RESUMEN
The translocator protein (TSPO) ligands affected inflammatory and immune responses. However, the exact effects of TSPO ligands on Th1 responses in vitro and in vivo are still unclear. In the present study, we found that TSPO ligands, FGIN1-27 and Ro5-4864, suppressed the cytokine production in a dose-dependent manner by purified human CD4+ T-cells from peripheral blood mononuclear cells (PBMCs) after stimulation. TSPO ligands inhibited the production of interferon γ (IFN-γ) by memory CD4+ T-cells and the differentiation of naïve CD4+ T-cells into Th1 cells via suppressing the activity of the corresponding transcription factors as indicated by reduced expression of T-bet and down-regulation of STAT1, STAT4 and STAT5 phosphorylation. TSPO ligands suppressed cell proliferation and activation of CD4+ T-cells by the inhibition of TCR signal transduction including membrane proteins: Zap, Lck, Src; cytoplasm proteins: Plcγ1, Slp-76, ERK, JNK and the nucleoproteins: c-Jun and c-Fos. In addition, FGIN1-27 inhibited mixed lymphocyte reactions by human or murine cells. After the transplantation of allogeneic murine skin, injection of FGIN1-27 into mice prevented graft rejection by inhibition of cell infiltration and IFN-γ production. Taken together, our data suggest that TSPO ligands inhibit Th1 cell responses and might be novel therapeutic medicine for the treatment of autoimmune diseases and prevention of transplant rejection.
Asunto(s)
Rechazo de Injerto/prevención & control , Ácidos Indolacéticos/uso terapéutico , Trasplante de Piel , Células TH1/inmunología , Adolescente , Adulto , Animales , Benzodiazepinonas/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Citocinas/biosíntesis , Evaluación Preclínica de Medicamentos/métodos , Femenino , Rechazo de Injerto/inmunología , Humanos , Ácidos Indolacéticos/inmunología , Ligandos , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosforilación/inmunología , Receptores de GABA/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/inmunología , Proteínas de Dominio T Box/metabolismo , Adulto JovenRESUMEN
A sensitive and specific enzyme immunoassay for FK480, a novel cholecystokinin type-A (CCK-A) receptor antagonist, was developed to study the pharmacokinetics of the drug at low-dose administration using a specific monoclonal antibody. The high performance liquid chromatography (HPLC) method had been used for studying toxicokinetics, but its determination limit (2.5 ng ml-1) was too high for use in clinical studies. Subsequently we developed an enzyme immunoassay (EIA) using rabbit anti-FK480 serum (polyclonal antibody). It had higher sensitivity (0.1 ng ml-1) when 0.5 ml of plasma was used but its specificity was low because of the cross-reactivity of the metabolites of FK480. Therefore we produced several monoclonal antibodies for FK480 by cell fusion, and selected the antibody which was least cross-reactive for the isolated metabolites of FK480. Finally we developed a sensitive and specific EIA using this monoclonal antibody. The lower limit of quantification of this method was 0.2 ng ml-1 when 0.2 ml of human plasma was used. The coefficient of variation over the calibration range (0.2-10 ng ml-1) was less than 15%. We used this method for clinical studies, and it showed a good correlation to the HPLC method when plasma concentration was 2.5 ng ml-1 or more.
Asunto(s)
Benzodiazepinonas/sangre , Antagonistas de Hormonas/sangre , Indoles/sangre , Receptores de Colecistoquinina/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales , Benzodiazepinonas/inmunología , Benzodiazepinonas/metabolismo , Benzodiazepinonas/farmacocinética , Cromatografía Líquida de Alta Presión , Reacciones Cruzadas , Perros , Femenino , Antagonistas de Hormonas/inmunología , Antagonistas de Hormonas/metabolismo , Antagonistas de Hormonas/farmacocinética , Humanos , Técnicas para Inmunoenzimas , Indoles/inmunología , Indoles/metabolismo , Indoles/farmacocinética , Masculino , Ratones , Ratones Endogámicos BALB C , Conejos , Ratas , Ratas Sprague-Dawley , Sensibilidad y EspecificidadRESUMEN
Treatment of pregnant Long Evans rats with a low dose of diazepam (1.25 mg/kg from gestational day 14-20) produced offspring suffering from suppression of cellular immune responses. Analogous effects were produced by clonazepam, a benzodiazepine (BDZ) with high affinity for the central-type, and Ro 5-4864, a BDZ with selective affinity for the peripheral-type BDZ receptor. Peripheral-type BDZ receptors are found to develop early in fetal life in peripheral organs including primary (thymus) and secondary (spleen) lymphoid organs, in the central nervous system and on immune cells (lymphocytes). In prenatally diazepam-exposed offspring the affinity constant is significantly changed. BDZ and PK 11195 also inhibit mitogen and alloantigen-induced T and B cell proliferation in vitro in adult murine lymphocytes. Diazepam, Ro 5-4864 and PK 11195 were found to be the most active compounds.
Asunto(s)
Benzodiazepinonas/farmacología , Clonazepam/farmacología , Convulsivantes/farmacología , Diazepam/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal , Receptores de GABA-A/efectos de los fármacos , Animales , Autorradiografía , Benzodiazepinonas/inmunología , Sitios de Unión , División Celular/efectos de los fármacos , Clonazepam/inmunología , Convulsivantes/inmunología , Diazepam/inmunología , Femenino , Inmunidad Celular/efectos de los fármacos , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Masculino , Intercambio Materno-Fetal , Embarazo , Ratas , Receptores de GABA-A/inmunologíaRESUMEN
Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Ro11-6896, and Ro5-3438 (the KD values are in the 10(-9) M range). However, these antibodies have low affinities for the benzodiazepine receptor inverse agonists (beta-carbolines) and antagonists (Ro15-1788 and CGS-8216).
Asunto(s)
Ansiolíticos , Anticuerpos Monoclonales/inmunología , Benzodiazepinas/inmunología , Animales , Benzodiazepinonas/inmunología , Diazepam/inmunología , Flumazenil , Flunitrazepam/inmunología , Flurazepam/análogos & derivados , Flurazepam/inmunología , Ratones , Ratones Endogámicos BALB C , Receptores de GABA-A/inmunología , Relación Estructura-ActividadRESUMEN
Antibodies specific for benzodiazepines were raised in rabbits by immunization with a conjugate of a benzodiazepine derivative, Ro 7-1986/1, with bovine serum albumin. The presence of anti-Ro 7-1986/1 antibodies in the sera was demonstrated by a radioimmunoassay using the radioligand [3H]flunitrazepam ([3h]FNZ). The antibodies displayed a high-affinity for [3H]FNZ (KD = 0.073 +/- 0.003 nM) and and cross-reacted with a broad spectrum of benzodiazepine derivatives. Benzodiazepine levels in samples of sera and urine of benzodiazepine-treated humans were determined. Due to the high sensitivity of the assay only minute volumes (microliter quantities) of body fluids are employed and, therefore, no extraction of the drugs is required. Nitrazepam and diazepam levels as low as 20 picograms can be easily observed. Intoxicating levels of benzodiazepines can be detected by a single measurement in less than 10 min. This radioimmunoassay is advantageous for pharmacokinetic studies, toxicological examinations and forensic medicine due to its high sensitivity, wide-range specificity and technical simplicity.