Asunto(s)
Cannabinoides/biosíntesis , Nociceptores/efectos de los fármacos , Dolor/tratamiento farmacológico , Dolor/fisiopatología , Amidas , Analgésicos/farmacología , Animales , Ácidos Araquidónicos/biosíntesis , Ácidos Araquidónicos/farmacología , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Interacciones Farmacológicas/fisiología , Endocannabinoides , Etanolaminas , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Glicéridos/biosíntesis , Humanos , Nociceptores/citología , Nociceptores/metabolismo , Dolor/patología , Ácidos Palmíticos/farmacología , Alcamidas Poliinsaturadas , Receptores de Cannabinoides , Receptores de Droga/efectos de los fármacos , Receptores de Droga/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The biological actions of the endogenous cannabinoid anandamide are terminated by carrier-mediated transport into neurons and astrocytes, followed by enzymatic hydrolysis. Anandamide transport is inhibited by the compound N-(4-hydroxyphenyl)arachidonylamide (AM404). AM404 potentiates several responses elicited by administration of exogenous anandamide, suggesting that it may also protect endogenous anandamide from inactivation. To test this hypothesis, we studied the effects of AM404 on the plasma levels of anandamide using high-performance liquid chromatography/mass spectrometry (HPLC/MS). Systemic administration of AM404 (10 mg kg(-1) intraperitoneal, i.p. ) caused a gradual increase of anandamide in rat plasma, which was significantly different from untreated controls at 60 and 120 min after drug injection. In plasma, both AM404 and anandamide were associated with a plasma protein, which we identified as albumin by non-denaturing polyacrylamide gel electrophoresis. AM404 (10 mg kg(-1), i.p.) caused a time-dependent decrease of motor activity, which was reversed by the cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.hydrochloride (SR141716A, 0.5 mg kg(-1), i.p). These results are consistent with the hypothesis that AM404 inhibits anandamide inactivation in vivo.
Asunto(s)
Ácidos Araquidónicos/sangre , Ácidos Araquidónicos/farmacología , Actividad Motora/efectos de los fármacos , Amidas , Animales , Ácidos Araquidónicos/química , Endocannabinoides , Etanolaminas , Masculino , Actividad Motora/fisiología , Ácidos Palmíticos/sangre , Ácidos Palmíticos/química , Alcamidas Poliinsaturadas , Ratas , Ratas Sprague-DawleyRESUMEN
Currently, few informations are available about spontaneous production of T cell cytokines in rheumatoid arthritis (RA) peripheral blood (PB), because these cytokines are generally under the detection threshold of ELISAs. Because the Th1/Th2 balance could help to determine the outcome of RA, we used a sensitive and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method to mesure spontaneous T cell production of IL-2, IL-4, IL-10, and IFN-gamma mRNAs using unstimulated PBMC from 25 active RA patients, not taking any DMARDs for at least 6 weeks, and 19 healthy controls. Spontaneous IL-2 and IL-4 mRNA expressions are significantly lower in RA patients compared to healthy controls. Levels of IL-10 and IFN-gamma are similar in the two groups. No correlation was found between cytokine mRNA levels and clinical parameters. Spontaneous IL-4 and IL-10 mRNA levels are respectively correlated to the number of CD4+ T cells and to the number of monocytes in PB. After in vitro stimulation, IFN-gamma mRNA production by RA PBMC is significantly decreased. Most of the patients cannot be classified as having a T cell cytokine type 1 or type 2 secretion pattern in PB. IL-2 and IL-4 mRNAs in PB of active RA are produced at a low spontaneous level and the response to in vitro activation by mitogen is weak.
Asunto(s)
Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Citocinas/genética , Linfocitos T/inmunología , Transcripción Genética , Adulto , Anciano , Artritis Reumatoide/fisiopatología , Células Cultivadas , Humanos , Interferón gamma/inmunología , Interleucina-10/genética , Interleucina-2/genética , Interleucina-4/genética , Persona de Mediana Edad , ARN Mensajero/genética , Valores de Referencia , Análisis de Regresión , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: To test if interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), IL-4, or IL-10 gene polymorphisms could be used as markers of susceptibility or severity in rheumatoid arthritis (RA). METHODS: The study included 108 patients with early RA followed up for 2 years and 128 healthy controls. From genomic DNA, 6 polymorphisms in genes for IL-1beta, IL-1Ra, IL-10, and IL-4 were typed. Allelic frequencies and carriage rates were compared between RA patients and controls, between patients with erosive and nonerosive RA, and between patients with or without sustained remission. RESULTS: The RP1 allele of the IL-4 gene was found with a significantly higher frequency in RA patients compared with controls. The combination of an RA-related HLA-DR allele expressing shared epitope and the presence of allele E2 in IL-1beta exon 5 was found to expose patients to an increased risk of erosive disease, with an odds ratio of 8.20 (95% confidence interval 2.59-25.84, P < 0.0001). No significant association was observed between polymorphisms and the occurrence of sustained remission. CONCLUSION: This report, for the first time, indicates an association between RA and a polymorphic IL-4 gene sequence located in 5q31-33. In addition, the results show the prognostic value of a polymorphism in IL-1beta exon 5, which allowed prediction of erosive disease with a specificity of 91.8% in 42.1% of patients. Although these observations are very interesting, they have to be considered preliminary and will need to be confirmed.
Asunto(s)
Artritis Reumatoide/genética , Interleucina-10/genética , Interleucina-1/genética , Interleucina-4/genética , Polimorfismo Genético , Sialoglicoproteínas/genética , Artritis Reumatoide/fisiopatología , ADN/análisis , Cartilla de ADN/química , Susceptibilidad a Enfermedades , Frecuencia de los Genes , Antígenos HLA-DR/genética , Cadenas HLA-DRB1 , Prueba de Histocompatibilidad , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Reacción en Cadena de la Polimerasa , Índice de Severidad de la EnfermedadRESUMEN
The binding ability of 23 overlapping peptides, all derived from the CB11 fragment of CII, was tested on several HLA-DR molecules associated or not with disease susceptibility. These experiments were performed on a variety of cells expressing different HLA-DR molecules, using both indirect and direct binding assays. The CII (256-271) fragment was shown to bind to a restricted population among which the HLA-DR molecules associated with susceptibility to rheumatoid arthritis. The results also clearly indicate that the binding specificity of CII (256-271), among the DR4 molecules, is controlled by the nature of the HLA-DR molecule beta-chain residues 71 and 74, residues previously shown by X-ray crystallography to be involved in the HLA-DR/peptide interaction. The human CII (256-271) peptide is thus likely to play a role in the disease process.
Asunto(s)
Artritis Reumatoide/inmunología , Enfermedades Autoinmunes/inmunología , Colágeno/metabolismo , Antígenos HLA-DR/metabolismo , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Artritis Reumatoide/genética , Enfermedades Autoinmunes/genética , Sitios de Unión , Unión Competitiva , Colágeno/inmunología , Predisposición Genética a la Enfermedad , Antígenos HLA-DR/genética , Humanos , Epítopos Inmunodominantes/inmunología , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Unión Proteica , Relación Estructura-ActividadRESUMEN
OBJECTIVE: To look for in vitro modulation of the main immunoregulatory and antiinflammatory cytokines by methotrexate (MTX) during the course of rheumatoid arthritis (RA). METHODS: We quantified interleukin-2 (IL-2), IL-4, IL-10, and interferon-gamma (IFNgamma) gene expression by peripheral blood mononuclear cells ex vivo under basal conditions and in vitro after stimulation with phytohemagglutinin (PHA) or PHA plus MTX, by competitive reverse transcriptase-polymerase chain reaction (RT-PCR), in 12 patients with untreated active RA (group 1), 10 patients with MTX-treated disease in partial remission (group 2), and 11 healthy control subjects. Simultaneously, under the same experimental conditions, we quantified cytokine production by specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: Under basal conditions, we found no differences in IL-2, IL-10, and IFNgamma gene expression in the 3 groups, while IL-4 gene expression was significantly decreased in RA patient group 1 compared with the control group. In vitro, under the action of MTX, IL-10 gene expression was significantly increased in the 3 groups, IL-4 gene expression was significantly increased in RA group 1 and in the control group, and IL-2 and IFNgamma gene expression was significantly decreased in RA group 1. Cytokine gene expression assessed by RT-PCR and cytokine production assessed by specific ELISAs were highly correlated. CONCLUSION: In vitro modulation of the cytokine network by MTX, increasing Th2 cytokines and decreasing Th1 cytokines, could explain its antiinflammatory and immunoregulatory actions in vivo during the treatment of RA.