RESUMEN
Fractalkine (FKN/CX3CL1) is a unique member of the chemokine gene family and contains a chemokine domain (CD), a mucin-like stalk, a single transmembrane region, and a short intracellular C terminus. This structural distinction affords FKN the property of mediating capture and firm adhesion of FKN receptor (CX3CR1)-expressing cells under physiological flow conditions. Shed forms of FKN also exist, and these promote chemotaxis of CX3CR1-expressing leukocytes. The goal of the present study was to identify specific residues within the FKN-CD critical for FKN-CX3CR1 interactions. Two residues were identified in the FKN-CD, namely Lys-7 and Arg-47, that are important determinants in mediating an FKN-CX3CR1 interaction. FKN-K7A and FKN-R47A mutants exhibited 30-60-fold decreases in affinity for CX3CR1 and failed to arrest efficiently CX3CR1-expressing cells under physiological flow conditions. However, these mutants had differential effects on chemotaxis of CX3CR1-expressing cells. The FKN-K7A mutant acted as an equipotent partial agonist, whereas the FKN-R47A mutant had marked decreased potency and efficacy in measures of chemotactic activity. These data identify specific structural features of the FKN-CD that are important in interactions with CX3CR1 including steady state binding, signaling, and firm adhesion of CX3CR1-expressing cells.
Asunto(s)
Adhesión Celular/fisiología , Quimiocinas CX3C/química , Quimiocinas CX3C/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Receptores de Citocinas/metabolismo , Receptores del VIH/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos Diaminos , Animales , Sitios de Unión , Receptor 1 de Quimiocinas CX3C , Calcio/metabolismo , Línea Celular , Quimiocina CX3CL1 , Quimiotaxis/fisiología , Humanos , Cinética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Cocaine abuse is a major medical and public health concern in the United States, with approximately 2.1 million people dependent on cocaine. Pharmacological approaches to the treatment of cocaine addiction have thus far been disappointing, and new therapies are urgently needed. This paper describes an immunological approach to cocaine addiction. Antibody therapy for neutralization of abused drugs has been described previously, including a recent paper demonstrating the induction of anti-cocaine antibodies. However, both the rapidity of entry of cocaine into the brain and the high doses of cocaine frequently encountered have created challenges for an antibody-based therapy. Here we demonstrate that antibodies are efficacious in an animal model of addiction. Intravenous cocaine self-administration in rats was inhibited by passive transfer of an anti-cocaine monoclonal antibody. To actively induce anti-cocaine antibodies, a cocaine vaccine was developed that generated a high-titer, long-lasting antibody response in mice. Immunized mice displayed a significant change in cocaine pharmacokinetics, with decreased levels of cocaine measured in the brain of immunized mice only 30 seconds after intravenous (i.v.) administration of cocaine. These data establish the feasibility of a therapeutic cocaine vaccine for the treatment of cocaine addiction.
Asunto(s)
Cocaína/inmunología , Haptenos/inmunología , Inmunoterapia Activa , Trastornos Relacionados con Opioides/terapia , Vacunas/inmunología , Animales , Cocaína/administración & dosificación , Condicionamiento Operante , Estudios de Evaluación como Asunto , Haptenos/administración & dosificación , Inyecciones Intravenosas , Masculino , Ratones , Ratones Endogámicos BALB C , Trastornos Relacionados con Opioides/inmunología , Ratas , Ratas Wistar , Autoadministración , Albúmina Sérica Bovina/administración & dosificación , Albúmina Sérica Bovina/inmunologíaRESUMEN
[DTrp6] LH-RH was prepared using urethane-protected alpha-amino and N-carboxyanhydrides (UNCAs) both in solution and by solid-phase methodologies. The yields and purities of the crude materials in both cases were high, as indicated by HPLC. The structures were confirmed by 1H NMR, FAB mass spectrometry and found to be identical to an authentic sample. Both synthetic samples were analytically pure by capillary zone electrophoresis.
Asunto(s)
Anhídridos , Química/métodos , Pamoato de Triptorelina/síntesis química , Secuencia de Aminoácidos , Electroforesis , Espectroscopía de Resonancia Magnética/métodos , Datos de Secuencia Molecular , Soluciones , Uretano/químicaRESUMEN
We studied the immunogenicity of human insulin in 11 diabetic mothers and their newborns. Serum antibody formation was assayed by two different methods. Upon switching four patients from beef/pork insulin to human insulin, we found that elevated baseline antibody levels in three women decreased, in two to undetectable levels at term. The fourth patient had undetectable antibody levels at baseline and borderline levels at term. Only one of their four newborns had antibodies. Upon initiation of insulin treatment in another five diabetics without detectable antibodies at baseline, only two developed antibodies, and only one of their newborns developed antibodies. Two other patients, initially not on insulin, had baseline elevations of antibody that decreased with administration of human insulin; both of their newborns had antibodies. Overt diabetes evolved subsequently in both mothers after pregnancy. We conclude the following: 1) Upon transfer from beef/pork insulin to human insulin, mothers and their newborns show a decrease in insulin antibodies; 2) new patients initiated on insulin develop low levels of antibodies, if any, and their newborns also have low levels of antibodies if any; and 3) the decreased or absent immunogenicity of human insulin supports its use in pregnant diabetics.