RESUMEN
Screening large numbers of plaques containing particular proteins is accomplished by techniques that are analogous to those described for screening with radioactive DNA probes. However, in the basic protocol described here, the plaques are screened with antibodies specific to the desired proteins. An alternate protocol provides a method for increasing the amount of recombinant protein in each plaque by inducing expression from the lac promoter that directs its expression.
Asunto(s)
Bacteriófago lambda/genética , Proteínas Recombinantes/análisis , Anticuerpos , Bacteriófago lambda/crecimiento & desarrollo , Sondas de ADN , ADN Viral/genética , Biblioteca de Genes , Indicadores y Reactivos , Radioisótopos de Yodo , Proteínas Recombinantes/inmunología , Ensayo de Placa Viral/métodosRESUMEN
OBJECTIVE: To evaluate the safety, pharmacokinetics, pharmacodynamics, and immunogenicity of a humanized anti-CD11/CD18 monoclonal antibody (Hu23F2G) in patients with multiple sclerosis. METHODS: In this phase I uncontrolled dose escalation study, patients (n = 24) with primary or secondary progressive multiple sclerosis received single intravenous infusions of Hu23F2G (0.01 to 4.0 mg/kg). Study parameters included safety, pharmacology, immunogenicity, and brain magnetic resonance imaging (MRI). RESULTS: Hu23F2G had few adverse effects, but 2 cases of urinary tract infection and 2 cases of gingivitis did occur. Transient leukocytes developed in some subjects receiving > or = 1.0 mg/kg. The pharmacokinetic response was nonlinear, with the area under the curve increasing out of proportion to the increase in dose. The mean terminal half-life increased with dose and was 21.9 (SD, 12.8) hours at the 4.0 mg/kg dose. High saturation (> 80%) of CD11/CD18 on circulating leukocytes was achieved with doses > or = 0.2 mg/kg. The duration of high leukocyte saturation was dose-dependent, persisting for more than a week at the 4.0 mg/kg dose. A marked decrease in leukocyte migration in response to cutaneous inflammation was observed. Antibodies against Hu23F2G were not detected. The neurologic examinations were stable except for 1 subject who had worsening weakness associated with an infection. No significant changes were noted on brain MRI scans. CONCLUSIONS: Hu23F2G was tolerated at doses that achieved high degrees of leukocyte CD11/CD118 saturation with in vivo inhibition of leukocyte migration. Because this phase I study was not designed to determine the clinical efficacy of Hu23F2G, further studies are needed.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD11/inmunología , Antígenos CD18/inmunología , Esclerosis Múltiple/terapia , Adulto , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/farmacocinética , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Infusiones Intravenosas , Leucocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Factores de Tiempo , Resultado del TratamientoAsunto(s)
Endotelio/fisiología , Ganglios Linfáticos/fisiología , Linfocitos/fisiología , Ganglios Linfáticos Agregados/fisiología , Receptores Inmunológicos/fisiología , Animales , Antígenos , Antígenos de Superficie/inmunología , Adhesión Celular , Diferenciación Celular , Movimiento Celular , Glicoproteínas/fisiología , Humanos , Lectinas/fisiología , Receptores de Antígenos/fisiología , Receptores Inmunológicos/genética , Ubiquitinas/metabolismo , Vénulas/fisiologíaRESUMEN
A cDNA library was prepared from a terminal deoxynucleotidyltransferase-containing thymoma in the lambda phage vector lambda gt11. By screening plaques with anti-terminal transferase antibody, positive clones were identified of which some had beta-galactosidase-cDNA fusion proteins identifiable after electrophoretic fractionation by immunoblotting with anti-terminal transferase antibody. The predominant class of cross-hybridizing clones was determined to represent cDNA for terminal transferase by showing that one representative clone hybridized to a 2200-nucleotide mRNA in close-matched enzyme-positive but not to enzyme-negative cells and that the cDNA selected a mRNA that translated to give a protein of the size and antigenic characteristics of terminal transferase. Only a small amount of genomic DNA hybridized to the longest available clone, indicating that the sequence is virtually unique in the mouse genome.
Asunto(s)
Clonación Molecular , ADN Nucleotidilexotransferasa/genética , ADN Nucleotidiltransferasas/genética , ADN/metabolismo , Escherichia coli/genética , Timoma/enzimología , Animales , Anticuerpos , Complejo Antígeno-Anticuerpo , Bacteriófago lambda/genética , ADN Nucleotidilexotransferasa/análisis , Vectores Genéticos , Ratones , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/genética , Neoplasias del Timo/enzimología , beta-Galactosidasa/genéticaAsunto(s)
Galactosa/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Bacteriófago lambda/genética , Secuencia de Bases , Deleción Cromosómica , Mapeo Cromosómico , Clonación Molecular , Enzimas de Restricción del ADN , ADN de Hongos , ADN Viral , Electroforesis en Gel de Poliacrilamida , Mutación , Hibridación de Ácido Nucleico , ARN de HongosAsunto(s)
Deleción Cromosómica , Galactosa/genética , Operón , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Bases , Clonación Molecular , ADN de Hongos , Hibridación de Ácido Nucleico , Plásmidos , Poliploidía , ARN de Hongos , Recombinación Genética , Transformación GenéticaAsunto(s)
Elementos Transponibles de ADN , ADN de Hongos/genética , ARN de Hongos/genética , Saccharomyces cerevisiae/genética , Regulación de la Expresión Génica , Ligamiento Genético , Histidina/genética , Hibridación de Ácido Nucleico , Poli A/genética , Recombinación Genética , Transformación GenéticaAsunto(s)
Enzimas de Restricción del ADN , ADN/aislamiento & purificación , Hibridación de Ácido Nucleico , Bacteriófago lambda/análisis , ADN Bacteriano/aislamiento & purificación , ADN de Hongos/aislamiento & purificación , ADN Recombinante/aislamiento & purificación , ADN Viral/aislamiento & purificación , PlásmidosRESUMEN
Multiple nitrocellulose DNA filter replicas of plaques of in vitro generated recombinants of phage lambda and Saccharomyces cerevisiae have been screened by hybridization with 32P-labeled cDNA probes. These probes were representative of total poly(A)-containing RNA of yeast cells grown on acetate, galactose, glucose or maltose. This approach allows the use of specific differences in total RNA populations as probes for gene isolation. Five "galactose-induced" clones have been isolated. Expression of the RNA coding regions on at least two cloned sequences, Sc481 and Sc482, is regulated by genes known to control the expression of the structural genes required for the conversion of exogenous galactose to endogenous glucose-1-phosphate. One cloned sequence, Sc484, is expressed during growth on all carbon sources except glucose, and is not under control by the galactose regulatory genes. This clone contains a sequence that is repeated 3 times in the yeast genome. The cloned fragment Sc481 contains coding regions for all or part of three galactose"induced RNAs and may correspond to the GAL 1, GAL 7, GAL 10 gene cluster region of chromosome II.