RESUMEN
Interleukin-25 (IL-25) is the only anti-inflammatory cytokine of the IL-17 family, and it has been shown to be efficacious in inhibiting neuroinflammation. Known for its effects on cells of the adaptive immune system, it has been more recently described to be effective also on cells of the innate immune system, namely macrophages. We used a lentiviral-mediated gene therapy approach to deliver IL-25 to the central nervous system (CNS) in two mouse models of neuroinflammation, entorhinal cortex lesion and experimental autoimmune encephalomyelitis. In both, we found that IL-25 gene therapy was able to modulate CNS myeloid cells, either infiltrating macrophages or resident microglia, towards an anti-inflammatory, tissue-protective phenotype, as testified by the increase in markers such as Arginase-1 (Arg1), Mannose receptor 1 (CD206) and Chitinase 3-like 3 (Ym1). As a consequence, neuroinflammation was partly inhibited and the CNS protected from immune-mediated damage. To our knowledge, this is the first example of M2 shift (alternative activation) induced in vivo on CNS-resident myeloid cells by gene therapy, and may constitute a promising strategy to investigate the potential role of protective microglia in neurological disorders.
Asunto(s)
Encefalomielitis Autoinmune Experimental/terapia , Corteza Entorrinal , Terapia Genética , Inflamación/terapia , Interleucina-17/genética , Animales , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/genética , Corteza Entorrinal/metabolismo , Corteza Entorrinal/patología , Humanos , Inflamación/genética , Interleucina-17/uso terapéutico , Lentivirus/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Microglía/patología , Microglía/trasplante , Células Mieloides/metabolismo , Células Mieloides/patologíaRESUMEN
BACKGROUND: Inflammation triggers secondary neurodegeneration in multiple sclerosis (MS). OBJECTIVES: It is unclear whether classical anti-inflammatory cytokines have the potential to interfere with synaptic transmission and neuronal survival in MS. METHODS: Correlation analyses between cerebrospinal fluid (CSF) contents of anti-inflammatory cytokines and molecular, imaging, clinical, and neurophysiological measures of neuronal alterations were performed. RESULTS: Our data suggest that interleukin-13 (IL-13) plays a neuroprotective role in MS brains. We found, in fact, that the levels of IL-13 in the CSF of MS patients were correlated with the contents of amyloid-ß(1-42). Correlations were also found between IL-13 and imaging indexes of axonal and neuronal integrity, such as the retinal nerve fibre layer thickness and the macular volume evaluated by optical coherence tomography. Furthermore, the levels of IL-13 were related to better performance in the low-contrast acuity test and Multiple Sclerosis Functional Composite scoring. Finally, by means of transcranial magnetic stimulation, we have shown that GABAA-mediated cortical inhibition was more pronounced in patients with high IL-13 levels in the CSF, as expected for a neuroprotective, anti-excitotoxic effect. CONCLUSIONS: The present correlation study provides some evidence for the involvement of IL-13 in the modulation of neuronal integrity and synaptic function in patients with MS.
Asunto(s)
Interleucina-13/líquido cefalorraquídeo , Corteza Motora/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Degeneración Nerviosa/inmunología , Neuronas/inmunología , Adulto , Péptidos beta-Amiloides/líquido cefalorraquídeo , Biomarcadores/líquido cefalorraquídeo , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Sensibilidad de Contraste , Evaluación de la Discapacidad , Potenciales Evocados Motores , Femenino , Humanos , Italia , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Corteza Motora/patología , Corteza Motora/fisiopatología , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Esclerosis Múltiple Recurrente-Remitente/patología , Esclerosis Múltiple Recurrente-Remitente/fisiopatología , Degeneración Nerviosa/líquido cefalorraquídeo , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Degeneración Nerviosa/prevención & control , Neuronas/patología , Fragmentos de Péptidos/líquido cefalorraquídeo , Transmisión Sináptica , Tomografía de Coherencia Óptica , Estimulación Magnética Transcraneal , Adulto JovenRESUMEN
Inflammation and immune reaction, or pre-existing immunity towards commonly used viral vectors for gene therapy severely impair long-term gene expression in the central nervous system (CNS), impeding the possibility to repeat the therapeutic intervention. Here, we show that injection of a helper-dependent adenoviral (HD-Ad) vector by lumbar puncture into the cerebrospinal fluid (CSF) of non-human primates allows long-term (three months) infection of neuroepithelial cells, also in monkeys bearing a pre-existing anti-adenoviral immunity. Intrathecal injection of the HD-Ad vector was not associated with any sign of systemic or local toxicity, nor by signs of a CNS-specific immune reaction towards the HD-Ad vector. Injection of HD-Ad vectors into the CSF circulation may thus represent a valuable approach for CNS gene therapy allowing for long-term expression and re-administration.
Asunto(s)
Adenoviridae/genética , Líquido Cefalorraquídeo/virología , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Virus Helper/genética , Enfermedad de Parkinson/terapia , Animales , Expresión Génica , Ingeniería Genética , Vectores Genéticos/inmunología , Interleucina-4/genética , Macaca fascicularis , Masculino , Modelos Animales , Células Neuroepiteliales/inmunología , Células Neuroepiteliales/virología , Enfermedad de Parkinson/inmunología , Punción Espinal , Transducción Genética/métodosRESUMEN
Central nervous system (CNS) delivery of anti-inflammatory cytokines, such as interleukin 4 (IL4), holds promise as treatment for multiple sclerosis (MS). We have previously shown that short-term herpes simplex virus type 1-mediated IL4 gene therapy is able to inhibit experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in mice and non-human primates. Here, we show that a single administration of an IL4-expressing helper-dependent adenoviral vector (HD-Ad) into the cerebrospinal fluid (CSF) circulation of immunocompetent mice allows persistent transduction of neuroepithelial cells and long-term (up to 5 months) CNS transgene expression without toxicity. Mice affected by chronic and relapsing EAE display clinical and neurophysiological recovery from the disease once injected with the IL4-expressing HD-Ad vector. The therapeutic effect is due to the ability of IL4 to increase, in inflamed CNS areas, chemokines (CCL1, CCL17 and CCL22) capable of recruiting regulatory T cells (CD4+CD69-CD25+Foxp3+) with suppressant functions. CSF delivery of HD-Ad vectors expressing anti-inflammatory molecules might represent a valuable therapeutic option for CNS inflammatory disorders.
Asunto(s)
Sistema Nervioso Central/inmunología , Terapia Genética/métodos , Interleucina-4/genética , Esclerosis Múltiple/terapia , Linfocitos T Reguladores/inmunología , Adenoviridae/genética , Animales , Sistema Nervioso Central/patología , Quimiocinas/inmunología , Quimiotaxis de Leucocito , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/administración & dosificación , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Virus Helper/genética , Humanos , Interleucina-4/análisis , Interleucina-4/inmunología , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción Genética/métodosRESUMEN
Multiple sclerosis (MS) is a T cell-mediated organ-specific inflammatory disease leading to central nervous system (CNS) demyelination. On the basis of results obtained in experimental autoimmune encephalomyelitis (EAE) models, MS treatment by administration of antiinflammatory cytokines such as interleukin 4 (IL-4) is promising but is hampered by the limited access of the cytokines to the CNS and by the pleiotropic effects of systemically administered cytokines. We established a cytokine delivery system within the CNS using non-replicative herpes simplex type 1 (HSV-1) viral vectors engineered with cytokine genes. These vectors injected into the cisterna magna (i.c.) of mice diffuse in all ventricular and subarachnoid spaces and infect with high efficiency the ependymal and leptomeningeal cell layers surrounding these areas, without obvious toxic effects. Heterologous genes contained in the vectors are efficiently transcribed in infected ependymal cells, leading to the production of high amounts of the coded proteins. For example, 4.5 ng of interferon gamma (IFN-gamma) per milliliter is secreted into the cerebrospinal fluid (CSF) up to day 28 postinjection (p.i.) and reaches the CNS parenchyma in bioactive form, as demonstrated by upregulation of MHC class I expression on CNS-resident cells. We then exploited the therapeutic potential of the vectors in EAE mice. An HSV-1-derived vector containing the IL-4 gene was injected i.c. in Biozzi AB/H mice at the time of EAE induction. We found the following in treated mice: (1) delayed EAE onset, (2) a significant decrease in clinical score, (3) a significant decrease in perivascular inflammatory infiltrates and in the number of macrophages infiltrating the CNS parenchyma and the submeningeal spaces, and (4) a reduction in demyelinated areas and axonal loss. Peripheral T cells from IL-4-treated mice were not affected either in their antigen-specific proliferative response or in cytokine secretion pattern. Our results indicate that CNS cytokine delivery with HSV-1 vectors is feasible and might represent an approach for the treatment of demyelinating diseases. Advantages of this approach over systemic cytokine administration are the high cytokine level reached in the CNS, the absence of effects on the peripheral immune system, and the long-lasting cytokine production in the CNS after a single vector administration.
Asunto(s)
Sistema Nervioso Central/metabolismo , Virus Defectuosos/genética , Enfermedades Desmielinizantes/prevención & control , Encefalomielitis Autoinmune Experimental/patología , Herpesvirus Humano 1/genética , Interleucina-4/administración & dosificación , Animales , División Celular , Sistema Nervioso Central/patología , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Vectores Genéticos , Inmunohistoquímica , Interferón gamma/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos BALB C , Linfocitos T/citologíaRESUMEN
Systemic administration of antiinflammatory molecules to patients affected by immune-mediated inflammatory demyelinating diseases of the central nervous system (CNS) has limited therapeutic efficacy due to the presence of the blood-brain barrier (BBB). We found that three of five rhesus monkeys injected intrathecally with a replication-defective herpes simplex virus (HSV) type 1-derived vector engineered with the human interleukin 4 (IL-4) gene were protected from an hyperacute and lethal form of experimental autoimmune encephalomyelitis induced by whole myelin. The intrathecally injected vector consistently diffused within the CNS via the cerebrospinal fluid and infected ependymal cells, which in turn sustained in situ production of IL-4 without overt immunological or toxic side effects. In EAE-protected monkeys, IL-4-gene therapy significantly decreased the number of brain as well as spinal cord inflammatory perivenular infiltrates and the extent of demyelination, necrosis, and axonal loss. The protective effect was associated with in situ downregulation of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein 1 (MCP-1), upregulation of transforming growth factor beta (TGF-beta), and preservation of BBB integrity. Our results indicate that intrathecal delivery of HSV-1-derived vectors containing antiinflammatory cytokine genes may play a major role in the future therapeutic armamentarium of inflammatory CNS-confined demyelinating diseases and, in particular, in the most fulminant forms where conventional therapeutic approaches have, so far, failed to achieve a satisfactory control of the disease evolution.
Asunto(s)
Enfermedades Autoinmunes/genética , Sistema Nervioso Central/metabolismo , Encefalomielitis/genética , Terapia Genética/métodos , Vectores Genéticos , Herpesvirus Humano 1/genética , Interleucina-4/genética , Animales , Enfermedades Autoinmunes/prevención & control , Barrera Hematoencefálica , Encéfalo/patología , División Celular , Quimiocina CCL2/biosíntesis , Citocinas/biosíntesis , Regulación hacia Abajo , Encefalomielitis/prevención & control , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-4/biosíntesis , Macaca mulatta , Imagen por Resonancia Magnética , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/inmunología , Linfocitos T/citología , Factores de Tiempo , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Regulación hacia ArribaRESUMEN
The indole-depleting effects of repeated subcutaneous doses of dexfenfluramine (D-F) (2.5, 5, 10, 20 and 40 mg/kg/day, for four days) in mice were examined with regard to the initial response and time-course of recovery and related to the pharmacokinetics of D-F and its active metabolite dexnorfenfluramine (D-NF). Steady-state plasma and brain concentrations of D-F rose dose-dependently with a metabolite-to-drug ratio averaging 0.4 in brain. This confirmed that in mice D-NF contributes less than in other species to the effects of D-F. Regional serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents were decreased dose-dependently 4 hr after the last injection of D-F. However, two weeks after D-F (2.5-10 mg/kg/day) brain indoles had almost totally recovered, and the long-term effects of the 20 mg/kg/day dose were completely reversed by six weeks, when significant effects are still observable in rats. Although substantial recovery was evident even at 40 mg/kg/day, 5-HT but not 5-HIAA was still slightly reduced nine weeks later. Comparative studies in rats given 2.5-20 mg/kg/day D-F indicated much more severe initial indole depletions than in mice. Brain levels of D-F and D-NF were much higher in rats than in mice. The total active drug brain concentration (D-F + D-NF) was significantly correlated with 5-HT content in both species, with approx 20 nmol/g of total drug causing 50% reduction. These findings point to species differences in D-F kinetics as a main reason for differences in the neurochemical response, supporting the view that the recovery of indoles over time is related to the extent of initial depletion, which in turn depends on critical drug brain concentrations. In view of the qualitative and quantitative species differences in the pharmacodynamics and pharmacokinetics of D-F neither of these rodent species is a suitable model for predicting potential drug toxicity in humans.
Asunto(s)
Encéfalo/efectos de los fármacos , Fenfluramina/administración & dosificación , Inhibidores Selectivos de la Recaptación de Serotonina/administración & dosificación , Serotonina/análisis , Animales , Corteza Cerebral/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fenfluramina/farmacocinética , Fenfluramina/farmacología , Hipocampo/efectos de los fármacos , Ácido Hidroxiindolacético/análisis , Masculino , Ratones , Ratas , Inhibidores Selectivos de la Recaptación de Serotonina/farmacocinética , Inhibidores Selectivos de la Recaptación de Serotonina/farmacologíaRESUMEN
Systemic administration of interferon (IFN)-beta has been recently approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). The immunological mechanism by which IFN-beta ameliorates MS is still partially unknown. We measured the number of blood circulating CD4(+), CD4(-), CD8(+), and CD8(-) T cells secreting IFN-gamma and IL-4 in 26 RRMS patients followed for up to 9 months of an alternate day s.c. treatment with 8x16 IU of IFN-beta1b. Compared to pre-treatment values, a significant (P<0.05) reduction of CD4(+), CD4(-), CD8(+) and CD8(-) cells producing IFN-gamma and of CD4(+) and CD4(-) cells producing IL-4 was observed in MS patients. The IFN-beta-associated effect was evident soon after the beginning of the treatment and persisted for the entire follow-up period. We did not observe any effect of IFN-beta treatment on the percentage of IL-4-producing CD8(+) and CD8(-) cells nor in that of natural killer (NK) cells producing IFN-gamma. Our results show that IFN-beta treatment in MS patients induces a profound and persistent down-regulation of the number of circulating T cells secreting IFN-gamma and IL-4 thus suggesting a broader rather than a specific immunomodulatory effect of IFN-beta in MS.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Interferón beta/administración & dosificación , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Adulto , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Femenino , Humanos , Interferón gamma/inmunología , Interleucina-4/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/inmunología , Receptores de IgG/análisis , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Multiple sclerosis (MS) is considered an immune-mediated disease of the central nervous system (CNS) sustained by a chronic inflammatory process leading to patchy demyelination and axonal loss. However, the inflammatory triggering event as well as the target of the pathogenic process in MS are still partially unknown. We report evidence that a 'local' inflammatory process occurring in the CNS (considered as a reaction of blood vessels in vascularized living tissue to a local injury leading to the accumulation of fluid and blood cells) along with a concomitant, but possibly unrelated, peripheral inflammatory event may trigger a CNS-specific autoimmune reaction cascade sustaining the MS pathogenesis. In the CNS, inflammatory mediators (mainly cytokines) act either as regulatory (i.e. activation of glial cells, shaping the autoimmune response) or effector molecules (i.e. myelinotoxicity, oligodendrotoxicity). In the periphery, inflammatory cytokines induce, in a bystander fashion, activation of monocytes and T cells. Among this latter cell population there are myelin-specific T cells belonging to the normal 'autoimmune' repertoire that home to the CNS where they may trigger the continuous recruitment of effector cells (macrophages) from the periphery. The concept that two concomitant, but possibly unrelated, inflammatory events, occurring in the CNS and in the periphery, represent the crucial elements sustaining MS, might reveal a more comprehensive view (dual signal hypothesis) of the entire etiopathogenic process underlying this disease.
Asunto(s)
Citocinas/fisiología , Esclerosis Múltiple/inmunología , Animales , Encéfalo/inmunología , Humanos , Inflamación/etiología , Mediadores de Inflamación/fisiología , Activación de Linfocitos , Esclerosis Múltiple/etiología , Linfocitos T/inmunologíaRESUMEN
The effects of pretreatment with inducers of hepatic cytochrome P450 isoenzymes (phenobarbital, dexamethasone and beta-naphthoflavone) on the metabolism of d-fenfluramine (d-F) and its acute and long-lasting indole-depleting effects were studied in rats, in an effort to obtain further information on the importance of hepatic drug metabolism in relation to its neurochemical actions. Twenty-four hours after the last dose of each inducer, rats were injected with d-F hydrochloride (5 mg/kg, IP) and killed at various times thereafter for parallel determination of indoles and drug concentrations in plasma and brain. Additional rats were treated as above and killed 1 week after d-F hydrochloride (5 and 10 mg/kg) to study the recovery of indole in the cortex, a particularly sensitive brain area. Phenobarbital and beta-naphthoflavone and, to a lesser degree, dexamethasone, stimulated the metabolism of d-F, as evidenced by a decrease in plasma and brain areas under the curve (AUC) compared to vehicle-treated rats. This indicated that multiple isoenzymes are capable of mediating the drug's metabolism, primarily by N-dealkylation to d-norfenfluramine (d-NF). None of the inducers raised plasma and brain AUC of the nor-derivative, and in fact phenobarbital and particularly beta-naphthoflavone reduced it. These different effects were even apparent in rats given d-NF (2.5 mg/kg), indicating that both phenobarbital and beta-naphthoflavone also stimulate the sequential metabolism of the nor-metabolite (by N-deamintaion) which, however, is apparently enhanced most actively by beta-naphthoflavone-inducible forms of P-450.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Química Encefálica/efectos de los fármacos , Encéfalo/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Fenfluramina/farmacología , Indoles/análisis , Animales , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Fenfluramina/metabolismo , Isoenzimas/metabolismo , Masculino , Fenobarbital/farmacología , Ratas , Factores de TiempoRESUMEN
No studies have examined the pharmacokinetics of isosorbide dinitrate (ISDN) after infusion of long duration, even though such infusions are used in patients. We therefore measured ISDN and its active metabolites, isosorbide-5-mononitrate (IS5MN) and isosorbide-2-mononitrate (IS2MN), in plasma of 9 healthy volunteers who received a continuous intravenous infusion of ISDN for 24 hours at a dose rate that lowered diastolic blood pressure by 10% during the first 30 minutes of infusion. All subjects tolerated the infusion except one who experienced intolerable headache. Five subjects received 1 microgram.min-1.kg-1, one 2 micrograms.min-1.kg-1, and two 4 micrograms.min-1.kg-1 ISDN, whereas the full rate of 6 micrograms.min-1.kg-1 was used continuously in one subject. At all infusion rates the plasma concentrations of ISDN were higher at 24 hours than at earlier times, suggesting that a steady-state condition had not been reached at that time. The same was true for the mononitrate metabolites, which reached higher plasma concentrations and were cleared more slowly than the parent compound after the end of the infusion. Apparent elimination half-lives of ISDN, IS2MN, and IS5MN were 67 +/- 10 minutes, 115 +/- 13 minutes, and 272 +/- 38 minutes, respectively. Comparison of low-rate infusions (1 and 2 micrograms.min-1.kg-1) with high-rate infusions (4 and 6 micrograms.min-1.kg-1) showed that the plasma concentration ratios at 24 hours of mononitrate metabolites to parent drug and apparent plasma clearance of ISDN were almost halved at the higher infusion rates.
Asunto(s)
Dinitrato de Isosorbide/farmacocinética , Vasodilatadores/farmacocinética , Adulto , Humanos , Infusiones Intravenosas , Dinitrato de Isosorbide/administración & dosificación , MasculinoRESUMEN
The effects of oral dexnorfenfluramine (DNF; 1-4 mg/kg, twice daily for 4 days), the active metabolite of dexfenfluramine, were examined on rat regional brain indole contents and [3H]citalopram binding. Two hours after the last dose, serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were dose-dependently lowered at doses above 1.5 mg/kg, with slight regional differences. Cortical 5-HT uptake sites were reduced only at the highest dose. Above 2 mg/kg DNF also caused a more lasting reduction (4 weeks) of regional indoles and cortical 5-HT uptake sites. At this longer time while the decrease in hippocampal 5-HT levels and cortical 5-HT uptake sites remained essentially constant, cortical and striatal 5-HT levels were lowered less than at 2 h, suggesting a return toward control values.
Asunto(s)
Encéfalo/efectos de los fármacos , Fenfluramina/farmacología , Serotonina/metabolismo , Administración Oral , Animales , Encéfalo/metabolismo , Citalopram/farmacología , Fenfluramina/metabolismo , Ácido Hidroxiindolacético/metabolismo , Masculino , RatasRESUMEN
The effects of repeated subcutaneous (s.c) injections of dexfenfluramine (d-F; 10 mg/kg, twice daily, for 4 days) on the contents of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the brain were assessed in primates (cynomolgus and rhesus monkeys) and compared with the regional brain concentrations of unchanged drug and its active metabolite, dexnorfenfluramine (d-NF). This four-day, high-dose, regimen caused a large depletion of 5-HT (more than 95%) and of 5-HIAA (80-90%) in all brain areas studied (cortex, hippocampus, putamen, caudate nucleus and hypothalamus) 2 h after the last injection of d-F. Analysis of the plasma and brain contents of d-F and d-NF confirmed that both compounds were concentrated as in other species, in regions of the primate brain. However, d-NF was concentrated to a greater extent than d-F, and there were differences between the two primate species. Unlike in the rat brain, concentrations of d-NF greatly exceeded those of d-F in the primate brain suggesting that in these primates the d-NF may play a major role in the overall neurochemical response. The effects of d-F and d-NF on different in vitro parameters of serotoninergic neuronal function did not show appreciable differences between cynomolgus or rhesus monkeys when compared to rats, the ability of the two compounds to inhibit 5-HT reuptake, to enhance its release, and to affect the binding of [3H] -d-F or of [3H] -mesulergine (a ligand for 5-HT2C receptors) being similar. Kinetic differences in the disposition of d-F appear to have more relevance than biochemical effects in providing an explanation for the more marked brain depletion induced by d-F in primates than in rodents.
Asunto(s)
Depresores del Apetito/farmacología , Química Encefálica/efectos de los fármacos , Fenfluramina/farmacología , Norfenfluramina/farmacología , Receptores de Serotonina/efectos de los fármacos , Agonistas de Receptores de Serotonina/análisis , Serotonina/análisis , Animales , Depresores del Apetito/metabolismo , Encéfalo/metabolismo , Fenfluramina/sangre , Fenfluramina/metabolismo , Técnicas In Vitro , Macaca fascicularis , Macaca mulatta , Masculino , Norfenfluramina/análogos & derivados , Norfenfluramina/sangre , Ratas , Receptores de Serotonina/metabolismo , Serotonina/análogos & derivados , Serotonina/metabolismo , Serotonina/farmacocinética , Especificidad de la EspecieRESUMEN
A method for intracellular iron labeling of human mononuclear cells (lymphocytes and monocytes) for magnetic resonance imaging (MRI) using simple incubation of cells with approved MRI iron contrast agents is presented. Labeled cells can be detected by MRI in vitro, and this suggests the possibility that the technique could become a marker for in vivo lymphocyte and monocyte trafficking studies in acute inflammatory lesions such as those in Multiple Sclerosis.
Asunto(s)
Medios de Contraste , Hierro , Linfocitos/patología , Imagen por Resonancia Magnética/métodos , Monocitos/patología , Esclerosis Múltiple/diagnóstico , Óxidos , Supervivencia Celular , Células Cultivadas , Dextranos , Óxido Ferrosoférrico , Humanos , Líquido Intracelular , Nanopartículas de Magnetita , Reproducibilidad de los Resultados , SuspensionesRESUMEN
The neuropharmacological effects of repeated oral doses of dexfenfluramine (DF; 1.25-10 mg/kg, twice daily for 21 days) were examined in rats and related to the drug brain levels. Results were compared with fluoxetine (FL) given at similar doses relative to its anorectic ED50. Both drugs dose-dependently slowed body weight gain and reduced brain serotonin (5-HT). However, at 1.25 mg/kg DF caused only a slight and transient decrease in cortical 5-HT. Comparable doses of FL (6.25-12.5 mg/kg) lowered 5-HT more than DF, besides slightly reducing striatal dopamine. At higher doses DF markedly reduced 5-HT in all regions, and to a lesser extent noradrenaline in hippocampus. There was a negative relationship between 5-HT and log total active drug levels and the indole was approximately halved at drug levels about 50 times lower with DF than FL. However, the ratio between drug levels causing marked 5-HT reductions and those considered anorectic was similar for DF and FL because brain levels at the anorectic ED50 were higher with FL than DF. Long-lasting reductions of 5-HT were also observed but recovery was only consistently slow beginning from 5 mg/ kg DF. Comparable doses of FL could not be used because its general toxicity leads to the death of rats after only 2-4 multiples of its anorectic ED50.
Asunto(s)
Depresores del Apetito/farmacología , Fenfluramina/farmacología , Fluoxetina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Administración Oral , Animales , Depresores del Apetito/administración & dosificación , Depresores del Apetito/análisis , Peso Corporal/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Catecolaminas/análisis , Relación Dosis-Respuesta a Droga , Fenfluramina/administración & dosificación , Fenfluramina/sangre , Fluoxetina/administración & dosificación , Fluoxetina/análogos & derivados , Fluoxetina/sangre , Hipocampo/química , Indoles/análisis , Masculino , Norfenfluramina/sangre , Ratas , Serotonina/análisis , Inhibidores Selectivos de la Recaptación de Serotonina/administración & dosificación , Inhibidores Selectivos de la Recaptación de Serotonina/sangre , Estereoisomerismo , Factores de TiempoRESUMEN
Oxygen-free radical formation from either the parent compound amphetamine, its metabolites or drug-released serotonin (5-HT) has been implicated in the reduction of serotoninergic markers caused by amphetamine derivatives. Therefore, the present study investigated the effects of the spin-trapping agent α-phenyl-tert-butyl nitrone (PBN) on the 5-HT-lowering action of dexfenfluramine (DF) in rats, compared with p-chloroamphetamine (PCA). PBN (150 mg/kg, i.p, divided in two doses) almost totally prevented the reduction of 5-HT in particularly sensitive regions of the rat brain (cortex and striatum) 1 and 7 days after DF (10 mg/kg, i.p.). It also provided complete protection against the acute 5-HT-depleting action of PCA (5 mg/kg, i.p.), reducing it at 7 days in striatum, although with the higher dose (300 mg/kg, divided in two doses) there was a tendency to antagonize the long-term effects in both regions. With DF, however, the antagonistic effect of PBN was associated with a marked reduction of the plasma and brain concentrations of the parent drug, but particularly its active metabolite dexnorfenfluramine (DNF). Thus, reduced brain availability of the total active drug (DF+DNF) may explain why PBN prevents the neurochemical effects of DF (but not PCA), including the long-term one which possibly depends on the extent of the initial 5-HT lowering.
RESUMEN
We here present data on immune gene expression of chemokines, chemokine receptors, cytokines and regulatory T-cell (T-reg) markers in chronic patients suffering from either schizophrenia (SCZ, N=20) or bipolar disorder (BD=20) compared with healthy controls (HCs, N=20). We extracted RNA from peripheral blood mononuclear cells and performed real-time (RT)-PCR to measure mRNA levels of chemokines, chemokine receptors, cytokines and T-reg markers. All the analyses were Bonferroni-corrected. The classical monocyte activation (M1) markers il6, ccl3 were significantly increased in BD as compared with both HC and SCZ patients (P=0.03 and P=0.002; P=0.024 and P=0.021, respectively), whereas markers of alternative (M2) monocyte activation ccl1, ccl22 and il10 were coherently decreased (controls: P=0.01, P=0.001 and P=0.09; SCZ subjects: P=0.02, P=0.05 and P=0.011, respectively). Concerning T-cell markers, BD patients had compared with HC downregulated ccr5 (P=0.02) and upregulated il4 (P=0.04) and compared with both healthy and SCZ individuals downregulated ccl2 (P=0.006 and P=0.003) and tgfß (P=0.004 and P=0.007, respectively). No significant associations were found between any immune gene expression and clinical variables (prior hospitalizations, Brief Psychiatric Rating Scale, medications' dosages and lifetime administration). Although some markers are expressed by different immune cell types, these findings suggest a coherent increased M1/decrease M2 signature in the peripheral blood of BD patients with potential Th1/Th2 shift. In contrast, all the explored immune marker levels were preserved in SCZ. Further larger studies are needed to investigate the relevance of inflammatory response in BD, trying to correlate it to psychopathology, treatment and outcome measures and, possibly, to brain connectivity.
Asunto(s)
Trastorno Bipolar/inmunología , Citocinas/inmunología , Monocitos/inmunología , Esquizofrenia/inmunología , Células TH1/inmunología , Células Th2/inmunología , Adulto , Biomarcadores/sangre , Enfermedad Crónica , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , ARN MensajeroRESUMEN
Alzheimer's disease (AD) is characterized by extracellular amyloid-ß (Aß) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Aß species, rather than insoluble fibrils, are the most toxic forms of Aß. Preventing soluble Aß formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of soluble Aß species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Aß forms trafficked to MVs after Aß internalization into microglia. MV neurotoxicity was neutralized by the Aß-interacting protein PrP and anti-Aß antibodies, which prevented binding to neurons of neurotoxic soluble Aß species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Microglía/metabolismo , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Vesículas Transportadoras/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Humanos , Interleucina-1beta/metabolismo , Masculino , Microglía/efectos de los fármacos , Neuronas/citología , Neuronas/metabolismo , Fragmentos de Péptidos/química , Proteínas PrPC/metabolismo , Ratas , Solubilidad , Vesículas Transportadoras/química , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Based on gene expression data, we tested the P8A-CCL2 variant of the chemokine CCL2, able to interfere with the chemotactic properties of the parental molecule, in relapsing-remitting (RR)-EAE SJL. Only preventive treatment significantly delayed disease onset in a dose dependent manner. P8A-CCL2 administration, however, decreased demyelination, axonal loss and number of CNS infiltrating T cells and macrophages. Immunological analysis revealed that P8A-CCL2 does not act on Ag-specific T cell proliferation and does not interfere with the differentiation of IFNgamma-releasing effectors T cells. These results suggest that the therapeutic mechanism of P8A-CCL2 may rely on interference with immune cell recruitment.
Asunto(s)
Quimiocina CCL2/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Vaina de Mielina/efectos de los fármacos , Adulto , Animales , Proliferación Celular/efectos de los fármacos , Quimiocina CCL2/síntesis química , Quimiocina CCL2/uso terapéutico , Quimiotaxis de Leucocito/inmunología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Humanos , Factores Inmunológicos/farmacología , Factores Inmunológicos/uso terapéutico , Interferón gamma/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Vaina de Mielina/inmunología , Vaina de Mielina/patología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Degeneración Walleriana/tratamiento farmacológico , Degeneración Walleriana/inmunología , Degeneración Walleriana/fisiopatologíaRESUMEN
Primary proinflammatory cytokines, such as IL-1beta, play a crucial pathogenic role in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE), and may represent, therefore, a suitable therapeutic target. We have previously established the delivery of anti-inflammatory cytokine genes within the central nervous system (CNS), based on intracisternal (i.c.) injection of non-replicative HSV-1-derived vectors. Here we show the therapeutic efficacy of i.c. administration of an HSV-1-derived vector carrying the interleukin-1receptor antagonist (IL-1ra) gene, the physiological antagonist of the proinflammatory cytokine IL-1, in C57BL/6 mice affected by myelin oligodendrocyte glycoprotein-induced EAE. IL-1ra gene therapy is effective preventively, delaying EAE onset by almost 1 week (22.4+/-1.4 days post-immunization vs 15.9+/-2.1 days in control mice; P=0.0229 log-rank test), and decreasing disease severity. Amelioration of EAE course was associated with a reduced number of macrophages infiltrating the CNS and in a decreased level of proinflammatory cytokine mRNA in the CNS, suggesting an inhibitory activity of IL-1ra on effector cell recruitment, as antigen-specific peripheral T-cell activation and T-cell recruitment to the CNS is unaffected. Thus, local IL-1ra gene therapy may represent a therapeutic alternative for the inhibition of immune-mediated demyelination of the CNS.