells. All the selected antibodies were of the IgG2A isotype and bound to protein A. They each recognized both native and recombinant receptors by indirect surface immunofluorescence and by immunoprecipitation. Several of the antibodies also recognized presumably denatured receptors as detected by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three different epitopes on the extracellular domain of the GM-CSF receptor alpha-chain were defined by these antibodies, and two of the epitopes did not appear to be involved in binding hGM-CSF or in interactions with the beta-chain of the GM-CSF receptor that are required for high-affinity binding of GM-CSF. On the other hand, the epitope recognized by antibody 2B7-17-A appeared to be critically involved in the binding of GM-CSF because this antibody completely abrogated both high- and low-affinity binding of GM-CSF to native and recombinant receptors. Antibody 2B7-17-A had a relatively high affinity for the GM-CSF receptor alpha-chain (kd = 3 nmol/L) and slow dissociation kinetics (kd = 0.002 min-1). These properties made the 2B7-17-A antibody a potent inhibitor of hGM-CSF biologic action in several different bioassays, with a half-maximal inhibitory dose of about 6 nmol/L (1 microgram/mL). This antibody could prove useful in alleviating any pathologic states mediated by excess GM-CSF levels and in defining the domains of the GM-CSF receptor required for ligand binding.
Asunto(s)
Anticuerpos Monoclonales , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Línea Celular , Estimulación Eléctrica , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Cinética , Sustancias Macromoleculares , Ratones , Peso Molecular , Pruebas de Neutralización , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/análisis , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , TransfecciónRESUMEN
Leukemia inhibitory factor (LIF) has many biological actions which parallel those of IL-1, IL-6 and tumor necrosis factor-alpha, but its role in the pathogenesis of human disease is unknown. A specific radioreceptor competition assay capable of detecting LIF at concentrations above 1 ng/ml (45 pM) was developed. To identify disease states in which LIF might be involved, a cross-sectional survey of serum and body fluids from approximately 1,500 subjects with a variety of diseases was performed using the LIF radioreceptor competition assay. Serum LIF concentrations were transiently elevated (2-200 ng/ml) in six subjects with meningococcal or Gram-negative septic shock, and in a subject with idiopathic fulminant hepatic failure. Moderately elevated LIF concentrations (> 10 ng/ml) were detected in cerebrospinal fluid from subjects with bacterial meningitis, in effusions associated with pneumonia and peritonitis, and in amniotic fluid from a woman with chorioamnionitis. Low LIF concentrations (1-10 ng/ml) were present in synovial fluid from subjects with inflammatory arthritis, amniotic fluid from women in labor, and some reactive, chronic inflammatory and malignant effusions and cyst fluids, but rarely in transudates. These initial findings suggest that LIF might be involved in the pathogenesis of inflammation and septic shock.
Asunto(s)
Líquidos Corporales/química , Inhibidores de Crecimiento/análisis , Inflamación/metabolismo , Interleucina-6 , Linfocinas/análisis , Choque Séptico/metabolismo , Líquido Amniótico/química , Animales , Quistes/metabolismo , Inhibidores de Crecimiento/fisiología , Humanos , Factor Inhibidor de Leucemia , Linfocinas/fisiología , Meningitis/metabolismo , Ratones , Ensayo de Unión Radioligante , Líquido Sinovial/químicaRESUMEN
Antigenic variation of infectious organisms is a major factor in evasion of the host immune response. However, there has been no definitive demonstration of this phenomenon in the malaria parasite Plasmodium falciparum. In this study, cloned parasites were examined serologically and biochemically for the expression of erythrocyte surface antigens. A cloned line of P. falciparum gave rise to progeny that expressed antigenically distinct forms of an erythrocyte surface antigen but were otherwise identical. This demonstrates that antigenic differences on the surface of P. falciparum-infected erythrocytes can arise by antigenic variation of clonal parasite populations. The antigenic differences were shown to result from antigenic variation of the parasite-encoded protein, the P. falciparum erythrocyte membrane protein 1.
Asunto(s)
Variación Antigénica , Antígenos de Protozoos/genética , Plasmodium falciparum/inmunología , Aglutinación , Animales , Antígenos de Protozoos/análisis , Antígenos de Protozoos/inmunología , Western Blotting , Eritrocitos/fisiología , Humanos , Sueros Inmunes , Immunoblotting , Plasmodium falciparum/genética , ConejosRESUMEN
The survival of Plasmodium falciparum-infected erythrocytes is enhanced by the sequestration of mature trophozoites and schizonts from the peripheral circulation. Cytoadherence of infected erythrocytes in vivo is associated with the presence of knobs on the erythrocyte surface, but we and others have shown recently that cytoadherence to C32 melanoma cells may occur in vitro in the absence of knobs. We show here that a knobless clone of P. falciparum adheres to the leukocyte differentiation antigen, CD36, suggesting that binding to CD36 is independent of the presence of knobs on the surface of the infected erythrocyte. This clone showed little cytoadherence to immobilized thrombospondin or to endothelial cells expressing the intercellular adhesion molecule 1. Furthermore, an Mr approximately 300-kD trypsin-sensitive protein doublet was immunoprecipitated from knobless trophozoite-infected erythrocytes. Finding a P. falciparum erythrocyte membrane protein 1 (PfEMP1)-like molecule on these infected erythrocytes is consistent with a role for PfEMP1 in cytoadherence to CD36 and C32 melanoma cells.