RESUMEN
BACKGROUND: Imatinib, a tyrosine kinase inhibitor, has been shown to restore blood-brain barrier integrity and reduce infarct size, haemorrhagic transformation and cerebral oedema in stroke models treated with tissue plasminogen activator. We evaluated the safety of imatinib, based on clinical and neuroradiological data, and its potential influence on neurological and functional outcomes. METHODS: A phase II randomized trial was performed in patients with acute ischaemic stroke treated with intravenous thrombolysis. A total of 60 patients were randomly assigned to four groups [3 (active): 1 (control)]; the active treatment groups received oral imatinib for 6 days at three dose levels (400, 600 and 800 mg). Primary outcome was any adverse event; secondary outcomes were haemorrhagic transformation, cerebral oedema, neurological severity on the National Institutes of Health Stroke Scale (NIHSS) at 7 days and at 3 months and functional outcomes on the modified Rankin scale (mRS). RESULTS: Four serious adverse events were reported, which resulted in three deaths (one in the control group and two in the 400-mg dose group; one patient in the latter group did not receive active treatment and the other received two doses). Nonserious adverse events were mostly mild, resulting in full recovery. Imatinib ameliorated neurological outcomes with an improvement of 0.6 NIHSS points per 100 mg imatinib (P = 0.02). For the 800-mg group, the mean unadjusted and adjusted NIHSS improvements were 4 (P = 0.037) and 5 points (P = 0.012), respectively, versus controls. Functional independence (mRS 0-2) increased by 18% versus controls (61 vs. 79; P = 0.296). CONCLUSION: This phase II study showed that imatinib is safe and tolerable and may reduce neurological disability in patients treated with intravenous thrombolysis after ischaemic stroke. A confirmatory randomized trial is currently underway.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Mesilato de Imatinib/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Accidente Cerebrovascular/tratamiento farmacológico , Terapia Trombolítica , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Isquemia Encefálica/mortalidad , Esquema de Medicación , Femenino , Humanos , Mesilato de Imatinib/administración & dosificación , Mesilato de Imatinib/efectos adversos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Accidente Cerebrovascular/mortalidad , Resultado del Tratamiento , Adulto JovenRESUMEN
Evidence from epidemiological, clinical and experimental studies favour the hypothesis that inflammatory events are part of the neuropathology in Alzheimer's disease. Proinflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) have been found in activated microglia in the vicinity of amyloid plaques in Alzheimer's disease brain. In the present study, the levels of soluble IL-1 receptor type II (sIL-1R type II), IL-1 receptor antagonist (IL-1ra), IL-1beta, IL-6 and TNF-alpha were analyzed in cerebrospinal fluid (CSF) samples from Alzheimer's disease patients and control subjects. The levels of sIL-1R type II were significantly higher in CSF from Alzheimer's disease patients than in CSF samples from control subjects (38.5+/-8 pg/ml (mean+/-S.E.M.) vs. 7.9+/-4 pg/ml, p<0.05). Measurements of the proinflammatory cytokines IL-6 and TNF-alpha showed no significant difference between the two groups, and the levels of IL-1beta and IL-1ra in the present material were too low to permit detection. The increased levels of sIL-1R type II may reflect a compensatory mechanism to balance an increased release of IL-1 receptor agonists in the Alzheimer's disease brain.
Asunto(s)
Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/diagnóstico , Receptores de Interleucina-1/metabolismo , Anciano , Anciano de 80 o más Años , Biomarcadores , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interleucina-6/líquido cefalorraquídeo , Masculino , Microglía/metabolismo , Persona de Mediana Edad , Solubilidad , Factor de Necrosis Tumoral alfa/líquido cefalorraquídeoRESUMEN
Glucose-induced insulin release is decreased in the spontaneously diabetic GK rat, a nonobese rodent model of type 2 diabetes. Forskolin restores the impaired insulin release in both the isolated perfused pancreas and isolated islets from these rats (Abdel-Halim et al., Diabetes 45:934-940, 1996). We demonstrate here that the insulinotropic effect of forskolin in the GK rat is due to increased generation of cAMP and that it is associated with overexpression of adenylyl cyclase (AC)-III mRNA and gene mutations. The AC-III mRNA overexpression was demonstrated by in situ hybridization using oligonucleotide probes binding to different regions of the rat AC-III mRNA. It was associated with the presence of two point mutations identified at positions -28 bp (A --> G) and -358 bp (A --> C) of the promoter region of the AC-III gene and was demonstrable in both GK rat islets and peripheral blood cells. Transfection of COS cells with a luciferase reporter gene system revealed up to 25-fold increased promoter activity of GK AC-III promoter when compared with normal rat promoter (P < 0.0001). In conclusion, forskolin restores the impaired insulin release in islets of the GK rat through enhanced cAMP generation. This is linked to overexpression of AC-III mRNA in GK islets due to two functional point mutations in the promoter region of the AC-III gene.
Asunto(s)
Adenilil Ciclasas/genética , AMP Cíclico/biosíntesis , Diabetes Mellitus Tipo 2/genética , Regulación Enzimológica de la Expresión Génica/genética , Insulina/metabolismo , Islotes Pancreáticos/química , Islotes Pancreáticos/metabolismo , Mutación/genética , Regiones Promotoras Genéticas/genética , Animales , Secuencia de Bases , Estudios de Cohortes , Cartilla de ADN/química , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Hibridación in Situ , Técnicas In Vitro , Secreción de Insulina , Islotes Pancreáticos/patología , Masculino , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas Endogámicas , Ratas WistarRESUMEN
Experimental autoimmune neuritis (EAN) is a CD4+ T cell-mediated monophasic inflammatory disorder of the peripheral nervous system (PNS). Cellular mechanisms, including macrophage and T cell infiltration, and cytokines like IFN-gamma and TNF-alpha are intimately involved in the pathogenesis of EAN. Interleukin 10 (IL-10) is a TH2-type cytokine that suppresses monocyte and TH1 cell functions. We examined the effect of recombinant human IL-10 (rHuIL-10) in EAN. When administered from the start of immunization with bovine peripheral myelin emulsified in Freund's complete adjuvant, IL-10 effectively suppressed and shortened clinical EAN. Even when given after Day 12 post immunization (pi) after clinical EAN had been established, IL-10 also effectively suppressed the severity of EAN. Pheripheral nerve myelin antigen-reactive IFN-gamma-secreting TH1-like cells were decreased in lymph nodes from IL-10-treated compared to control EAN rats. PNS autoantigen-induced T cell proliferation and B cell responses were not affected. P2 protein-reactive IFN-gamma, TNF-alpha, IL-1 beta, and IL-6 mRNA-expressing lymph node cells were also downregulated in IL-10-treated compared to control EAN rats at Day 14 and 26 pi, while P2-reactive IL-4 mRNA-expressing cells were upregulated throughout treatment. Also, in IL-10-treated EAN rats, upregulated anti-P2 IgG1 and downregulated IgG2a were observed. Our results clearly show that rHuIL-10 can suppress clinical EAN, and this suppression is associated with downregulation of TH1 responses and macrophage function and upregulated TH2 responses.
Asunto(s)
Interleucina-10/fisiología , Interleucina-10/uso terapéutico , Neuritis Autoinmune Experimental/prevención & control , Células TH1/inmunología , Animales , Formación de Anticuerpos/efectos de los fármacos , Autoantígenos/farmacología , Linfocitos B/inmunología , Citocinas/genética , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Cambio de Clase de Inmunoglobulina/efectos de los fármacos , Masculino , Sistema Nervioso Periférico/inmunología , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/uso terapéutico , Linfocitos T/inmunologíaRESUMEN
Proinflammatory cytokines mediate brain injury in experimental studies. This study was undertaken to analyze the production of proinflammatory cytokines in experimental contusion. A brain contusion causing delayed edema was mimicked experimentally in rats using a weight-drop model. Intracerebral expression of the cytokines interleukin (IL)-1 beta, tumor necrosis factor-alpha (TNF alpha), IL-6, and interferon-gamma (IFN gamma) was studied by in situ hybridization and immunohistochemistry. The animals were killed at 6 hours or 1, 2, 4, 6, 8, or 16 days postinjury. In the injured area, no messenger (m)RNA expression was seen during the first 2 days after the trauma. On Days 4 to 6 posttrauma, however, strong IL-1 beta, TNF alpha, and IL-6 mRNA expression was detected in mononuclear cells surrounding the contusion. Expression of IFN gamma was not detected. Immunohistochemical double labeling confirmed the in situ hybridization results and demonstrated that mononuclear phagocytes and astrocytes produced IL-1 beta and that mainly astrocytes produced TNF alpha. The findings showed, somewhat unexpectedly, a late peak of intracerebral cytokine production in the injured area and in the contralateral corpus callosum, allowing for both local and global effects on the brain. An unexpected difference in the cellular sources of TNF alpha and IL-1 beta was detected. The cytokine pattern differs from that seen in other central nervous system inflammatory diseases and trauma models, suggesting that the intracerebral immune response is not a uniform event. The dominance of late cytokine production indicates that many cytokine effects are late events in an experimental contusion: Different pathogenic mechanisms may thus be operative at different times after brain injury.
Asunto(s)
Conmoción Encefálica/metabolismo , Encéfalo/metabolismo , Citocinas/análisis , Animales , Astrocitos/metabolismo , Conmoción Encefálica/genética , Edema Encefálico/genética , Edema Encefálico/metabolismo , Cuerpo Calloso/metabolismo , Citocinas/genética , Femenino , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente Directa , Colorantes Fluorescentes , Regulación de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Interferón gamma/análisis , Interferón gamma/genética , Interleucina-1/análisis , Interleucina-1/genética , Interleucina-6/análisis , Interleucina-6/genética , Monocitos/metabolismo , Fagocitos/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Exposure of peripheral blood mononuclear cells to promastigotes of Leishmania aethiopica derived from patients with the self-limiting, localized form of the disease (local cutaneous leishmaniasis; LCL) preferentially induced mRNA for interferon (IFN)-gamma but little for interleukin (IL)-10. In contrast, stimulation of the same cells with promastigotes derived from patients with the persistent, disseminated form of the disease (diffuse cutaneous leishmaniasis; DCL) stimulated the expression of IL-10 rather than IFN-gamma. In general, parasites derived from LCL patients induced more expression of other cytokines tested, including IL-4, IL-6, and transforming growth factor-beta, although tumor necrosis factor-alpha was equivalent in cultures stimulated with LCL or DCL promastigotes. The results suggest that the antigen-specific immunosuppression observed in DCL patients and the resulting clinical picture could in part be due to the properties of the infecting parasite to induce more IL-10 than IFN-gamma.
Asunto(s)
Interferón gamma/genética , Interleucina-10/genética , Leishmaniasis Cutánea/inmunología , Animales , Células Cultivadas , Expresión Génica , Humanos , Hibridación in Situ , Leishmania/inmunología , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/patología , ARN Mensajero/genética , Factores de TiempoRESUMEN
Experimental autoimmune encephalomyelitis (EAE) in rats is typically a brief and monophasic disease with sparse demyelination. However, inbred DA rats develop a demyelinating, prolonged and relapsing encephalomyelitis after immunization with rat spinal cord in incomplete Freund's adjuvant. This model enables studies of mechanisms related to chronicity and demyelination, two hallmarks of multiple sclerosis (MS). Here we have investigated, in situ, the dynamics of cytokine mRNA expression in the central nervous system (CNS) and peripheral lymphoid organs (lymph node cells and splenocytes) of diseased DA rats. We demonstrate that peripheral lymphoid cells stimulated in vitro with encephalitogenic peptides 69-87 and 87-101 of myelin basic protein responded with high mRNA expression for proinflammatory cytokines; interferon-gamma, interleukin-12 (IL-12), tumour necrosis factors alpha and beta, IL-1 beta and cytolysin. A high expression of mRNA for these proinflammatory cytokines was also observed in the CNS where it was accompanied by classical signs of inflammation such as expression of major histocompatibility complex class I and II, CD4, CD8 and IL-2 receptor. The expression of mRNA for proinflammatory cytokines was remarkably long-lasting in DA rats as compared to LEW rats which display a brief and monophasic EAE. Furthermore, mRNAs for putative immunodownmodulatory cytokines, i.e. transforming growth factor-beta (TGF-beta), IL-10 and IL-4 were almost absent in DA rats, in both the CNS and in vitro stimulated peripheral lymphoid cells, while their levels were elevated in the CNS of LEW rats during the recovery phase. We conclude that the MS-like prolonged and relapsing EAE in DA rats is associated with a prolonged production of proinflammatory cytokines and/or low or absent production of immunodownmodulatory cytokines.
Asunto(s)
Citocinas/genética , Encefalomielitis Autoinmune Experimental/inmunología , Interleucina-10/genética , Médula Espinal/metabolismo , Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Citocinas/metabolismo , Expresión Génica , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Proteína Básica de Mielina/química , Proteína Básica de Mielina/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , ARN Mensajero/genética , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
In this study, cytokine mRNA profiles in microglia from newborn rats were detected by in situ hybridization. Under natural culture conditions, microglia expressed the immunosuppressive transforming growth factor-beta 1 (TGF-beta 1) and interleukin (IL) 10 to a greater degree than the pro-inflammatory cytokines IL-1 beta, IL-6, IL-12, interferon-gamma (IFN-gamma) and TNF-alpha. High TGF-beta 1 and IL-10 levels could reflect one mechanism for immune privilege within the CNS under physiological conditions. Stimulation of microglia with LPS or IFN gamma resulted in strong up-regulation of proinflammatory cytokines, while TGF-beta 1 and IL-10 were down-regulated. These effects of LPS or IFN-gamma are anticipated to reflect immunopathogenic processes within the CNS.
Asunto(s)
Animales Recién Nacidos/metabolismo , Recuento de Células/efectos de los fármacos , Citocinas/metabolismo , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Hibridación in Situ , Microglía/metabolismo , Ratas , Ratas Endogámicas LewRESUMEN
A Trypanosoma brucei brucei-derived lymphocyte triggering factor (TLTF) induced CD8+ T cells to produce IFN-gamma, which in turn stimulates parasite growth. This parasite-host interaction was studied in mouse strains that are either relatively susceptible (C3H/He) or resistant (C57Bl/6J) to infection, as well as in athymic nude mice. In all mouse strains, T. b. brucei infection caused a strong induction of IFN-gamma production by spleen mononuclear cells (MNC). In vivo blocking of IFN-gamma by intraperitoneal injection of mouse monoclonal anti-IFN-gamma antibody suppressed parasite growth and increased survival of both C3H/H3 and C57Bl/6J animals, suggesting that, irrespective of strain-related disease susceptibility, IFN-gamma is a growth-promoting stimulus for T. b. brucei. Spleen MNC from noninfected mice of all strains were in vitro like-wise strongly induced to IFN-gamma production when exposed to TLTF. This suggests that CD8+ expressing T cell receptor (TCR) alpha/beta, gamma/delta-bearing T cells and NK cells may all be triggered to IFN-gamma production by TLTF. In all mouse strains, TLTF also caused an increase in the number of cells expressing mRNA for TGF-beta in vitro. However, significant triggering to IL-4 mRNA expression only occurred in the relatively disease-resistant C57Bl/6J strain. As IL-4 is required for the synthesis and class switches of immunoglobulins, which are essential host immune defenses against T. b. brucei, the degree of resistance may be related to inherent strain ability to produce IL-4 in response to TLTF.
Asunto(s)
Factores Biológicos/farmacología , Inductores de Interferón/farmacología , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Trypanosoma brucei brucei/fisiología , Animales , Susceptibilidad a Enfermedades , Femenino , Interacciones Huésped-Parásitos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Desnudos , Especificidad de la EspecieRESUMEN
The increased intrathecal production of immunoglobulins within the cerebrospinal fluid (CSF) compartment commonly observed in multiple sclerosis (MS) implicates participation of B cell activating factors. One effect of the cytokine interleukin (IL)-6 is induction of immunoglobulin production by activated B cells. Employing in situ hybridization (ISH) with synthetic oligonucleotide probes, we measured numbers of IL-6 mRNA-expressing mononuclear cells (MNC) in blood and CSF from patients with MS, aseptic meningo-encephalitis (AM), and in blood from patients with other neurological diseases (OND) and healthy subjects. Numbers of IL-6 mRNA-expressing MNC were elevated in blood (mean frequency 1 per 33,000 MNC) and even further enriched in the CSF (1 per 10,000 MNC) of MS patients, and to a similar extent in AM patients' blood. Cultivation in the presence of myelin basic protein and proteolipid protein revealed strong augmentation of IL-6 mRNA-positive cells in MS but not in OND. The results suggest that IL-6 is one of several cytokines which are upregulated in MS, in particular locally in the CSF. A role of IL-6 in MS, whether disease- promoting or protective, remains unclear.
Asunto(s)
Interleucina-6/genética , Leucocitos Mononucleares/metabolismo , Esclerosis Múltiple/metabolismo , ARN Mensajero/análisis , Adulto , Anciano , Anciano de 80 o más Años , Líquido Cefalorraquídeo/citología , Líquido Cefalorraquídeo/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The kinetics of mRNA expression in the central nervous system (CNS) for a series of putatively disease-promoting and disease-limiting cytokines during the course of experimental autoimmune encephalomyelitis (EAE) in Lewis rats were studied. Cytokine mRNA-expressing cells were detected in cryosections of spinal cords using in situ hybridization technique with synthetic oligonucleotide probes. Three stages of cytokine mRNA expression could be distinguished: (i) interleukin (IL)-12, tumor necrosis factor (TNF)-beta (= lymphotoxin-alpha) and cytolysin appeared early and before onset of clinical signs of EAE; (ii) TNF-alpha peaked at height of clinical signs of EAE; (iii) IL-10 appeared increasingly at and after clinical recovery. The early expression of IL-12 prior to the expression of interferon-gamma (IFN-gamma) mRNA shown previously is consistent with a role of IL-12 in promoting proliferation and activation of T helper 1 (Th1) type cells producing IFN-gamma. The TNF-beta mRNA expression prior to onset of clinical signs favours a role for this cytokine in disease initiation. A pathogenic effector role of TNF-alpha was suggested from these observations that TNF-alpha mRNA expression roughly paralleled the clinical signs of EAE. This may be the case also for cytolysin. IL-10-expressing cells gradually increased to high levels in the recovery phase of EAE, consistent with a function in down-regulating the CNS inflammation. From these data we conclude that there is an ordered appearance of putative disease-promoting and -limiting cytokines in the CNS during acute monophasic EAE.
Asunto(s)
Encefalomielitis Autoinmune Experimental/genética , Interleucina-10/genética , Interleucina-12/genética , Linfotoxina-alfa/genética , Glicoproteínas de Membrana/genética , Médula Espinal/metabolismo , Factor de Necrosis Tumoral alfa/genética , Animales , Expresión Génica , Hibridación in Situ , Masculino , Perforina , Proteínas Citotóxicas Formadoras de Poros , ARN Mensajero/genética , Ratas , Ratas Endogámicas LewRESUMEN
Evidence has been presented for the involvement of various cytokines, including interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-alpha, in the pathogenesis of human immunodeficiency virus (HIV) infection. Since measured plasma levels may poorly reflect in vivo production of cytokines, we adopted in situ hybridization with cDNA oligonucleotide probes to enumerate blood mononuclear cells (MNCs) expressing mRNA for IL-6, IL-10, TNF-alpha, and perforin. The HIV-infected patients had elevated levels of MNCs expressing mRNA for all four cytokines compared to healthy controls. Numbers of IL-6 mRNA-expressing cells were higher in patients with clinical AIDS than in asymptomatic seropositive patients, and correlated inversely with CD4+ cell counts in blood, reflecting the involvement of IL-6 in later stages of HIV infection. The described approach could be an alternative way to study cytokines in HIV infection.
Asunto(s)
Citocinas/biosíntesis , Infecciones por VIH/sangre , VIH-1 , Leucocitos Mononucleares/metabolismo , Glicoproteínas de Membrana/biosíntesis , ARN Mensajero/biosíntesis , Síndrome de Inmunodeficiencia Adquirida/sangre , Adulto , Citocinas/genética , ADN Complementario/análisis , Femenino , Infecciones por VIH/inmunología , Seropositividad para VIH/sangre , Humanos , Hibridación in Situ , Interleucina-10/biosíntesis , Interleucina-10/genética , Interleucina-6/biosíntesis , Interleucina-6/genética , Masculino , Glicoproteínas de Membrana/genética , Persona de Mediana Edad , Sondas de Oligonucleótidos , Perforina , Proteínas Citotóxicas Formadoras de Poros , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The potential role of certain important immunoregulatory and effector cytokines in autoimmune neuroinflammation have been studied. We have examined the expression of mRNA, with in situ hybridization, of interferon gamma (IFN-gamma), interleukin 4 (IL-4) and transforming growth factor beta (TGF-beta) both in sections of spinal cords and the antigen-induced expression of these cytokines by lymphoid cells after stimulation with a dominant encephalitogenic peptide of MBP (MBP 63-88) during the course of actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. In spinal cords, the target organ in EAE, cells expressing mRNA for IFN-gamma, first appeared at the onset of clinical signs, i.e., day 10 postimmunization (p.i.), peaked at the height of disease (day 13 p.i.) and then gradually decreased concomitant with recovery. Very few IL-4 mRNA-expressing cells appeared in the spinal cord with no clear relation to clinical signs or histopathology. In contrast, expression of mRNA for TGF-beta did not increase until day 13 p.i., at height of the disease, shortly preceding recovery. These data are consistent with a disease upregulating role of IFN-gamma, while TGF-beta may act to limit central nervous system (CNS) inflammation. In lymphoid organs, primed MBP 63-88 reactive T cells showed an interesting time-dependent evolution of their cytokine production in vitro. Thus, early after immunization there was a conspicuous MBP 63-88-induced production of both IFN-gamma and IL-4. Such cells may act in the initiation and promotion of the disease. Later, in the recovery phase, MBP 63-88 induced lymphoid cells to TGF-beta production. Thus, an autoantigen-specific production of TGF-beta occurred during EAE and hypothetically such a mechanism may serve to downregulate aggressive autoimmunity systemically.
Asunto(s)
Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Tejido Linfoide/metabolismo , ARN Mensajero/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Secuencia de Aminoácidos , Animales , Inmunohistoquímica , Hibridación in Situ , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Tejido Linfoide/citología , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas Lew , Bazo/citología , Bazo/metabolismo , Linfocitos T/metabolismoRESUMEN
Oral administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with Torpedo AChR+complete Freund's adjuvant (CFA) results in the prevention of experimental autoimmune myasthenia gravis (EAMG) and the suppression of AChR-specific B cell responses and counteracts the development of AChR-reactive interferon-gamma (IFN-gamma) secreting T cells. To study the involvement of the T helper type 1 (Th1) cell-related lymphokine IFN-gamma, the Th2 cell-related interleukin-4 (IL-4), and transforming growth factor beta (TGF-beta) that suppresses the synthesis of IFN-gamma and IL-4, we used in situ hybridization with complementary DNA oligonucleotide probes to enumerate mononuclear cells (MNC) expressing mRNA for the cytokines IFN-gamma, IL-4, and TGF-beta. Upon in vivo recognition of AChR, popliteal, inguinal, and mesenteric lymph nodes, spleen and thymus of rats with EAMG contained higher levels of IFN-gamma, IL-4, and TGF-beta mRNA-expressing cells compared to CFA-injected control rats, implicating the involvement in EAMG of AChR-reactive Th1 and Th2 cells in parallel. TGF-beta was also upregulated in EAMG. Oral tolerance to EAMG was characterized by suppression of the levels of MNC expressing IFN-gamma and IL-4, but augmentation of cells expressing TGF-beta. The results suggest that IFN-gamma, IL-4, and TGF-beta are involved in the development of EAMG, and that TGF-beta is important in the induction of oral tolerance to EAMG.
Asunto(s)
Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Miastenia Gravis/inmunología , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Expresión Génica , Tolerancia Inmunológica , Tejido Linfoide/inmunología , Proteína Básica de Mielina/inmunología , ARN Mensajero/genética , Ratas , Ratas Endogámicas Lew , Receptores Nicotínicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Factores de Tiempo , Distribución TisularRESUMEN
The inflammatory nature of multiple sclerosis (MS) implicates the participation of immunoregulatory cytokines, including the T-helper type 1 (Th1) cell-associated interferon-gamma (IFN-gamma), the Th2 cell-related interleukin-4 (IL-4), and the immune response-downregulating cytokine transforming growth factor-beta (TGF-beta), but proof for their involvement in MS has been lacking. By adopting in situ hybridization with complementary DNA oligonucleotide probes for human IFN-gamma IL-4, and TGF-beta, the expression of mRNA for these cytokines was detected in mononuclear cells (MNC) from blood and cerebrospinal fluids. Strongly elevated levels of MNC expressing all three cytokines were found in peripheral blood and at even higher frequencies in cerebrospinal fluid from untreated patients with MS and optic neuritis, i.e., a common first manifestation of MS, compared with patients with other neurological diseases and healthy subjects. In MS and optic neuritis, IL-4 mRNA expressing cells predominated, followed by TGF-beta- and IFN-gamma-positive cells. Control patients with myasthenia gravis had similarly elevated levels of IFN-gamma and TGF-beta and TGF-beta mRNA expressing blood MNC but lower numbers of IL-4-positive cells. No or slight disability of MS was associated with high levels of TGF-beta mRNA expressing cells, while MS patients with moderate or severe disability had high levels of IFN-gamma-positive cells. IFN-gamma and TGF-beta may have opposing effects in MS, and treatments inhibiting IFN-gamma and/or promoting TGF-beta might ameliorate MS.
Asunto(s)
Interferón gamma/metabolismo , Interleucina-4/metabolismo , Esclerosis Múltiple/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Femenino , Humanos , Hibridación in Situ , Masculino , Persona de Mediana Edad , ARN Mensajero/análisisRESUMEN
T cells recognizing the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) are increased in multiple sclerosis (MS), and there are elevated numbers of T cells recognizing the nicotinic acetylcholine receptor (AChR) in myasthenia gravis (MG). However, the cytokine repertoires in these diseases are largely unknown. We adopted in situ hybridization with radiolabeled complementary DNA oligonucleotide probes to enumerate mononuclear cells that expressed the T-helper type 1 (Th1) cell-related interferon-gamma (IFN-gamma) and Th2-associated interleukin-4 (IL-4) after short-term culture in the presence of autoantigen. High numbers of IFN-gamma and IL-4 mRNA-expressing cells in response to MBP and PLP were detected in patients with untreated MS, and to AChR in MG. The levels of IFN-gamma and IL-4 mRNA-positive cells in MS after culture in the presence of AChR, and in MG after culture in the presence of MBP or PLP, did not differ from those detected after culture without antigen. The CSF of MS patients contained four- to eightfold more myelin protein-reactive IFN-gamma and IL-4 expressing cells. The findings imply that MS and MG are associated with mixed Th1- and Th2-like cell responses directed to organ-specific target antigens.
Asunto(s)
Expresión Génica , Interferón gamma/genética , Interleucina-4/genética , Esclerosis Múltiple/genética , Miastenia Gravis/genética , Adulto , Autoantígenos/inmunología , Autoantígenos/fisiología , Epítopos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Esclerosis Múltiple/inmunología , Miastenia Gravis/inmunología , Proteína Básica de Mielina/inmunología , Proteínas de la Mielina/inmunología , Proteína Proteolipídica de la Mielina , ARN Mensajero/metabolismo , Receptores Colinérgicos/fisiologíaRESUMEN
Multiple sclerosis (MS) is characterized by patchy accumulations of inflammatory cells combined with demyelination. There are mononuclear cells in blood and cerebrospinal fluid of patients with MS that produce interferon-gamma and interleukin-4 in response to myelin basic protein (MBP) and proteolipid protein (PLP). Here we describe autoantigen-induced production of transforming growth factor-beta (TGF-beta). This multifunctional cytokine has inhibitory effects on the growth, differentiation, and effector functions of activated T cells. Blood and cerebrospinal fluid cells were exposed in short-term cultures to MBP and PLP and, after hybridization with complementary DNA oligonucleotide probes, they were evaluated for TGF-beta mRNA expression. Patients with MS had higher numbers of MBP- and PLP-responsive TGF-beta mRNA expressing cells in blood compared with control patients with other neurological diseases or myasthenia gravis and a five- and threefold further increment in their cerebrospinal fluid. In blood of patients with myasthenia gravis, where the acetylcholine receptor (AChR) is a target for autoaggressive immunity, there were increased levels of AChR-responsive TGF-beta mRNA expressing cells. Thymectomized myasthenia gravis patients showed higher levels of TGF-beta mRNA expressing cells compared with patients not thymectomized. Numbers of cells responding to AChR in MS and MBP in myasthenia gravis did not differ from numbers found in absence of antigen. Patients with other neurological diseases showed infrequent and low responses to MBP, PLP, and AChR. Diseases with presumed autoimmune pathogenesis are associated with organ-specific autoantigen-induced TGF-beta production, which is increased after thymectomy.
Asunto(s)
Esclerosis Múltiple/genética , Miastenia Gravis/genética , Proteínas de la Mielina/farmacología , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/genética , Adulto , Autoantígenos/farmacología , Líquido Cefalorraquídeo/citología , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Miastenia Gravis/inmunología , Proteína Proteolipídica de la Mielina , ARN Mensajero/inmunología , Receptores Colinérgicos/metabolismo , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
An interferon (IFN)-gamma immunoreactive molecule, localized to small neurons in peripheral sensory ganglia (N-IFN-gamma), has been detected with two mouse monoclonal antibodies (DB1 and DB16) directed against different epitopes of rat IFN-gamma. To define N-IFN-gamma with regard to its protein characteristics and bioactivities, DB1 and DB16 were used to purify N-IFN-gamma from rat trigeminal ganglia in a two-step sequential antibody-affinity procedure. Sodium dodecylsulfate polyacrylamide gel electrophoresis (PAGE) and silver staining of purified N-IFN-gamma displayed three bands with an approximate molecular mass of 66, 62 and 54 kDa. The N-IFN-gamma bioactivity was confined to the protein stained on gel when native material was run on PAGE. Biological effects of pure N-IFN-gamma were examined and compared with those of lymphocyte-derived recombinant IFN-gamma. N-IFN-gamma had antiviral effects in vitro and induced major histocompatibility complex class I and II antigens on macrophages and in cells in skeletal muscle cell cultures. N-IFN-gamma also stimulated myoblast proliferation and affected cholinergic receptor distribution on myotubes similar to recombinant IFN-gamma. Both molecules potently stimulated Trypanosoma brucei brucei growth. These data suggest that, although N-IFN-gamma is a protein distinct from lymphocyte-derived IFN-gamma, the two molecules have enough structural similarities to allow for antibody recognition of at least two epitopes, and action on similar target structures on both parasite and mammalian cells.
Asunto(s)
Interferón gamma/aislamiento & purificación , Neuronas/química , Animales , Antivirales , Bioensayo , Células Cultivadas , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Técnicas In Vitro , Interferón gamma/inmunología , Macrófagos/inmunología , Músculos/citología , Ratas , Ratas Sprague-Dawley , Agregación de Receptores/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Ganglio del Trigémino/química , Trypanosoma/crecimiento & desarrolloRESUMEN
Studies on patients with monosymptomatic optic neuritis (ON) should give opportunities to identify features typical for early multiple sclerosis (MS). There are increased T and B cell responses to the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) in both ON and MS, but there is little information on the types of cytokines produced by such cells. We describe the use of in situ hybridization with complementary DNA oligonucleotide probes to detect and enumerate mononuclear cells expressing mRNA for the cytokines interferon-gamma (IFN-gamma) which augments cell-mediated immunity; interleukin-4 (IL-4) which promotes the B cell response; and transforming growth factor beta (TGF-beta) that in many cases downregulates immune responses. Expression of these cytokines was studied in mononuclear cells from peripheral blood and cerebrospinal fluid (CSF) from patients with ON and MS after in vitro exposure to MBP and PLP, and in absence of antigen. There were elevated levels of cells that in response to MBP and PLP expressed IFN-gamma, IL-4 and TGF-beta mRNA in blood and further enriched in CSF in both ON and MS, compared to patients with other neurological diseases. The results suggest that IFN-gamma, IL-4 as well as TGF-beta are involved in both ON and MS, and that the cytokine profile in early MS as reflected by ON is not different from that in clinically definite MS.
Asunto(s)
Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Proteína Básica de Mielina/farmacología , Proteínas de la Mielina/farmacología , Neuritis Óptica/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Adulto , Femenino , Humanos , Interferón gamma/genética , Interleucina-4/genética , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/metabolismo , Proteína Proteolipídica de la Mielina , ARN Mensajero/análisis , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Polymorphism of the major histocompatibility complex (MHC) influences susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP) in rats. Current concepts relate such influences to the capacity of class II molecules to present relevant peptides to autoreactive T cells. We have here analyzed the MHC influence on the immune response and the development of EAE after immunization with the immunodominant peptide MBP-63-88. Analysis of MHC-congenic LEWIS strains showed that RT1a, RT1c and RT1(1) haplotypes are permissive for disease induction, whereas RT1d and RT1u are resistant. All EAE responding strains showed peptide-specific proliferation and interferon (IFN)-gamma secretion, but no early significant tendency to express interleukin (IL-4) or transforming growth factor (TGF)-beta mRNA in lymphocytes in response to the MBP 63-88, 7 days post immunization (p.i.). Later, 14 days p.i., peptide-specific induction of IL-4 and TGF-beta occurred in RT1(1) rats. Among the EAE non-responders strains, only the RT1u rats showed an immune response to MBP 63-88. This response, however, was qualitatively different from the immune response in the EAE-susceptible strains. Thus, there was no proliferation and only moderate IFN-gamma production in response to peptide, but in contrast, a significant and early peptide-induced IL-4 and TGF-beta response was observed. The data suggest that the MHC-associated susceptibility to EAE is partly related to the ability to mount a TH1-like immune response while the MHC-associated EAE resistance may either be related to MBP peptide non-responsiveness or to peptide recognition and induction of a qualitatively different and disease down-regulatory immune response.