RESUMEN
Organic acids play a pivotal role in improving plant response to long-term drought stress. External application of organic acids has been reported to improve drought resistance in several species. However, whether organic acids have similar effects in tobacco remains unknown. A screening study of the protective function of organic acids in tobacco and understanding the underlying molecular mechanism would be useful in developing a strategy for drought tolerance. Several physiological and molecular adaptations to drought including abscisic acid, stomatal closure, reactive oxygen species homeostasis, amino acid accumulation, and drought-responsive gene expression were observed by exogenous citric acid in tobacco plants. Exogenous application of 50 mm citric acid to tobacco plants resulted in higher chlorophyll content, net photosynthesis, relative water content, abscisic acid content and lower stomatal conductance, transpiration and water loss under drought conditions. Moreover, reactive oxygen species homeostasis was better maintained through increasing activity of antioxidant enzymes and decreasing hydrogen peroxide content after citric acid pretreatment under drought. Amino acids involved in the TCA cycle accumulated after external application of citric acid under drought stress. Furthermore, several drought stress-responsive genes also dramatically changed after application of citric acid. These data support the idea that external application of citric acid enhances drought resistance by affecting physiological and molecular regulation in tobacco. This study provides clear insights into mechanistic details of regulation of amino acid and stress-responsive gene expression by citric acid in tobacco in response to drought, which is promising for minimizing growth inhibition in agricultural fields.
Asunto(s)
Sequías , Nicotiana , Ácido Abscísico , Ácido Cítrico , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Estrés Fisiológico , Nicotiana/genéticaRESUMEN
Juvenile amyotrophic lateral sclerosis (JALS) occurs at an age of onset below 25 years with a heterogeneous disease onset location, variable progression and survival time. To investigate whether an ALS gene profile could resolve any aspects of clinical symptom heterogeneity, we have used targeted sequencing technology in a cohort of 12 JALS patients of Chinese descent. We detected 5 likely pathogenic mutations, 2 in familial probands and 3 in sporadic patients. One was a known TARDBP mutation (p.G348V) and 4 were FUS frameshift mutations including a known p.Gln519Ilefs*9 mutation and 3 novel mutations, p.Gly515Valfs*14, p.Gly486Profs*30, and p.Arg498Alafs*32. Of the 4 FUS mutations, 2 were able to be confirmed as de novo mutations. The TARDBP mutation carrier showed a classic ALS phenotype. All patients with FUS mutations experienced limb weakness at an early age and developed bulbar symptoms during the disease course. FUS mutations have previously been associated with increased JALS disease progression, however, we found a large range 12 to 84 months in disease survival (mean 58.2 months). Our results justify future screening for variants in FUS as it remains the most frequent genetic determinant of early onset, JALS (found in 30% of our patients).
Asunto(s)
Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/genética , Pueblo Asiatico/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Adolescente , Adulto , Edad de Inicio , Esclerosis Amiotrófica Lateral/epidemiología , Niño , China , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Masculino , Mutación , Linaje , Fenotipo , Proteína FUS de Unión a ARN/genética , Evaluación de Síntomas , Adulto JovenRESUMEN
In this multicenter, open-label pilot study, the efficacy, safety, and immunological impact of tacrolimus in Chinese patients with generalized myasthenia gravis are assessed. Forty-seven generalized myasthenia gravis (MG) patients were enrolled into this study and given 3mg/day tacrolimus for 24 weeks. The primary efficacy measurements used to monitor response to tacrolimus in MG patients were the Osserman grade, the quantitative MG score (QMGS) recommended by the MGFA, the MG-specific manual muscle testing (MMT) score, and the MG-related activities of daily living (MG-ADL) scale. Also, reduction in steroid doses was used to monitor the effect of tacrolimus. Clinical evaluations were conducted at weeks 4, 8, 12, 16, 20, and 24, while immunological parameters were measured at weeks 4, 12, and 24. Measurements of the Osserman grade, QMGS, MMT, and MG-ADL all suggested improvement in patient health by the fourth week of treatment. Steroid dosage was reduced during the course of the study in 74.2% of the forty-three patients who completed the study. There were thirty-one reported adverse events in the study. Only one was considered serious. We found that tacrolimus reduced levels of the IFN-γ, IL-2, IL-10, and IL-13 cytokines and induced the proliferation of tolerogenic plasmacytoid dendritic cells after treatment. Tacrolimus did not change the population of T cell subtypes but did steadily reduce the population of BAFF-R(+) CD19(+) B cells over the course of the study. Our results show that tacrolimus improves the clinical condition of MG patients and is well tolerated. The decrease in IL-13 and reduction of BAFF-R(+) CD19(+) B cells may be related to the therapeutic effect of tacrolimus.
Asunto(s)
Inmunosupresores/uso terapéutico , Miastenia Gravis/tratamiento farmacológico , Miastenia Gravis/inmunología , Tacrolimus/uso terapéutico , Adolescente , Corticoesteroides/administración & dosificación , Corticoesteroides/efectos adversos , Corticoesteroides/uso terapéutico , Adulto , Anciano , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/efectos adversos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Tacrolimus/administración & dosificación , Tacrolimus/efectos adversos , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
The basis for the reduced relapse rate of multiple sclerosis (MS) during pregnancy remains unexplained but, if defined, could create novel treatment options. Estrogen constitutes one candidate molecule, but the mechanism by which estrogen may affect MS during pregnancy is unclear. In this study, we used monocyte-derived dendritic cells (DCs) from MS patients to explore the estrogen (17-beta-estradiol)-related pathway of immune modulation. Estrogen induced the expression of indoleamine 2,3-dioxygenase (IDO) on DCs, limiting T-cell proliferation and both Th1 and Th2 cytokine production. The suppression of T-cell proliferation mediated by estrogen-exposed DCs was partly abolished by the IDO-inhibitor, 1-methyl-dl-tryptophan, indicating that estrogen-exposed DCs induced IDO-dependent T-cell suppression. Our data support the hypothesis that the change in the clinical course of MS observed in pregnancy may be related to the estrogen-DC-IDO axis, which could represent a novel target for MS therapy.
Asunto(s)
Células Dendríticas/enzimología , Estrógenos/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Esclerosis Múltiple/inmunología , Complicaciones del Embarazo/inmunología , Adulto , División Celular/inmunología , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/inmunología , Femenino , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Monocitos/citología , Esclerosis Múltiple/metabolismo , Embarazo , Complicaciones del Embarazo/metabolismo , Remisión Espontánea , Células TH1/citología , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/citología , Células Th2/inmunología , Células Th2/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
In several experimental studies of cerebral ischaemia, G-CSF (granulocyte colony-stimulating factor) exerted neuroprotective effects through different mechanisms, including mobilization of haemopoietic stem cells, anti-apoptosis, neuronal differentiation, angiogenesis and anti-inflammation. Hence, G-CSF not only inhibits neuron death, but also generates 'new' neural tissue formation. A small pilot trial reports on the safety and feasibility of G-CSF therapy in stroke patients. According to this evidence, we can speculate that G-CSF, being used either alone or in combination with another agent, should have a dual activity beneficial both to acute neuronal protection and long-term plasticity after cerebral ischaemia, thus proposing that G-CSF is an ideal new drug for stroke and neurodegenerative diseases.
Asunto(s)
Apoptosis/efectos de los fármacos , Isquemia Encefálica/tratamiento farmacológico , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Movilización de Célula Madre Hematopoyética , Fármacos Neuroprotectores/uso terapéutico , Encéfalo/efectos de los fármacos , Encéfalo/patología , Isquemia Encefálica/mortalidad , Isquemia Encefálica/patología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Humanos , Inflamación/prevención & control , Neovascularización Fisiológica/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/mortalidad , Accidente Cerebrovascular/patología , Análisis de Supervivencia , SobrevivientesRESUMEN
This study was designed to investigate the therapeutic effects of interferon (IFN)-gamma-modulated dendritic cells (DC) in experimental autoimmune myasthenia gravis (EAMG). We induced EAMG in Lewis rats by immunization with Torpedo nicotinic acetylcholine receptor (nAChR) and adjuvant. On day 33 post-immunization (p.i.), splenic DC were prepared, exposed to IFN-gamma alone (IFN-gamma-DC) or to IFN-gamma in combination with 1-methyl-DL-tryptophan (1-MT), the specific inhibitor of indoleamine 2,3-dioxygenase (IDO) (IFN-gamma + 1-MT-DC), and injected subcutaneously into rats with incipient EAMG on day 5 p.i. A control group of EAMG rats received naive DC on day 5 p.i., while another group received 1-MT every other day, intraperitoneally (p.i.), from days 5 to 41 p.i. The severity of clinical signs of EAMG was reduced dramatically in IFN-gamma-DC-treated rats compared to rats receiving naive DC, IFN-gamma + 1-MT-DC or 1-MT alone. The number of plasma cells secreting nAChR antibodies was reduced and the expression of B cell activation factor (BAFF) on splenic and lymph node mononuclear cells (MNC) was down-regulated in rats treated with IFN-gamma-DC. In vitro co-culture of MNC derived from EAMG rats with IFN-gamma-DC produced relatively few cells secreting nAChR antibodies. Addition of 1-MT to the co-culture significantly increased the number of cells secreting nAChR antibodies. We conclude that IFN-gamma-DC reduced the number of plasma cells secreting nAChR antibodies in an IDO-dependent manner and ameliorated the development of EAMG in Lewis rats.
Asunto(s)
Linfocitos B/inmunología , Células Dendríticas/inmunología , Interferón gamma/inmunología , Miastenia Gravis Autoinmune Experimental/inmunología , Triptófano/análogos & derivados , Animales , Factor Activador de Células B , División Celular/inmunología , Femenino , Indolamina-Pirrol 2,3,-Dioxigenasa , Leucocitos Mononucleares/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Proteínas de la Membrana/inmunología , Ratas , Ratas Endogámicas Lew , Receptores Colinérgicos/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T/inmunología , Triptófano/inmunología , Triptófano Oxigenasa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Dendritic cells (DC) represent a phenotypically heterogeneous population endowed with two important biological functions, immunity and tolerance. Here we report that the injection of splenic CD8alpha(+) DC, derived from rats with experimental allergic encephalomyelitis (EAE), delayed the onset and suppressed the severity of EAE in Lewis rats. This was accompanied by the lack of magnetic resonance imaging (MRI) lesions in the brain and spinal cord and by reduced numbers of inflammatory cells within the central nervous system. Injection of CD8(alpha+) DC inhibited T cell proliferation that may relate to increased interferon (IFN)-gamma and nitric oxide production. Although CD8(+)CD28(-) suppressor T cells, apoptotic cells and co-stimulatory molecules were not altered, CD4(+) T cells expressing interleukin (IL)-10 were augmented in rats receiving CD8alpha(+) DC compared to rats receiving total DC or medium. These results demonstrate that rat splenic CD8alpha(+) DC could provide a cellular basis for a novel, individualized immunotherapy using autologous DC as a complement to conventional therapy in diseases with an autoimmune background such as multiple sclerosis.
Asunto(s)
Antígenos CD8/inmunología , Células Dendríticas/trasplante , Encefalomielitis Autoinmune Experimental/inmunología , Animales , Encéfalo/patología , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Tolerancia Inmunológica , Inmunización Pasiva/métodos , Inmunofenotipificación , Interferón gamma/inmunología , Interleucina-10/inmunología , Imagen por Resonancia Magnética , Ratas , Ratas Endogámicas Lew , Médula Espinal/patología , Subgrupos de Linfocitos T/inmunologíaAsunto(s)
Proteínas Bacterianas , Síndrome de Behçet/etiología , Síndrome de Behçet/inmunología , Chaperoninas/inmunología , Toxina del Cólera/inmunología , Infecciones Estreptocócicas/complicaciones , Síndrome de Behçet/prevención & control , Chaperonina 60 , Reacciones Cruzadas , Herpes Simple/inmunología , Herpesvirus Humano 1 , Humanos , Inmunización , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Streptococcus sanguis/inmunología , Uveítis/etiología , Uveítis/prevención & controlRESUMEN
Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG), characterized by an autoaggressive T-cell-dependent antibody-mediated immune response directed against the acetylcholine receptor (AChR) of the neuromuscular junction. Dendritic cells (DC) are unique antigen-presenting cells which control T- and B-cell functions and induce immunity or tolerance. Here, we demonstrate that DC exposed to TGF-beta1 in vitro mediate protection against EAMG. Freshly prepared DC from spleen of healthy rats were exposed to TGF-beta1 in vitro for 48 h, and administered subcutaneously to Lewis rats (2 x 10(6)DC/rat) on day 5 post immunization with AChR in Freund's complete adjuvant. Control EAMG rats were injected in parallel with untreated DC (naive DC) or PBS. Lewis rats receiving TGF-beta1-exposed DC developed very mild symptoms of EAMG without loss of body weight compared with control EAMG rats receiving naive DC or PBS. This effect of TGF-beta1-exposed DC was associated with augmented spontaneous and AChR-induced proliferation, IFN-gamma and NO production, and decreased levels of anti-AChR antibody-secreting cells. Autologous DC exposed in vitro to TGF-beta1 could represent a new opportunity for DC-based immunotherapy of antibody-mediated autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes/terapia , Células Dendríticas/efectos de los fármacos , Inmunoterapia , Miastenia Gravis Autoinmune Experimental/terapia , Factor de Crecimiento Transformador beta/farmacología , Animales , Presentación de Antígeno , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Diferenciación Celular/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/trasplante , Femenino , Humanos , Inyecciones Subcutáneas , Miastenia Gravis Autoinmune Experimental/inmunología , Ratas , Ratas Endogámicas Lew , Receptores Colinérgicos/inmunología , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta1 , Pérdida de PesoRESUMEN
We have previously shown that dendritic cells (DC), upon being pulsed in vitro with encephalitogenic myelin basic protein peptide 68-86 (MBP 68-86) and injected subcutaneously (s.c.) back to healthy Lewis rats, transfer immune tolerance to experimental allergic encephalomyelitis (EAE) induced by immunization with MBP 68-86 and Freund's complete adjuvant (FCA). We here assumed that DC become pulsed in EAE rats, and that expansion in vitro of such 'in vivo pulsed EAE-DC' might also have the capacity to induce immune tolerance to EAE, thereby eliminating the need for in vitro pulsing of DC with autoantigens which are still unknown in many autoimmune diseases in the human. In the present study, EAE-DC were generated from bone marrow of Lewis rats, with EAE induced with MBP 68-86 + FCA, and expanded in vitro by culture with GM-CSF and IL-4. In comparison with DC from normal rats, EAE-DC exhibited higher viability in the absence of growth factors, and presented specific antigen to naïve T cells in vitro. The DC derived from both EAE and healthy rats stimulated strong proliferation in an antigen-independent manner, lasting for 4 weeks after DC were s.c. injected into healthy rats. During this time, injection of EAE-DC did not induce clinical EAE. However, when these rats were immunized with MBP 68-86 + FCA, subsequent EAE was dramatically suppressed, and was associated with increased IFN-gamma expression, nitric oxide production, gradually reduced proliferation and cell apoptosis, compared with PBS-injected control EAE rats. LPS-treated DC did not induce tolerance, suggesting that the tolerance is mediated by an immature stage of DC. These observations support the hypothesis that EAE-DC can transfer immune tolerance to EAE, thereby omitting the step of characterizing specific autoantigen. Omitting the step of loading DC with antigen not only eliminates the extremely complex procedure of defining pathogenically-relevant autoantigens, but also avoids the risk of inducing immunogenicity of DC in the treatment of autoimmune diseases.
Asunto(s)
Células Dendríticas/trasplante , Encefalomielitis Autoinmune Experimental/inmunología , Autotolerancia , Traslado Adoptivo , Animales , Presentación de Antígeno , Apoptosis , Autoantígenos/inmunología , Células de la Médula Ósea/inmunología , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/inmunología , Inmunoglobulina G/biosíntesis , Interferón gamma/biosíntesis , Activación de Linfocitos , Masculino , Proteína Básica de Mielina/inmunología , Óxido Nítrico/biosíntesis , Fragmentos de Péptidos/inmunología , Ratas , Ratas Endogámicas Lew , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Multiple sclerosis (MS) is assumed to result from autoaggressive T cell-mediated immune responses, in which T helper type 1 (Th1) cells producing cytokines, e.g. IFN-gamma and lymphotoxin promote damage of oligodendrocyte-myelin units. Dendritic cells (DCs) as potent antigen presenting cells initiate and orchestrate immune responses. Whether phenotype and function of DCs with respect to Th1 cell promotion are altered in MS, are not known. This study revealed that blood-derived DCs from MS patients expressed low levels of the costimulatory molecule CD86. In addition, production of IFN-gamma by blood mononuclear cells (MNCs) was strongly enhanced by DCs derived from MS patients. IFN-beta and IL-10 inhibited the costimulatory capacity of DCs in mixed lymphocyte reaction (MLR) and showed additive effects on suppression of IL-12 production by DCs. Correspondingly, DCs pretreated with IFN-beta and IL-10 significantly suppressed IFN-gamma production by MNCs. IFN-beta in vitro also upregulated CD80 and, in particular, CD86 expression on DCs. In vitro, anti-CD80 antibody remarkably increased, while anti-CD86 antibody inhibited DC-induced IL-4 production in MLR. We conclude that DC phenotype and function are altered in MS, implying Th1-biased responses with enhanced capacity to induce Th1 cytokine production. In vitro modification of MS patients' DCs by IFN-beta and IL-10 could represent a novel way of immunomodulation and of possible usefulness for future immunotherapy of MS.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Interferón beta/farmacología , Interleucina-10/farmacología , Esclerosis Múltiple/sangre , Adulto , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2 , Células Dendríticas/inmunología , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Interleucina-4/metabolismo , Leucocitos Mononucleares/inmunología , Prueba de Cultivo Mixto de Linfocitos , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Fenotipo , Regulación hacia ArribaRESUMEN
Linomide (roquinimex, LS 2616) is a quinoline-3-carboxamide with pleiotropic immune modulating capacity and it has therapeutic effects in several experimental animal models of autoimmune diseases. Linomide has been evaluated in clinical trials for multiple sclerosis, and was indeed shown to have disease inhibitory effects. However, due to unexpected side effects recorded in patients treated with Linomide, premature termination of clinical trials was required. The basic mechanism(s) of action of Linomide in inducing beneficial effects in autoimmune diseases is still elusive. Some experimental evidence indicates that Linomide influences the regulation of the cytokine profile, resulting in the inhibition of autoimmune and inflammation pathologies. This review focuses on Linomide applied in models for autoimmune and inflammation pathologies of the central and the peripheral nervous system, and summarises its very encouraging disease inhibitory effects and their potential pharmacological basis. The beneficial effects recorded with Linomide in both experimental and clinical trials emphasise the possible value of substances with Linomide-like activity for clinical use in autoimmune and inflammation pathologies in the near future.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Enfermedades Autoinmunes/tratamiento farmacológico , Sistema Nervioso Central/patología , Citocinas/biosíntesis , Hidroxiquinolinas/farmacología , Inmunidad Celular/efectos de los fármacos , Sistema Nervioso Periférico/patología , Adyuvantes Inmunológicos/uso terapéutico , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Humanos , Hidroxiquinolinas/uso terapéuticoRESUMEN
Brain ischemia is characterized by local inflammation reflected by accumulation of inflammatory cells and a multitude of mediators. Among them, cytokines and chemokines may influence the inflammatory cascade that follows cerebral ischemia. Here we report on brain hemispheric and systemic increase of pro-inflammatory IL-17 and IFN-gamma, the anti-inflammatory cytokines IL-4 and IL-10, and the chemokines IP-10, IL-8 and MIP-2, 1 h to 6 days after permanent middle cerebral artery occlusion (pMCAO). IL-17 and IFN-gamma mRNA levels were elevated in the ischemic hemispheres of pMCAO-operated rats compared with corresponding hemispheres of sham-operated rats. Levels were slightly elevated at 1 h, and peaked at 6 days after pMCAO. IL-8 and MIP-2 levels in the ischemic hemispheres peaked at 24 h, whereas IP-10 showed a biphasic profile with two peaks at 6 h and 6 days after pMCAO. IL-4 peaked in the ischemic hemispheres at 6 h, when IL-10 levels were lower than in sham-operated rats, and IL-10 levels peaked at 2 days after pMCAO. Systemically, the numbers of IL-17 and IFN-gamma mRNA expressing blood mononuclear cells were elevated already at 1 h after pMCAO, preceding the changes in the ischemic hemispheres. Altered levels of IL-17 and IFN-gamma after pMCAO may affect outcome of brain ischemia.
Asunto(s)
Arteriopatías Oclusivas/metabolismo , Encéfalo/metabolismo , Arterias Cerebrales , Interferón gamma/genética , Interleucina-17/genética , ARN Mensajero/metabolismo , Animales , Arteriopatías Oclusivas/patología , Encéfalo/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Quimiocina CXCL10 , Quimiocina CXCL2 , Quimiocinas/genética , Quimiocinas CXC/genética , Interleucina-10/genética , Interleucina-4/genética , Interleucina-8/genética , Ganglios Linfáticos/metabolismo , Masculino , Monocitos/metabolismo , Neuronas/patología , ARN Mensajero/sangre , Ratas , Ratas Sprague-Dawley , Bazo/metabolismoRESUMEN
NO is involved in the regulation of immune responses. The role of NO in the pathogenesis of experimental allergic encephalomyelitis (EAE) is controversial. In this study, 3-morpholinosydnonimine (SIN-1), an NO donor, was administered to Lewis rats on days 5-7 postimmunization, i.e., during the incipient phase of EAE. SIN-1 reduced clinical signs of EAE compared with those in PBS-treated control rats and was accompanied by reduced ED1(+) macrophages and CD4(+) T cell infiltration within the CNS. Blood mononuclear cells (MNC) obtained on day 14 postimmunization revealed that SIN-1 administration enhanced NO and IFN-gamma production by blood MNC and suppressed Ag- and mitogen-induced proliferative responses. MHC class II, B7-1 and B7-2 were down-regulated in SIN-1-treated EAE rats. Simultaneously, frequencies of apoptotic cells among blood MNC were increased. In vivo, SIN-1 is likely to behave as an NO donor. Administration of SIN-1 induced NO production, but did not affect superoxide and peroxynitrite formation. Enhanced NO production during the priming phase of EAE thus promotes apoptosis, down-regulates disease-promoting immune reactivities, and ameliorates clinical EAE, mainly through SIN-1-derived NO, without depending on NO synthase.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Molsidomina/administración & dosificación , Donantes de Óxido Nítrico/administración & dosificación , Adyuvantes Inmunológicos/uso terapéutico , Secuencia de Aminoácidos , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/uso terapéutico , Antígenos CD/biosíntesis , Antígeno B7-1/biosíntesis , Antígeno B7-2 , Membrana Celular/inmunología , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Regulación hacia Abajo/inmunología , Esquema de Medicación , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Cobayas , Antígenos de Histocompatibilidad Clase II/biosíntesis , Inmunosupresores/administración & dosificación , Inmunosupresores/uso terapéutico , Inyecciones Intraperitoneales , Interferón gamma/biosíntesis , Interferón gamma/metabolismo , Recuento de Leucocitos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/efectos de los fármacos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/biosíntesis , Datos de Secuencia Molecular , Molsidomina/análogos & derivados , Molsidomina/uso terapéutico , Proteína Básica de Mielina/inmunología , Óxido Nítrico/biosíntesis , Donantes de Óxido Nítrico/uso terapéutico , Fragmentos de Péptidos/inmunología , Ratas , Ratas Endogámicas Lew , Médula Espinal/patologíaRESUMEN
Autoimmune diseases such as multiple sclerosis (MS) are characterized by the loss of tolerance to self-determinants, activation of autoreactive lymphocytes and subsequent damage to single or multiple organs. The mechanisms by which autoimmune responses are triggered, and how activation of autoreactive lymphocytes is initiated and maintained, are not fully understood. Therapeutic approaches in autoimmune diseases have so far concentrated on antigens and T cells. Given the exceptional capacity of dendritic cells (DCs) to induce immunity in vivo, recent reports of the first successful clinical trials based on vaccination of tumor patients with autologous blood DCs pulsed in vitro with tumor antigen come as no surprise. The recent identification of tolerogenic subsets of DCs and their generation in culture may allow a novel approach to induce tolerance in autoimmune diseases. By selective in vitro manipulation of DCs and their subsequent reinfusion, DC-mediated tolerance has been achieved in animal models of human autoimmune diseases, including experimental autoimmune encephalomyelitis in Lewis rats and SJL/J mice and spontaneous diabetes in NOD mice. In vitro observations of human blood DCs are promising for DC-based treatment of MS and other diseases with an autoimmune component. Data from animal models and human materials suggest that DC-based immunotherapy could be beneficial at least as a complement to conventional therapy. Molecular-biological approaches to tolerogenic DCs could provide a rationale for designing immunotherapeutic strategies in autoimmune diseases.
Asunto(s)
Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Inmunoterapia Activa/métodos , Esclerosis Múltiple/inmunología , Animales , Encefalomielitis Autoinmune Experimental/terapia , Humanos , Esclerosis Múltiple/terapiaRESUMEN
Suppression of interleukin 12 (IL-12) production by dendritic cells (DCs) has been hypothesized to be a principal mechanism underlying the biological action of interferon (IFN)-beta used for treatment of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system with possible autoimmune origin. How IFN-beta interacts with DCs to inhibit IL-12 production remains unclear. In this study, we found that DCs derived from human blood monocytes, upon culture in the presence of IFN-beta with granulocyte-macrophage colony- stimulating factor (GM-CSF) and IL-4, differentiated into a population expressing CD14- CD1a- HLA-DR+. This population expressed CD123 (IL-3Ralpha). IFN-beta dose-dependently increased IL-3Ralpha+ DCs and decreased CD1a+ DCs. After 7 days' culture with IFN-beta at a concentration of 10 000 U/ml, more than 40% of DCs expressed IL-3Ralpha. IFN-beta, together with GM-CSF and IL-4, also induced maturation of IL-3Ralpha-expressing cells, as reflected by upregulation of HLA-DR and of the costimulatory molecules CD40, CD80 and CD86. In contrast to control DCs, IFN-beta-treated DCs produced predominantly IL-10 but only low levels of IL-12p40. Correspondingly, IFN-beta-treated DCs strongly suppressed IFN-gamma production but enhanced IL-10 production by allogeneic blood mononuclear cells. Our data suggest that IFN-beta in vitro can induce the development of DC2, which provide a permissive environment for Th2 differentiation. This finding represents a novel mechanism for action of IFN-beta in MS.
Asunto(s)
Células Dendríticas/metabolismo , Células Dendríticas/fisiología , Interferón beta/metabolismo , Antígenos CD1/metabolismo , Diferenciación Celular , División Celular , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interferón beta/farmacología , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Interleucina-12/antagonistas & inhibidores , Interleucina-12/biosíntesis , Interleucina-4/farmacología , Leucocitos/metabolismo , Monocitos/metabolismo , Fenotipo , Proteínas Recombinantes/metabolismo , Células TH1/metabolismo , Células Th2/metabolismo , Factores de TiempoRESUMEN
The damage of acetylcholine receptor (AChR) at neuromuscular junctions of experimental autoimmune myasthenia gravis (EAMG), an animal model of human MG, is mediated by B cells which require T cell help. The Th2 associated cytokine IL-10 suppresses production of cytokines released by Th1 cells and is considered for treatment of human autoimmune diseases. To evaluate the role of IL-10 in EAMG, rhIL-10 was administered daily to Lewis rats by the subcutaneous route starting at the day of immunization and continued for 7 weeks. IL-10 failed to abrogate EAMG at low dose (0.1 or 1 microg/day) and at the dose of 3 microg/day caused earlier onset and aggravated clinical signs of EAMG when compared to EAMG rats injected with PBS only. Although Th1 responses reflected by AChR-induced lymphocyte proliferation and levels of IFN-gamma secreting cells, as well as AChR-induced Th1 cytokine mRNA expression was suppressed, augmented IL-4 mRNA expression and AChR-specific B cell responses may play an important role in the failure of IL-10 to abrogate EAMG. This study implicates a critical precaution in planning immunotherapy of IL-10 in antibody-mediated autoimmune diseases, e.g. MG.
Asunto(s)
Acetilcolina/inmunología , Linfocitos B/inmunología , Interleucina-10/farmacología , Miastenia Gravis Autoinmune Experimental/inmunología , Células Th2/inmunología , Animales , Linfocitos B/efectos de los fármacos , División Celular/inmunología , Femenino , Expresión Génica/inmunología , Humanos , Inmunoglobulina G/sangre , Interleucina-10/genética , Interleucina-10/inmunología , Debilidad Muscular/inmunología , ARN Mensajero/análisis , Ratas , Ratas Endogámicas Lew , Receptores Colinérgicos/inmunología , Células Th2/efectos de los fármacos , TorpedoRESUMEN
Rat models (experimental autoimmune encephalomyelitis, EAE; myasthenia gravis, EAMG) have been developed that mimic certain aspects of the pathophysiology of multiple sclerosis (MS) and myasthenia gravis (MG) in humans. Rat EAE and EAMG are, therefore, widely used to evaluate immunotherapeutic strategies. Mucosal tolerance induced by oral or nasal administration of autoantigen effectively suppresses rat EAE and EAMG. Nasal administration of the cytokines interleukin (IL)-4, IL-10 or transforming growth factor (TGF)-beta1 also ameliorates clinical signs of rat EAE. However, neither mucosal tolerance nor cytokines affect clinical disease if administered during ongoing rat EAE or EAMG. Dendritic cells (DC) pulsed in vitro with autoantigen can mediate peripheral tolerance against rat EAE and EAMG, but do not affect ongoing EAE or EAMG. DC from healthy rats, modified in vitro by exposure to IFN-gamma or TGF-beta1, effectively suppress ongoing EAE and EAMG when given by the subcutaneous route. Most importantly, DC from EAMG rats, after being modified in vitro with IL-10, also inhibit ongoing rat EAMG. These results demonstrate that different strategies influencing T- and/or B-cell responses in rat EAE or EAMG are available, but that DC-based immunotherapies are the most promising, in order to ameliorate ongoing organ-specific autoaggressive immunity Based on these observations, autologous DC, after being modified in vitro with cytokines, constitute a basis for new immunotherapeutic strategies in MS, MG and other diseases with autoimmune background.
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Encefalomielitis Autoinmune Experimental/terapia , Inmunoterapia , Miastenia Gravis/terapia , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Miastenia Gravis/inmunología , RatasRESUMEN
Dendritic cells (DC) can modulate the nature of immune responses in a stimulatory or tolerogenic fashion. Great attention has been given to the induction of immunity to tumour and infection. In this study, bone marrow-derived DC from healthy Lewis rats were pulsed in vitro with encephalitogenic myelin basic protein peptide 68-86 (MBP 68-86), and injected subcutaneously (1 x 106/rat) into normal Lewis rats. Upon observation of the rats pretreated in this way for 4 weeks, when no clinical signs of EAE occurred, these rats were immunized with MBP 68-86 and Freund's complete adjuvant. The pretreated rats failed to develop clinical EAE. This tolerance was associated with augmented proliferative responses, interferon-gamma secretion, inducible nitric oxide synthase (iNOS) expression and NO production. The frequency of apoptotic cells was increased in the rats receiving MBP 68-86-pulsed DC compared with unpulsed control DC. Few infiltrating inflammatory cells were observed in spinal cord sections from rats that had received MBP 68-86-pulsed DC. The data are compatible with the interpretation that MBP 68-86-pulsed DC induce tolerance to EAE possibly through up-regulation of iNOS expression and NO production, which mediate cell apoptosis, thereby reducing infiltration of inflammatory cells within the central nervous system.
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Autoantígenos/inmunología , Células de la Médula Ósea/inmunología , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Proteína Básica de Mielina/inmunología , Fragmentos de Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Apoptosis , Linfocitos T CD4-Positivos/fisiología , División Celular , Trasplante de Células , Susceptibilidad a Enfermedades , Expresión Génica , Inmunidad Innata/inmunología , Interferón gamma/metabolismo , Interleucina-10/genética , Interleucina-12/genética , Macrófagos/fisiología , Masculino , Datos de Secuencia Molecular , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero , Ratas , Ratas Endogámicas Lew , Médula Espinal/citología , Bazo/citologíaRESUMEN
We have previously shown that tolerance can be induced against acute experimental autoimmune encephalomyelitis (EAE) in Lewis rats by bone marrow-derived dendritic cells (DC) that have been pulsed in vitro with encephalitogenic myelin basic protein peptide 68-86 (MBP 68-86), and injected subcutaneously into healthy rats prior to immunization with MBP 68-86 plus complete Freund's adjuvant. To elucidate better the properties of tolerogenic DC, we here compared plastic-adherent DC with floating, non-adherent DC, which were cultured for 7 days in the presence of granulocyte-macrophage colony-stimulating factor plus interleukin-4 (IL-4). Adherent DC expressed high levels of IL-10 mRNA and protein, and low levels of IL-12 mRNA and showed high expression of CD54 compared with floating DC. Proliferation, nitrite concentration and capacity for antigen presentation were lower in adherent DC than in floating DC. There were no differences between adherent and floating DC regarding expression of CD11c, OX62, major histocompatibility complex class II, CD80, or CD86. Most importantly, we observed that adherent DC induced tolerance to EAE in vivo when injected subcutaneously into Lewis rats prior to immunization, while floating DC did not. Adherent DC-mediated tolerance to EAE was associated with augmented proliferation, nitric oxide production and frequency of apoptotic cells as well as with up-regulation of transforming growth factor-beta (TGF-beta) -expressing cells in T-cell areas of lymph nodes. Tolerance induction by adherent DC seems to be related to a nitric oxide-apoptosis pathway and to up-regulation of TGF-beta-expressing cells.