RESUMEN
Peripheral viral infection can substantially alter brain function. We have previously shown that intraperitoneal (i.p.) injection of a viral mimetic, polyinosinic-polycytidylic acid (PIC), engenders hyperexcitability of cerebral neurons. Because neuronal activity is invariably associated with their expression of the Cfos gene, the present study was undertaken to determine whether PIC challenge also increases neuronal c-fos protein level. Female C57BL/6 mice were i.p. injected with PIC, and neuronal c-fos was analyzed in the motor cortex by immunohistochemistry. PIC challenge instigated a robust increase in the number of c-fos-positive neurons. This increase reached approximately tenfold over control at 24 h. Also, the c-fos staining intensity of individual neurons increased. AMG-487, a specific inhibitor of the chemokine receptor CXCR3, profoundly attenuated the accumulation of neuronal c-fos, indicating the activation of CXCL10/CXCR3 axis as the trigger of the process. Together, these results show that the accumulation of c-fos is a viable readout to assess the response of cerebral neurons to peripheral PIC challenge, and to elucidate the underlying molecular mechanisms.
Asunto(s)
Neuronas , Proteínas Proto-Oncogénicas c-fos , Animales , Corteza Cerebral/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Poli I-C/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de Quimiocina/metabolismoRESUMEN
Peripheral infections can potently exacerbate neuropathological conditions, though the underlying mechanisms are poorly understood. We have previously demonstrated that intraperitoneal (i.p.) injection of a viral mimetic, polyinosinic-polycytidylic acid (PIC) induces a robust generation of CXCL10 chemokine in the hippocampus. The hippocampus also features hyperexcitability of neuronal circuits following PIC challenge. The present study was undertaken to determine the role of CXCL10 in mediating the development of hyperexcitability in response to PIC challenge. Briefly, young female C57BL/6 mice were i.p. injected with PIC, and after 24 h, the brains were analyzed by confocal microscopy. CXCL10 staining of neuronal perikarya and a less intense staining of the neuropil was observed in the hippocampus and cortex. CXCL10 staining was also evident in a subpopulation of astrocytes, whereas microglia were CXCL10 negative. CXCR3, the cognate receptor of CXCL10 was present exclusively on neurons, indicating that the CXCL10/CXCR3 axis operates through an autocrine/paracrine neuronal signaling. Blocking cerebral CXCR3 through intracerebroventricular injection of a specific inhibitor, AMG487, abrogated PIC challenge-induced increase in basal synaptic transmission and long-term potentiation (LTP), as well as the reduction of paired-pulse facilitation (PPF), in the hippocampus. The PIC-mediated abolishment of hippocampal long-term depression (LTD) was also restored after administration of AMG487. Moreover, CXCR3 inhibition attenuated seizure hypersensitivity induced by PIC challenge. The efficacy of AMG487 strongly strengthens the notion that CXCL10/CXCR3 axis mediates the induction of cerebral hyperexcitability by PIC challenge.
RESUMEN
Peripheral viral infections are potent triggers of exacerbation in multiple sclerosis (MS). Here, we used a preclinical model of MS, the experimental autoimmune encephalomyelitis (EAE) to corroborate this comorbidity in an experimental setting. EAE was induced by immunization of mice with MOG peptide, and paralysis was scored using a 5-point scale. At the onset of the chronic phase of the disease (Days 42-58 after MOG injection) the animals were divided into low responders (LR) and high responders (HR) with the mean score of 1.5 and 2.5, respectively. The acute phase response (APR) was induced by intraperitoneal injections of a viral mimetic, polyinosinic-polycytidylic acid (PIC). Two daily injections were performed on Days 42 and 44 (PIC42,44 challenge) and on Days 54, 55 and 56 (PIC54,55,56 challenge). PIC42,44 challenge had no effect of EAE disease, whereas PIC54,55,56 challenge rapidly increased paralysis but only in HR group. This exacerbation ultimately led to animal death by Day 58. These results demonstrate that antiviral APR is a potent exacerbator of EAE, and that this activity directly correlates with the severity of the disease. This in turn, indicates that antiviral APR might play a pivot role in linking peripheral viral infections with MS exacerbations.
Asunto(s)
Reacción de Fase Aguda/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/inmunología , Glicoproteína Mielina-Oligodendrócito/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Ratones Endogámicos C57BL , Fragmentos de Péptidos/inmunologíaRESUMEN
Peripheral viral infections increase seizure propensity and intensity in susceptible individuals. We have modeled this comorbidity by demonstrating that the acute phase response instigated by an intraperitoneal (i.p.) injection of a viral mimetic, polyinosinic-polycytidylic acid (PIC), induces protracted hypersusceptibility to kainic acid-induced seizures. We have further demonstrated that PIC challenge robustly increases the level of tonic extracellular glutamate and neuronal excitability in the hippocampus. This study was undertaken to determine a relationship between tonic glutamate and seizure susceptibility following PIC challenge. Briefly, glutamate-sensing microelectrodes were permanently implanted into the CA1 of 8-week-old female C57BL/6 mice. Following a 3-day recovery, acute phase response was induced by i.p. injection of 12 mg/kg of PIC, while saline-injected mice served as controls. Tonic glutamate was measured at 1, 2, 3 and 4 days after PIC challenge. PIC challenge induced an approximately fourfold increase in tonic glutamate levels measured after 24 h. The levels gradually declined to the baseline values within 4 days. Twenty-four hours after PIC challenge, the mice featured an approximately threefold increase in cumulative seizure scores and twofold increase in the duration of status epilepticus induced by subcutaneous injection of 12 mg/kg of kainic acid. Seizure scores positively correlated with pre-seizure tonic glutamate. Moreover, seizures resulted in a profound (76%) elevation of extracellular glutamate in the CA1 of PIC-challenged but not saline-injected mice. Our results implicate the increase in extracellular glutamate as a mediator of seizure hypersusceptibility induced by peripheral viral challenge.
Asunto(s)
Espacio Extracelular/metabolismo , Glutamatos/metabolismo , Hipocampo/metabolismo , Hipocampo/virología , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/virología , Poli I-C/toxicidad , Convulsiones/metabolismo , Convulsiones/virología , Reacción de Fase Aguda , Animales , Peso Corporal/efectos de los fármacos , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Electrodos Implantados , Agonistas de Aminoácidos Excitadores , Femenino , Ácido Kaínico , Ratones , Ratones Endogámicos C57BL , Convulsiones/inducido químicamente , Estado Epiléptico/inducido químicamente , Estado Epiléptico/metabolismo , Estado Epiléptico/virologíaRESUMEN
Peripheral infections increase the propensity and severity of seizures in susceptible populations. We have previously shown that intraperitoneal injection of a viral mimic, polyinosinic-polycytidylic acid (PIC), elicits hypersusceptibility of mice to kainic acid (KA)-induced seizures. This study was undertaken to determine whether this seizure hypersusceptibility entails alterations in glutamate signaling. Female C57BL/6 mice were intraperitoneally injected with PIC, and after 24 h, glutamate homeostasis in the hippocampus was monitored using the enzyme-based microelectrode arrays. PIC challenge robustly increased the level of resting extracellular glutamate. While pre-synaptic potassium-evoked glutamate release was not affected, glutamate uptake was profoundly impaired and non-vesicular glutamate release was augmented, indicating functional alterations of astrocytes. Electrophysiological examination of hippocampal slices from PIC-challenged mice revealed a several fold increase in the basal synaptic transmission as compared to control slices. PIC challenge also increased the probability of pre-synaptic glutamate release as seen from a reduction of paired-pulse facilitation and synaptic plasticity as seen from an enhancement of long-term potentiation. Altogether, our results implicate a dysregulation of astrocytic glutamate metabolism and an alteration of excitatory synaptic transmission as the underlying mechanism for the development of hippocampal hyperexcitability, and consequently seizure hypersusceptibility following peripheral PIC challenge. Peripheral infections/inflammations enhance seizure susceptibility. Here, we explored the effect of peritoneal inflammation induced by a viral mimic on glutamate homeostasis and glutamatergic neurotransmission in the mouse hippocampus. We found that peritoneal inflammation elevated extracellular glutamate concentration and enhanced the probability of pre-synaptic glutamate release resulting in hyperexcitability of neuronal networks. These mechanisms are likely to underlie the enhanced seizure propensity.
Asunto(s)
Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Transmisión Sináptica/fisiología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Hipocampo/efectos de los fármacos , Ácido Kaínico/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Ratones Endogámicos C57BL , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
Peripheral challenge with a viral mimetic, polyinosinic-polycytidylic acid (PIC) induces hippocampal hyperexcitability in mice. Here, we characterized this hippocampal response through a whole genome transcriptome analysis. Intraperitoneal injection of PIC resulted in temporal dysregulation of 625 genes in the hippocampus, indicating an extensive genetic reprogramming. The bioinformatics analysis of these genes revealed the complement pathway to be the most significantly activated. The gene encoding complement factor B (CfB) exhibited the highest response, and its upregulation was commensurate with the development of hyperexcitability. Collectively, these results suggest that the induction of hippocampal hyperexcitability may be mediated by the alternative complement cascades.
Asunto(s)
Antivirales/farmacología , Activación de Complemento/fisiología , Hipocampo/fisiología , Imitación Molecular/fisiología , Poli I-C/farmacología , Regulación hacia Arriba/fisiología , Animales , Activación de Complemento/efectos de los fármacos , Regulación de la Expresión Génica , Hipocampo/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Imitación Molecular/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
We have previously shown that peripherally restricted acute phase response (APR) elicited by intraperitoneal (i.p.) injection of a viral mimic, polyinosinic-polycytidylic acid (PIC), renders the brain hypersusceptible to excitotoxic insult as seen from profoundly exacerbated kainic acid (KA)-induced seizures. In the present study, we found that this hypersusceptibility was protracted for up to 72 h. RT-PCR profiling of hippocampal gene expression revealed rapid upregulation of 23 genes encoding cytokines, chemokines and chemokine receptors generally within 6 h after PIC challenge. The expression of most of these genes decreased by 24 h. However, two chemokine genes, i.e., Ccl19 and Cxcl13 genes, as well as two chemokine receptor genes, Ccr1 and Ccr7, remained upregulated for 72 h suggesting their possible involvement in the induction and sustenance of seizure hypersusceptibility. Also, 12 genes encoding proteins related to glutamatergic and GABAergic neurotransmission featured initial upregulation or downregulation followed by gradual normalization. The upregulation of the Gabrr3 gene remained upregulated at 72 h, congruent with its plausible role in the hypersusceptible phenotype. Moreover, the expression of ten microRNAs (miRs) was rapidly affected by PIC challenge, but their levels generally exhibited oscillating profiles over the time course of seizure hypersusceptibility. These results indicate that protracted seizure susceptibility following peripheral APR is associated with a robust polygenic response in the hippocampus.
Asunto(s)
Reacción de Fase Aguda , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Proteínas del Tejido Nervioso/biosíntesis , Reacción de Fase Aguda/genética , Citocinas/biosíntesis , Citocinas/sangre , Citocinas/genética , Citocinas/metabolismo , Susceptibilidad a Enfermedades , Relación Dosis-Respuesta a Droga , Conducta Exploratoria/efectos de los fármacos , Hipocampo/metabolismo , Conducta de Enfermedad/efectos de los fármacos , Inflamación/genética , Ácido Kaínico , MicroARNs/biosíntesis , Imitación Molecular , Proteínas del Tejido Nervioso/genética , Neuroinmunomodulación/genética , Neurotoxinas/toxicidad , Poli I-C/farmacología , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/genética , Receptores de Citocinas/biosíntesis , Receptores de Citocinas/genética , Convulsiones/sangre , Convulsiones/inducido químicamente , Convulsiones/genética , Transmisión Sináptica/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Inflammatory factors associated with immune challenge during early brain development are now firmly implicated in the etiologies of schizophrenia, autism and mood disorders later in life. In rodent models, maternal injections of inflammagens have been used to induce behavioral, anatomical and biochemical changes in offspring that are congruent with those found in human diseases. Here, we studied whether inflammatory challenge during the early postnatal period can also elicit behavioral alterations in adults. At postnatal day 14, rats were intraperitoneally injected with a viral mimic, polyinosinic:polycytidylic acid (PIC). Two months later, these rats displayed remarkably robust and consistent anxiety-like behaviors as evaluated by the open field/defensive-withdrawal test. These results demonstrate that the window of vulnerability to inflammatory challenge in rodents extends into the postnatal period and offers a means to study the early sequelae of events surrounding immune challenge to the developing brain.
Asunto(s)
Trastornos de Ansiedad , Ansiedad , Inflamación/metabolismo , Poli I-C/administración & dosificación , Animales , Animales Lactantes , Ansiedad/inducido químicamente , Ansiedad/inmunología , Trastornos de Ansiedad/etiología , Trastornos de Ansiedad/inmunología , Trastorno Autístico/etiología , Trastorno Autístico/inmunología , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/inmunología , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Esquizofrenia/etiología , Esquizofrenia/inmunologíaRESUMEN
Clinical evidence implicates peripheral inflammatory diseases as comorbid factors in epilepsy. The present study was designed to determine the effect of the acute phase of antiviral response on seizure susceptibility. Young adult mice were intraperitoneally injected with 12 mg/kg of a viral mimic, polyinosinic:polycytidylic acid (PIC). After 48 h, seizures were induced by subcutaneous injection of kainic acid (KA). PIC-pretreatment profoundly enhances vulnerability to excitotoxic insult as evidenced by increased seizure intensity and extended duration of status epilepticus. These results support the notion that peripheral viral infections may alter brain function resulting in enhanced predilection to seizures.
Asunto(s)
Encéfalo/metabolismo , Ácido Kaínico/administración & dosificación , Poli I-C/administración & dosificación , ARN Bicatenario/administración & dosificación , Convulsiones , Estado Epiléptico , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Convulsiones/inducido químicamente , Convulsiones/inmunología , Estado Epiléptico/inducido químicamente , Estado Epiléptico/inmunologíaRESUMEN
Previously, we have shown that peripheral challenge of mice with double stranded RNA (dsRNA), a viral mimic, evokes global upregulation of cerebral inflammatory genes and, particularly, genes encoding chemokines. Because chemokine networks are potent modulators of brain function, the present study was undertaken to comprehensively characterize the cerebral response of chemokine ligand and receptor genes to peripheral immune system stimulation. Briefly, C57BL/6 mice were intraperitoneally injected with 12 mg/kg of polyinosinic-polycytidylic acid (PIC) and the expression of 39 mouse chemokine ligand and 20 receptor genes was monitored in the cerebellum by real time quantitative RT-PCR within 24 h. Almost half of the ligand genes featured either transient or sustained upregulation from several- to several thousand-fold. Five CXC type genes, i.e., Cxcl9, Cxcl11, Cxcl10, Cxcl2 and Cxcl1, were the most robustly upregulated, and were followed by six CC type genes, i.e., Ccl2, Ccl7, Ccl5, Ccl12, Ccl4 and Ccl11. Seven genes showed moderate upregulation, whereas the remaining genes were unresponsive. Six receptor genes, i.e., Cxcr2, Ccr7, Cxcr5, Ccr6, Ccr1 and Ccr5, featured a several-fold upregulation. Similar chemokine gene response was observed in the forebrain and brainstem. This upregulation of chemokine genes could be induced in naïve mice by transfer of blood plasma from PIC-challenged mice. Employing oligodeoxynucleotide-labeled PIC we further showed that intraperitoneally injected PIC was not transferred to the blood. In conclusion, peripheral PIC challenge elicits a broad upregulation of cerebral chemokine genes, and this upregulation is mediated by blood-borne agents.
Asunto(s)
Encéfalo/metabolismo , Quimiocinas CC/metabolismo , Quimiocinas CXC/metabolismo , Mediadores de Inflamación , Poli I-C/administración & dosificación , Animales , Quimiocinas CC/genética , Quimiocinas CXC/genética , Modelos Animales de Enfermedad , Femenino , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Cholesterol and sulfatides play many important roles in learning and memory. To date, our observations about the effects of cholesterol on learning have been assessed during response acquisition; that is, the learning of a new memory. Here, we report for the first time to our knowledge, on the effect of a cholesterol diet on a previously formed memory. Rabbits were given trace conditioning of the nictitating membrane response for 10 days, then fed a 2% cholesterol diet for 8 weeks, and then assessed for memory recall of the initially learned task. We show that dietary cholesterol had an adverse effect on memory recall. Second, we investigated whether dietary cholesterol caused an increase in brain cholesterol and sulfatide levels in four major brain structures (hippocampus, frontal lobe, brainstem, and cerebellum) using a technique for analyzing myelin and myelin-free fractions separately. Although our data confirm previous findings that dietary cholesterol does not directly affect cholesterol and establish that it does not affect sulfatide levels in the brain, these levels did increase rather significantly in the hippocampus and frontal lobe as a function of learning and memory.
Asunto(s)
Encéfalo/metabolismo , Colesterol en la Dieta/efectos adversos , Colesterol/metabolismo , Trastornos de la Memoria/etiología , Memoria/fisiología , Sulfoglicoesfingolípidos/metabolismo , Animales , Condicionamiento Clásico/fisiología , Masculino , Memoria/efectos de los fármacos , Trastornos de la Memoria/patología , Membrana Nictitante/fisiopatología , Conejos , Fracciones Subcelulares/metabolismoRESUMEN
Intensive lifestyle changes have been shown to regress atherosclerosis, improve cardiovascular risk profiles, and decrease angina pectoris and cardiac events. We evaluated the influence of the Multisite Cardiac Lifestyle Intervention Program, an ongoing health insurance-covered lifestyle intervention conducted at our site, on endothelial function and inflammatory markers of atherosclerosis in this pilot study. Twenty-seven participants with coronary artery disease (CAD) and/or risk factors for CAD (nonsmokers, 14 men; mean age 56 years) were enrolled in the experimental group and asked to make changes in diet (10% calories from fat, plant based), engage in moderate exercise (3 hours/week), and practice stress management (1 hour/day). Twenty historically (age, gender, CAD, and CAD risk factors) matched participants were enrolled in the control group with usual standard of care. At baseline endothelium-dependent brachial artery flow-mediated dilatation (FMD) was performed in the 2 groups. Serum markers of inflammation, endothelial dysfunction, and angiogenesis were performed only in the experimental group. After 12 weeks, FMD had improved in the experimental group from a baseline of 4.23 + or - 0.13 to 4.65 + or - 0.15 mm, whereas in the control group it decreased from 4.62 + or - 0.16 to 4.48 + or - 0.17 mm. Changes were significantly different in favor of the experimental group (p <0.0001). Also, significant decreases occurred in C-reactive protein (from 2.07 + or - 0.57 to 1.6 + or - 0.43 mg/L, p = 0.03) and interleukin-6 (from 2.52 + or - 0.62 to 1.23 + or - 0.3 pg/ml, p = 0.02) after 12 weeks. Significant improvement in FMD, C-reactive protein, and interleukin-6 with intensive lifestyle changes in the experimental group suggests > or = 1 potential mechanism underlying the clinical benefits seen in previous trials.
Asunto(s)
Aterosclerosis/sangre , Aterosclerosis/terapia , Proteína C-Reactiva/metabolismo , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/prevención & control , Endotelio Vascular/metabolismo , Conducta de Reducción del Riesgo , Angina de Pecho/sangre , Angina de Pecho/prevención & control , Biomarcadores/sangre , Arteria Braquial/fisiopatología , Estudios de Casos y Controles , Ejercicio Físico , Femenino , Conductas Relacionadas con la Salud , Humanos , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo III/sangre , Proyectos Piloto , Calidad de Vida , Factores de Riesgo , Estrés Psicológico/prevención & control , Factores de Tiempo , VasodilataciónRESUMEN
Cyclic AMP is part of an endogenous mechanism that downregulates inflammatory response, and its intracellular concentration is regulated chiefly by cyclic nucleotide phosphodiesterases type 4. The goal of the present study was to determine whether phosphodiesterases 4 are involved in the inflammatory response of astrocytes mediated by Toll-like receptors. Astrocyte cultures established from newborn rat brain were challenged with lipoteichoic acid, a ligand of Toll-like receptor 2, polyinosinic-polycytidylic acid, a ligand of Toll-like receptor 3, or lipopolysaccharide, a ligand of Toll-like receptor 4. After 24 h the expression of genes encoding phosphodiesterase 4A, phosphodiesterase 4B and phosphodiesterase 4D was determined by real time reverse transcription polymerase chain reaction. The challenge of astrocytes with the ligands profoundly up-regulated expression of the phosphodiesterase 4B mRNA, while the phosphodiesterase 4A and 4D mRNA was either unaffected or downregulated. Moreover, Toll-like receptor ligation specifically up-regulated expression of the phosphodiesterase 4B2 transcriptional variant. Thus, polyinosinic-polycytidylic acid, lipopolysaccharide and lipoteichoic acid induced approximately 7-, 5- and 4-fold up-regulation of the message, respectively. Toll-like receptor ligation also led to an over 2-fold increase in the protein level of phosphodiesterase 4B2 as revealed by immunoblot analysis. The inactivation of Rho proteins by pretreatment with toxin B form C. difficile enhanced ligation-induced up-regulation of the phosphodiesterase 4B2 message by 4-9-fold. However, in spite of this increase in the message abundance, there was no increase in the protein level compared to cells challenged with the ligands alone. These results demonstrate that the phosphodiesterase 4B2 gene is an effector of Toll-like receptor signaling in astrocytes, and that its up-regulation at the protein level is controlled by complex mechanisms.
Asunto(s)
Astrocitos/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/fisiología , Receptores Toll-Like/fisiología , Animales , Western Blotting , Clostridium botulinum tipo B , Femenino , Variación Genética , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/fisiología , Isoenzimas/genética , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/fisiología , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Endogámicas Lew , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/fisiología , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/fisiologíaRESUMEN
The family of Toll-like receptors (TLRs) expressed by innate immune cells recognizes a spectrum of microbial components as well as molecules released from injured tissues. TLR ligation activates intracellular signaling cascades that culminate in the up-regulation of proinflammatory genes. We have recently demonstrated that the up-regulation of inflammatory cytokines mediated by TLR4 in astrocytes is negatively controlled by the monomeric GTPases of Rho subfamily. The present study was undertaken to examine further the involvement of Rho proteins in the inflammatory response of astrocytes elicited by the ligation of three TLRs that use divergent signaling pathways. Astrocyte cultures established from newborn rat brains were challenged with ligands of TLR2, TLR3, and TLR4. The expression of genes encoding interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNFalpha), interferon-beta (IFNbeta), and inducible nitric oxide synthase (NOS2) was up-regulated 24 hr after the challenge as determined by real-time RT-PCR. Pretreatment of the cells with toxin B, which specifically inactivates Rho proteins, enhanced the up-regulation of gene expression. The extent of this enhancement was both receptor and gene dependent. The enhancing effect of Rho protein inactivation was also evident at the protein level of IL-6 and NOS2 as revealed by ELISA and immunoblot analyses, respectively. These results suggest that Rho proteins control TLR-mediated up-regulation of inflammatory genes in astrocytes by interfering with multiple events along the signaling pathways.
Asunto(s)
Astrocitos/fisiología , Transducción de Señal/fisiología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Proteínas Bacterianas/farmacología , Toxinas Bacterianas/farmacología , Células Cultivadas , Interferón beta/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Mensajero/metabolismo , Ratas , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Proteínas de Unión al GTP rho/antagonistas & inhibidoresRESUMEN
It is well established that mediators of peripheral inflammation are relayed to the brain and elicit sickness behavior via neuroinflammatory agents that target neuronal substrates. In the present study, we used double-stranded RNA (dsRNA), a viral replication intermediate, to mimic the acute phase of viral infection. C57BL/6 mice were injected intraperitoneally with 12 mg/kg of synthetic dsRNA, i.e., polyinosinic-polycytidylic acid (PIC). The treatment induced severe sickness behavior in the animals as revealed by the burrowing test performed 6 hr postinjection. PIC challenge also induced up-regulation of mRNA for several cytokines in the brain as determined by real-time quantitative RT-PCR. In all brain regions, i.e., the forebrain, brainstem, and cerebellum, the gene encoding the CXCL2 chemokine featured the most robust up-regulation over the basal level (saline-injected animals), followed by the genes encoding the CCL2 chemokine, interferon-beta (IFNbeta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFalpha), and interleukin-1beta (IL-1beta). The forebrain featured the highest extent of up-regulation of the Ifnb gene, whereas the other genes attained the highest expression in the cerebellum. Most of the genes featured transient up-regulation, with peaks occurring 3-6 hr after PIC challenge. The TNFalpha, CCL2, CXCL2, IFNbeta, and IL-1beta messages remained profoundly up-regulated even at 24 hr. The expression of genes encoding inducible and neuronal nitric oxide synthase (NOS) in the brain was not affected by the peripheral PIC challenge. However, the endothelial NOS message was initially down-regulated and subsequently up-regulated, indicating stimulation of cerebral vasculature.
Asunto(s)
Encéfalo/metabolismo , Citocinas/genética , Poli I-C/administración & dosificación , ARN Bicatenario/administración & dosificación , Regulación hacia Arriba , Animales , Enfermedades Virales del Sistema Nervioso Central/inmunología , Quimiocina CCL2/metabolismo , Quimiocina CXCL2/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Interferón beta/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
This investigation is a follow-up to our previous in vivo studies revealing that rapid stretch increases tissue insulin in murine skin flaps, coincident with the up-regulation of key angiogenic effectors and enhanced vascularization. In the present study, we used human umbilical vein endothelial cells (HUVECs) as an in vitro model system to determine the role of insulin in the chemical signals regulating the processes of proliferation and viability (survival). MTT-based colorimetric methods demonstrated that insulin enhances proliferation and survival of HUVECs. Western blot analysis revealed that protein kinase B (pAkt [Thr(308)]) and vascular endothelial growth factor (VEGF) were the insulin-responsive intermediates in proliferating endothelial cells (ECs). In insulin-enhanced survival, both pAkt (Thr(308)) and pAkt (Ser(473)) were activated in HUVECs. However, no change in VEGF expression accompanied pAkt activation. The beneficial effects of insulin were abrogated by insulin receptor (IR)/insulin-like growth factor receptor (IGFR) or phosphoinositide-3 kinase (PI3-K) blockade, suggesting that insulin-induced EC proliferation and viability are mediated through pIR/pIGFR and PI3-K effectors. These data provide new insights into the beneficial effects of insulin on vascularization and tissue viability, providing a mechanistic link to the enhancement of healing in acutely stretched skin.
Asunto(s)
Proliferación Celular , Endotelio Vascular/citología , Insulina/fisiología , Venas Umbilicales/citología , Supervivencia Celular , Células Cultivadas , Endotelio Vascular/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Somatomedina/metabolismo , Transducción de Señal/fisiología , Venas Umbilicales/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Elimination of hydrogen sulfide from glutathione (GSH) converts a well known cellular nucleophile to an electrophilic species, gamma-glutamyldehydroalanylglycine (EdAG). We have found that a sulfonium metabolite formed from GSH and busulfan undergoes a facile beta-elimination reaction to give EdAG, which is an alpha,beta-unsaturated dehydroalanyl analog of GSH. EdAG was identified as a metabolite of busulfan in a human liver cytosol fraction. EdAG condenses with GSH in a Michael addition reaction to produce a lanthionine thioether [(2-amino-5-[[3-[2-[[4-amino-5-hydroxy-5-oxopentanoyl]amino]-3-(carboxymethylamino)-3-oxopropyl]sulfanyl-1-(carboxymethylamino)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid); GSG], which is a nonreducible analog of glutathione disulfide. EdAG was less cytotoxic than busulfan to C6 rat glioma cells. GSH and EdAG were equally effective in displacing a glutathione S-transferase (GST) isozyme (human GSTA1-1) from a GSH-agarose column. The finding of an electrophilic metabolite of GSH suggests that alteration of cellular GSH concentrations, irreversible nonreducible glutathionylation of proteins, and interference with GST function may contribute to the toxicity of busulfan.
Asunto(s)
Busulfano/metabolismo , Glutatión Transferasa/metabolismo , Glutatión/análogos & derivados , Glutatión/metabolismo , Alanina/análogos & derivados , Animales , Antineoplásicos Alquilantes , Busulfano/farmacología , Línea Celular Tumoral , Glioma/tratamiento farmacológico , Disulfuro de Glutatión/análogos & derivados , Humanos , Hígado , RatasRESUMEN
Although cognitive dysfunction manifested by severe memory and attention deficits has been reported in up to 70% of cancer patients undergoing chemotherapy, the mechanisms of this serious side effect have not been defined. In particular, it has not been decisively resolved whether the dysfunction is attributable to the chemotherapy or to the malignancy itself. In the present study we tested whether cognitive dysfunction can be induced in an experimental setting by the administration of commonly used chemotherapeutics to rats. Female 10 month old Sprague-Dawley rats were injected intraperitoneally with a combination of 2.5 mg/kg of adriamycin (ADR) and 25 mg/kg of cytoxan (CTX). A total of four doses were given at weekly intervals. The control group was treated with saline only. No mortality and no apparent morbidity were observed in either group. However, the chemotherapeutic treatment severely impaired memory function of rats as measured by a passive avoidance test. This memory deficiency was fully prevented by the administration of an antioxidant, N-acetyl cysteine (NAC) injected subcutaneously three times a week at 200 mg/kg in the course of chemotherapeutic treatment. These results indicate that chemotherapeutic agents alone, i.e., in the absence of malignancy, damage the brain resulting in memory dysfunction. Moreover, the results strongly indicate that the damaging effect is mediated by oxidative stress, as memory dysfunction is preventable by the co-administration of NAC.
Asunto(s)
Antineoplásicos/toxicidad , Trastornos del Conocimiento/inducido químicamente , Trastornos del Conocimiento/psicología , Animales , Antibióticos Antineoplásicos/toxicidad , Antineoplásicos Alquilantes/toxicidad , Antioxidantes/uso terapéutico , Reacción de Prevención/efectos de los fármacos , Trastornos del Conocimiento/prevención & control , Ciclofosfamida/toxicidad , Doxorrubicina/toxicidad , Femenino , Memoria a Corto Plazo/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Toll-like receptors (TLRs) are sentinels of innate immunity that recognize pathogenic molecules and trigger inflammatory response. Because inflammatory mediators are detrimental to the host, the TLR response is regulated by feedback inhibition. Statins, the inhibitors of isoprenoid biosynthesis, have been shown to be potent modulators of TLR activity, and this modulation may provide insight regarding mechanisms of the feedback inhibition. In the present study, we examined feedback mechanisms that regulate TLR4 activity in astrocytes using statins to perturb postligational signaling. Astrocytic cultures established from newborn rat brains were exposed to lipopolysaccharide (LPS), the ligand for TLR4. The up-regulation of expression of genes encoding interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNFalpha) was determined by real-time RT-PCR. Pretreatment of the cells with either atorvastatin or simvastatin enhanced the LPS-induced up-regulation of cytokine gene expression. The most profound enhancement of approximately 17-fold was observed for the Il-6 gene. The enhancements for the Tnfa and Il-1b genes were approximately 5- and 3.5-fold, respectively. Mevalonate fully reversed the effects of statins, indicating that these drugs act through the inhibition of isoprenoid synthesis. The inhibition of protein geranylgeranylation, but not protein farnesylation, mimicked the effects of statins, strongly indicating that the enhancement is mediated by the Rho proteins. In support of this notion, pretreatment of cells with toxin B, a specific inhibitor of the Rho proteins, also enhanced LPS-triggered up-regulation of the cytokine genes. These results indicate that the Rho proteins are involved in the activation of negative feedback inhibition of TLR4 signaling in astrocytes.
Asunto(s)
Astrocitos/metabolismo , Citocinas/genética , Expresión Génica/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Receptor Toll-Like 4/metabolismo , Animales , Astrocitos/efectos de los fármacos , Proteínas Bacterianas/farmacología , Toxinas Bacterianas/farmacología , Células Cultivadas , Retroalimentación Fisiológica , Lipopolisacáridos/farmacología , Ácido Mevalónico/farmacología , Prenilación de Proteína/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Tumor suppressor gene BRCA1 is frequently mutated in familial breast and ovarian cancer. BRCA1 plays pivotal roles in maintaining genomic stability by interacting with numerous proteins in cell cycle control and DNA repair. Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863) is one of a new class of anticancer agents that are analogs of mushroom-derived illudin toxins. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor cell types. The exact nature of irofulven-induced DNA damage is not completely understood. We demonstrated previously that irofulven activates ATM and its targets, NBS1, SMC1, CHK2, and p53. In this study, we hypothesize that irofulven induces DNA double-strand breaks and that BRCA1 may affect chemosensitivity by controlling cell cycle checkpoints, DNA repair, and genomic stability in response to irofulven treatment. We observed that irofulven induces the formation of chromosome breaks and radials and the activation and foci formation of gamma-H2AX, BRCA1, and RAD51. We also provided evidence that irofulven induces the generation of DNA double-strand breaks. By using BRCA1-deficient or -proficient cells, we demonstrated that in response to irofulven, BRCA1 contributes to the control of S and G(2)/M cell cycle arrest and is critical for repairing DNA double-strand breaks and for RAD51-dependent homologous recombination. Furthermore, we found that BRCA1 deficiency results in increased chromosome damage and chemosensitivity after irofulven treatment.