RESUMEN
Melanoma growth stimulating activity (MGSA) and IL-8 are related chemokines that are potent chemoattractants and activators of neutrophils both in vitro and in vivo. Increasing evidence suggests that these molecules play an important role in inflammation; thus, antagonists of their action could be useful therapeutically as antiinflammatory agents. We have generated an MGSA mutant, H19A, that shows a dissociation between receptor binding and biologic activity. The biologic activity of the H19A mutant is between 133-fold and 282-fold less potent than that of wild-type MGSA measured by three independent assays of neutrophil function, i.e., elastase release chemotaxis and the up-regulation of CD18. In addition, pretreatment of cells with the H19A mutant inhibited the ability of MGSA to induce elastase release and chemotaxis and to increase intracellular calcium. However, competition binding studies in cells transfected with the CXCR2 receptor and in neutrophils demonstrate that the receptor affinity of the H19A mutant is only 13-fold less than that of wild-type MGSA. These studies suggest that the mutant MGSA is defective in activating signaling through the receptor and indicate that binding to the receptor is not sufficient to activate a biologic response. The dissociation between receptor binding and activation for this mutant suggests that it should be possible to design antagonists of MGSA that may be of clinical utility.
Asunto(s)
Sustitución de Aminoácidos , Quimiocinas CXC , Factores Quimiotácticos/farmacología , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Interleucina/antagonistas & inhibidores , Antígenos CD/genética , Antígenos CD/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Quimiocina CXCL1 , Factores Quimiotácticos/genética , Quimiotaxis de Leucocito/efectos de los fármacos , Citocalasina B/farmacología , Sustancias de Crecimiento/genética , Humanos , Interleucina-8/farmacología , Elastasa de Leucocito/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-8A , Receptores de Interleucina-8B , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , TransfecciónRESUMEN
A polyclonal chicken antiserum against purified 32-kilodalton (kDa) recombinant inhibin-A (rh-InhA) and two monoclonal antibodies (mAb) against either rh-InhA (11B5) or 28-kDa recombinant activin-A (rh-ActA; 9A9) were used to develop three sensitive InhA enzyme-linked immunosorbent assays (ELISAs). The sensitivity of an ELISA using affinity-purified chicken anti-rh-InhA (Ck) for both coat and capture (Ck/Ck) averaged 78 +/- 3 pg/ml, while the mAb/Ck ELISAs (11B5/Ck or 9A9/Ck) averaged 100 +/- 6 pg/ml in a 10% serum matrix, with intra-and interassay coefficients of variation of 2-5% and 8-10%, respectively, for all assays. The ELISA formats did not cross-react with purified rh-ActA or recombinant human transforming growth factor-beta 1 or detect any immunoreactive proteins in medium conditioned by cell lines expressing rh-ActA or recombinant human transforming growth factor-beta 1. The Ck/Ck ELISA detected significant amounts of immunoreactivity in medium from cells expressing the free alpha-subunit of inhibin and recombinant inhibin-B (rh-InhB). In contrast, the mAb/Ck ELISAs showed no cross-reactivity to medium conditioned by these two cell lines. All three ELISA formats detected rh-InhA added to either human or rat serum in vitro or serum from rats injected with rhInhA. The Ck/Ck and 9A9/Ck ELISAs successfully quantitated inhibin in sera from patients undergoing ovulation induction and in rats (with or without sc administration of pregnant female serum gonadotropin). The 11B5/Ck ELISA appeared to be specific for the 32-kDa form of inhibin, while the 9A9/Ck ELISA was useful in quantitating inhibin-A in biological fluids, with little cross-reactivity to free alpha-chain or inhibin-B.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Inhibinas/sangre , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Pollos/inmunología , Femenino , Humanos , Inhibinas/inmunología , Masculino , Inducción de la Ovulación , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/inmunología , Sensibilidad y EspecificidadRESUMEN
The serum pharmacokinetics of recombinant human inhibin A (rh-inhibin A) and rh-activin A were examined in immature female Sprague Dawley-derived rats after iv and sc injection of the drugs. After iv administration of rh-inhibin A (120 micrograms/kg), the serum concentrations were described by a biexponential equation. The weight-normalized clearance was 21.3 ml/min.kg, and the initial (t1/2 alpha) and terminal (t1/2 beta) half-lives were 2.9 min and 37.9 min, respectively. Subcutaneous administration of 120 micrograms/kg rh-inhibin A resulted in a peak serum concentration of 10.6 ng/ml at 30.8 min after injection. Approximately 24% of the sc administered material was absorbed. Serum concentrations of rh-activin A also declined biexponentially after iv injection of the drug (120 micrograms/kg). The clearance of rh-activin A was 5.1 ml/min.kg, the t1/2 alpha was 6.1 min, and the t1/2 beta was 46.3 min. The peak serum concentration of rh-activin A (104.7 ng/ml) was achieved 24.7 min after sc delivery of the drug. The bioavailability of the sc dose was 38%. Iodinated rh-inhibin A and rh-activin A were used to examine the serum forms and metabolites of the drugs. [125I]rh-inhibin A and [125I]rh-activin A associated with two serum-binding proteins. Within 2 min of iv injection, the labeled hormones bound follistatin and alpha-2-macroglobulin. Even though rh-inhibin A and rh-activin A are structurally similar and appear to bind to the same serum proteins, their disposition in the immature rat differ.
Asunto(s)
Sustancias de Crecimiento/farmacocinética , Inhibinas/farmacocinética , Activinas , Envejecimiento/metabolismo , Animales , Proteínas Sanguíneas/metabolismo , Western Blotting , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inhibinas/sangre , Tasa de Depuración Metabólica , Unión Proteica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacocinética , Distribución TisularRESUMEN
"Death rates from natural causes and from cardiovascular diseases, age 35-74, age-adjusted, by sex and race, by county and State Economic Area [of the United States] for 1968-1972 have been calculated...." The counties with extremely high or low death rates for white males are identified, and the reasons for the variations in mortality observed are considered