RESUMO
OBJECTIVES: We have described cerebrospinal fluid (CSF) myeloid microvesicles (MVs) as a marker of microglia activation during neuroinflammation in Alzheimer disease (AD), and characterized their ability to produce toxic amyloid ß1-42 (Aß1-42 ) oligomers from aggregated or soluble substrate. The aim of this study is to investigate the association of CSF myeloid MVs with neuroimaging, clinical, and paraclinical data in AD and mild cognitive impairment (MCI). METHODS: We collected CSF from 106 AD patients, 51 MCI patients, and 29 neurologically healthy controls. We examined CSF myeloid MV content and AD markers. A subgroup of 34 AD and 21 MCI patients underwent structural and diffusion tensor MRI. RESULTS: Higher levels of myeloid MVs were found in the CSF of AD patients and MCI patients converting within 3 years relative to controls, but also, at a lower level, in MCI patients not converting to AD. CSF myeloid MVs were associated with Tau but not with Aß1-42 CSF levels. CSF MVs levels correlated with white matter (WM) tract damage in MCI, and with hippocampal atrophy in AD. INTERPRETATION: Microglial MVs are neurotoxic and myelinotoxic in the presence of Aß1-42 . CSF myeloid MVs, mirroring microglia activation and MV release, are associated with WM damage in MCI and hippocampal atrophy in AD. This suggests that hippocampal microglia activation, in the presence of Aß1-42 in excess, produces neurotoxic and oligodendrotoxic oligomers that, through WM tract damage, spread disease to neighboring and connected areas, causing local microglia activation and propagation of disease through the same sequence of events. Ann Neurol 2014;76:813-825.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Disfunção Cognitiva/líquido cefalorraquidiano , Bainha de Mielina/metabolismo , Células Mieloides/metabolismo , Neurônios/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico , Biomarcadores/líquido cefalorraquidiano , Contagem de Células/métodos , Disfunção Cognitiva/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Bainha de Mielina/patologia , Células Mieloides/patologia , Neurônios/patologiaRESUMO
Neuroplasticity is essential to prevent clinical worsening despite continuing neuronal loss in several brain diseases, including multiple sclerosis (MS). The precise nature of the adaptation mechanisms taking place in MS brains, ensuring protection from disability appearance and accumulation, is however unknown. Here, we explored the hypothesis that long-term synaptic potentiation (LTP), potentially able to minimize the effects of neuronal loss by providing extra excitation of denervated neurons, is the most relevant form of adaptive plasticity in stable MS patients, and it is disrupted in progressing MS patients. We found that LTP, explored by means of transcranial magnetic theta burst stimulation over the primary motor cortex, was still possible, and even favored, in stable relapsing-remitting (RR-MS) patients, whereas it was absent in individuals with primary progressive MS (PP-MS). We also provided evidence that platelet-derived growth factor (PDGF) plays a substantial role in favoring both LTP and brain reserve in MS patients, as this molecule: (1) was reduced in the CSF of PP-MS patients, (2) enhanced LTP emergence in hippocampal mouse brain slices, (3) was associated with more pronounced LTP in RR-MS patients, and (4) was associated with the clinical compensation of new brain lesion formation in RR-MS. Our results show that brain plasticity reserve, in the form of LTP, is crucial to contrast clinical deterioration in MS. Enhancing PDGF signaling might represent a valuable treatment option to maintain brain reserve and to attenuate the clinical consequences of neuronal damage in the progressive phases of MS and in other neurodegenerative disorders.
Assuntos
Esclerose Múltipla/fisiopatologia , Plasticidade Neuronal/fisiologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Transdução de Sinais/fisiologia , Sinapses/fisiologia , Adulto , Animais , Encéfalo/fisiologia , Córtex Cerebral/fisiologia , Progressão da Doença , Estimulação Elétrica , Fenômenos Eletrofisiológicos , Potenciais Evocados/fisiologia , Feminino , Humanos , Potenciação de Longa Duração/fisiologia , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla Crônica Progressiva/líquido cefalorraquidiano , Esclerose Múltipla Crônica Progressiva/fisiopatologia , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/fisiopatologia , Fator de Crescimento Derivado de Plaquetas/líquido cefalorraquidiano , Ritmo Teta/fisiologia , Estimulação Magnética TranscranianaRESUMO
BACKGROUND: Chronic inflammation leads to gray matter damage in progressive multiple sclerosis (MS), but the mechanism linking inflammation and neurodegeneration is unclear. OBJECTIVE: The objective of this paper is to investigate the synaptic mechanism of inflammatory neurodegeneration in progressive forms of MS. METHODS: Cytokine and neurofilament-light were determined in cerebrospinal fluid (CSF) of MS patients. In vitro electrophysiology and cell swelling experiments were performed to measure the effects of inflammatory cytokines in the CSF of MS patients on synaptic transmission and neuronal integrity. RESULTS: Tumor necrosis factor-α (TNF) was higher in CSF of progressive MS subjects, and caused excitotoxic neuronal death in vitro. In murine brain slices incubated in the presence of CSF from progressive MS, in fact, we observed increased spontaneous excitatory postsynaptic currents (sEPSCs) and glutamate-mediated neuronal swelling through a mechanism dependent on enhanced TNF signaling. We also suggested a pathogenic role of B cells in TNF CSF increase, exacerbation of glutamatergic transmission and neuronal damage, since CNS depletion of B cells with intrathecal rituximab caused a dramatic reduction of TNF levels, of TNF-induced sEPSC alterations, and of neurofilament CSF concentrations in a patient with progressive MS. CONCLUSION: Our results point to TNF as a primary neurotoxic molecule in progressive forms of MS.
Assuntos
Encéfalo/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Esclerose Múltipla/metabolismo , Degeneração Neural/metabolismo , Neurônios/metabolismo , Fator de Necrose Tumoral alfa/líquido cefalorraquidiano , Adulto , Animais , Feminino , Humanos , Inflamação/líquido cefalorraquidiano , Inflamação/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Esclerose Múltipla/líquido cefalorraquidiano , Degeneração Neural/líquido cefalorraquidiano , Adulto JovemRESUMO
OBJECTIVE: The frequency of inflammatory episodes in the early stages of multiple sclerosis (MS) has been correlated with late neurodegeneration, but the mechanism by which inflammation gives rise to delayed neuronal damage is unknown. Increased activity of the neurotransmitter glutamate is thought to play a role in the inflammation-driven neurodegenerative process of MS, and therefore we tested whether inflammatory cytokines released during acute MS attacks have the property of enhancing glutamate-mediated transmission and excitotoxicity in central neurons. METHODS: We compared the effect of cerebrospinal fluid (CSF) from active and quiescent MS patients on glutamate-mediated excitatory postsynaptic currents (EPSCs) and excitotoxic damage in rodent brain slices. We also measured CSF concentrations of tumor necrosis factor-α, of interleukin-1ß (IL-1ß), and of IL-1 receptor antagonist (IL-1ra), and correlated cytokine levels with cortical excitability assessed in MS patients by means of paired-pulse transcranial magnetic stimulation (TMS). RESULTS: CSF from MS patients with enhanced brain lesions at magnetic resonance imaging was able to increase spontaneous EPSC frequency and glutamate-mediated neuronal swelling in vitro, through a mechanism dependent on enhanced IL-1ß signaling and increased glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor stimulation. Furthermore, IL-1ß/IL-1ra ratio was significantly higher in the CSF of active MS subjects, and correlated with intracortical facilitation, an accredited TMS measure of glutamate transmission. Finally, we identified for the first time transient receptor potential vanilloid 1 channels as essential intermediates for the synaptic action of IL-1ß on central glutamatergic synapses. INTERPRETATION: Our results provide compelling evidence of the synaptic mechanism linking inflammation and excitotoxic neurodegeneration in MS.
Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Interleucina-1beta/fisiologia , Esclerose Múltipla/fisiopatologia , Transmissão Sináptica/fisiologia , Adulto , Animais , Feminino , Humanos , Interleucina-1beta/efeitos adversos , Interleucina-1beta/líquido cefalorraquidiano , Masculino , Camundongos , Pessoa de Meia-Idade , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/patologia , Técnicas de Cultura de Órgãos , Sinapses/fisiologia , Estimulação Magnética Transcraniana/métodos , Adulto JovemRESUMO
OBJECTIVE: Microvesicles (MVs) have been indicated as important mediators of intercellular communication and are emerging as new biomarkers of tissue damage. Our previous data indicate that reactive microglia/macrophages release MVs in vitro. The aim of the study was to evaluate whether MVs are released by microglia/macrophages in vivo and whether their number varies in brain inflammatory conditions, such as multiple sclerosis (MS). METHODS: Electron and fluorescence microscopy and flow cytometry were used to detect myeloid MVs in the cerebrospinal fluid (CSF) of healthy controls, MS patients, and rodents affected by experimental autoimmune encephalomyelitis (EAE), the animal model of MS. RESULTS: Myeloid MVs were detected in CSF of healthy controls. In relapsing and remitting EAE mice, the concentration of myeloid MVs in the CSF was significantly increased and closely associated with disease course. Analysis of MVs in the CSF of 28 relapsing patients and 28 patients with clinical isolated syndrome from 2 independent cohorts revealed higher levels of myeloid MVs than in 13 age-matched controls, indicating a clinical value of MVs as a companion tool to capture disease activity. Myeloid MVs were found to spread inflammatory signals both in vitro and in vivo at the site of administration; mice impaired in MV shedding were protected from EAE, suggesting a pathogenic role for MVs in the disease. Finally, FTY720, the first approved oral MS drug, significantly reduced the amount of MVs in the CSF of EAE-treated mice. INTERPRETATION: These findings identify myeloid MVs as a marker and therapeutic target of brain inflammation.
Assuntos
Biomarcadores/líquido cefalorraquidiano , Doenças do Sistema Nervoso Central/líquido cefalorraquidiano , Doenças do Sistema Nervoso Central/tratamento farmacológico , Inflamação/líquido cefalorraquidiano , Inflamação/tratamento farmacológico , Medula Espinal/metabolismo , Animais , Western Blotting , Sinalização do Cálcio/fisiologia , Comunicação Celular , Células Cultivadas , Encefalite/líquido cefalorraquidiano , Encefalite/patologia , Citometria de Fluxo , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Microscopia de Fluorescência , Esclerose Múltipla/patologia , Doença Autoimune do Sistema Nervoso Experimental/líquido cefalorraquidiano , Doença Autoimune do Sistema Nervoso Experimental/tratamento farmacológico , Neuroglia/metabolismo , Neuroglia/fisiologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/fisiologiaRESUMO
CD4(+)Foxp3(+) regulatory T cells (Tregs) have been considered crucial in controlling immune system homeostasis, and their derangement is often associated to autoimmunity. Tregs identification is, however, difficult because most markers, including CD25 and Foxp3, are shared by recently activated T cells. We show in this paper that CD4(+)Foxp3(+) T cells are generated in peripheral lymphoid organs on immunization and readily accumulate in the target organ of an autoimmune reaction, together with classical inflammatory cells, constituting up to 50% of infiltrating CD4(+) T cells. Most CD4(+)Foxp3(+) T cells are, however, CD25(-) and express proinflammatory cytokines such as IL-17 and IFN-γ, questioning their suppressive nature. Moreover, in vitro CD4(+) T lymphocytes from naive and autoimmune mice, stimulated to differentiate into Th1, Th2, Th17, and induced Tregs, display early mixed expression of lineage-specific markers. These results clearly point to an unprecedented plasticity of naive CD4(+) T cells, that integrating inflammatory signals may change their fate from the initial lineage commitment to a different functional phenotype.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Fatores de Transcrição Forkhead/imunologia , Interferon gama/imunologia , Interleucina-17/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Fatores de Transcrição Forkhead/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Ativação Linfocitária/genética , Camundongos , Camundongos Transgênicos , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologiaRESUMO
In degenerative disorders of the central nervous system (CNS), transplantation of neural multipotent (stem) precursor cells (NPCs) is aimed at replacing damaged neural cells. Here we show that in CNS inflammation, NPCs are able to promote neuroprotection by maintaining undifferentiated features and exerting unexpected immune-like functions. In a mouse model of chronic CNS inflammation, systemically injected adult syngeneic NPCs use constitutively activated integrins and functional chemokine receptors to selectively enter the inflamed CNS. These undifferentiated cells survive repeated episodes of CNS inflammation by accumulating within perivascular areas where reactive astrocytes, inflamed endothelial cells and encephalitogenic T cells produce neurogenic and gliogenic regulators. In perivascular CNS areas, surviving adult NPCs induce apoptosis of blood-borne CNS-infiltrating encephalitogenic T cells, thus protecting against chronic neural tissue loss as well as disease-related disability. These results indicate that undifferentiated adult NPCs have relevant therapeutic potential in chronic inflammatory CNS disorders because they display immune-like functions that promote long-lasting neuroprotection.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Células-Tronco Multipotentes/imunologia , Células-Tronco Multipotentes/transplante , Fármacos Neuroprotetores/metabolismo , Transplante de Células-Tronco , Animais , Apoptose , Transplante de Tecido Encefálico , Adesão Celular , Diferenciação Celular , Sistema Nervoso Central/irrigação sanguínea , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Quimiotaxia , Doença Crônica , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Inflamação/imunologia , Inflamação/patologia , Integrina alfa4beta1/metabolismo , Camundongos , Microesferas , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/fisiologia , Receptores de Quimiocinas/metabolismo , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The peri-ventricular area of the forebrain constitutes a preferential site of inflammation in multiple sclerosis, and the sub-ventricular zone (SvZ) is functionally altered in its animal model experimental autoimmune encephalomyelitis (EAE). The reasons for this preferential localization are still poorly understood. We show here that, in EAE mice, blood-derived macrophages, T and B cells and microglia (Mg) from the surrounding parenchyma preferentially accumulate within the SvZ, deranging its cytoarchitecture. We found that the chemokine Cxcl10 is constitutively expressed by a subset of cells within the SvZ, constituting a primary chemo-attractant signal for activated T cells. During EAE, T cells and macrophages infiltrating the SvZ in turn secrete pro-inflammatory cytokines such as TNFalpha and IFNgamma capable to induce Mg cells accumulation and SvZ derangement. Accordingly, lentiviral-mediated over-expression of IFNgamma or TNFalpha in the healthy SvZ mimics Mg/microglia recruitment occurring during EAE, while Cxcl10 over-expression in the SvZ is able to increase the frequency of peri-ventricular inflammatory lesions only in EAE mice. Finally, we show, by RT-PCR and in situ hybridization, that Cxcl10 is expressed also in the healthy human SvZ, suggesting a possible molecular parallelism between multiple sclerosis and EAE.
Assuntos
Movimento Celular/fisiologia , Quimiocina CXCL10/imunologia , Encefalomielite Autoimune Experimental/imunologia , Prosencéfalo/anatomia & histologia , Animais , Transplante de Medula Óssea , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Quimiocinas/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/imunologia , Esclerose Múltipla/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Quimeras de Transplante , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Neurodegeneration is the irremediable pathological event occurring during chronic inflammatory diseases of the CNS. Here we show that, in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, inflammation is capable in enhancing glutamate transmission in the striatum and in promoting synaptic degeneration and dendritic spine loss. These alterations occur early in the disease course, are independent of demyelination, and are strongly associated with massive release of tumor necrosis factor-alpha from activated microglia. CNS invasion by myelin-specific blood-borne immune cells is the triggering event, and the downregulation of the early gene Arc/Arg3.1, leading to the abnormal expression and phosphorylation of AMPA receptors, represents a culminating step in this cascade of neurodegenerative events. Accordingly, EAE-induced synaptopathy subsided during pharmacological blockade of AMPA receptors. Our data establish a link between neuroinflammation and synaptic degeneration and calls for early neuroprotective therapies in chronic inflammatory diseases of the CNS.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Degeneração Neural/patologia , Sinapses/patologia , Animais , Linhagem Celular Transformada , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/genética , Degeneração Neural/metabolismo , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/genética , Sinapses/metabolismoRESUMO
ß-thalassemia is caused by ß-globin gene mutations resulting in reduced (ß+) or absent (ß0) hemoglobin production. Patient life expectancy has recently increased, but the need for chronic transfusions in transfusion-dependent thalassemia (TDT) and iron chelation impairs quality of life1. Allogeneic hematopoietic stem cell (HSC) transplantation represents the curative treatment, with thalassemia-free survival exceeding 80%. However, it is available to a minority of patients and is associated with morbidity, rejection and graft-versus-host disease2. Gene therapy with autologous HSCs modified to express ß-globin represents a potential therapeutic option. We treated three adults and six children with ß0 or severe ß+ mutations in a phase 1/2 trial ( NCT02453477 ) with an intrabone administration of HSCs transduced with the lentiviral vector GLOBE. Rapid hematopoietic recovery with polyclonal multilineage engraftment of vector-marked cells was achieved, with a median of 37.5% (range 12.6-76.4%) in hematopoietic progenitors and a vector copy number per cell (VCN) of 0.58 (range 0.10-1.97) in erythroid precursors at 1 year, in absence of clonal dominance. Transfusion requirement was reduced in the adults. Three out of four evaluable pediatric participants discontinued transfusions after gene therapy and were transfusion independent at the last follow-up. Younger age and persistence of higher VCN in the repopulating hematopoietic cells are associated with better outcome.
Assuntos
Transfusão de Sangue , Osso e Ossos/patologia , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Talassemia beta/genética , Talassemia beta/terapia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Resultado do TratamentoRESUMO
Multiple Sclerosis (MS) therapies approved so far are unable to effectively reverse the chronic phase of the disease or improve the remyelination process. Here our aim is to evaluate the effects of C-Phycocyanin (C-Pc), a biliprotein from Spirulina platensis with anti-oxidant, anti-inflammatory and cytoprotective properties, in a chronic model of experimental autoimmune encephalomyelitis (EAE) in mice. C-Pc (2, 4 or 8 mg/kg i.p.) or IFN-beta (2000 IU, s.c.) was administered daily once a day or every other day, respectively, starting at disease onset, which differ among EAE mice between 11 and 15 days postinduction. Histological and immunohistochemistry (anti-Mac-3, anti-CD3 and anti-APP) assessments were performed in spinal cord in the postinduction time. Global gene expression in the brain was analyzed with the Illumina Mouse WG-6_V2 BeadChip microarray and the expression of particular genes, assessed by qPCR using the Fast SYBR Green RT-PCR Master Mix. Oxidative stress parameters (malondialdehyde, peroxidation potential, CAT/SOD ratio and GSH) were determined spectrophoto-metrically. Results showed that C-Pc ameliorates the clinical deterioration of animals, an effect that expresses the reduction of the inflammatory infiltrates invading the spinal cord tissue, the axonal preservation and the down-regulation of IL-17 expression in brain tissue and serum. C-Pc and IFN-beta improved the redox status in mice subjected to EAE, while microarray analysis showed that both treatments shared a common subset of differentially expressed genes, although they also differentially modulated another subset of genes. Specifically, C-Pc mainly modulated the expression of genes related to remyelination, gliogenesis and axon-glia processes. Taken together, our results indicate that C-Pc has significant therapeutic effects against EAE, mediated by the dynamic regulation of multiple biological processes.
Assuntos
Anti-Inflamatórios/farmacologia , Encefalomielite Autoimune Experimental/patologia , Interferon beta/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Ficocianina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Feminino , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologiaRESUMO
Reliable, and easy to measure, immunological markers able to denote disease characteristics in multiple sclerosis (MS) patients are still lacking. We applied a multivariate statistical analysis on results obtained by measuring-by real-time RT-PCR-mRNA levels of 25 immunological relevant molecules in PBMCs from 198 MS patients. The combined measurement of mRNA levels of IL-1beta, TNF-alpha, TGF-beta, CCL20 and CCR3 was able to distinguish MS patients from healthy individuals. CXCR5, CCL5, and CCR3 combined mRNA levels identify primary progressive MS patients while TNF-alpha, IL-10, CXCL10 and CCR3 differentiate relapsing MS patients. Our results indicate that multi-parametric analysis of mRNA levels of immunological relevant molecules in PBMCs may represent a successful strategy for the identification of putative peripheral markers of disease state and disease activity in MS patients.
Assuntos
Esclerose Múltipla/diagnóstico , Esclerose Múltipla/imunologia , Adulto , Biomarcadores , Quimiocina CCL20 , Quimiocina CCL5 , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Progressão da Doença , Feminino , Humanos , Interleucina-1/biossíntese , Modelos Lineares , Proteínas Inflamatórias de Macrófagos/genética , Proteínas Inflamatórias de Macrófagos/metabolismo , Imageamento por Ressonância Magnética/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Modelos Imunológicos , Esclerose Múltipla Crônica Progressiva/diagnóstico , Esclerose Múltipla Crônica Progressiva/imunologia , Análise Multivariada , RNA Mensageiro/metabolismo , Receptores CCR3 , Receptores CXCR5 , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Nitric oxide (NO)-releasing non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to have a safer profile and additional anti-inflammatory and immuno-modulatory properties compared to parent compounds. Preventive treatment of experimental autoimmune encephalomyelitis (EAE)-induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55-with the NO-releasing derivative of flurbiprofen HCT1026 delayed disease onset and significantly decreased disease severity. HCT1026 treatment was associated to (i) decreased mRNA levels of pro-inflammatory cytokines, caspase-1, and iNOS in blood cells; (ii) decreased ability of encephalitogenic T cells to proliferate; (iii) reduced number of central nervous system (CNS)-infiltrating T cells; (iv) decreased axonal loss and demyelination; (v) increased CD4(+) CD69(-) CD25(+) regulatory T cells in the spleen.
Assuntos
Encefalomielite Autoimune Experimental/prevenção & controle , Flurbiprofeno/análogos & derivados , Flurbiprofeno/administração & dosagem , Imunossupressores/administração & dosagem , Óxido Nítrico/metabolismo , Administração Oral , Transferência Adotiva , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Citocinas/antagonistas & inibidores , Citocinas/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Epitopos de Linfócito T/imunologia , Feminino , Glicoproteínas/administração & dosagem , Glicoproteínas/imunologia , Inibidores do Crescimento/administração & dosagem , Interleucina-10/deficiência , Interleucina-10/genética , Lectinas Tipo C , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , RNA Mensageiro/antagonistas & inibidores , Receptores de Interleucina-2/biossíntese , Baço/imunologia , Baço/patologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologiaRESUMO
In multiple sclerosis (MS), pathological white matter damage in the central nervous system is sustained by immune-inflammatory response. Caspase-1 plays a pivotal role in immune-mediated inflammation, as it regulates the cellular export of IL-1beta and IL-18. We carried out a preliminary in vitro study of the kinetics of extracellular caspase-1 release. We then measured caspase-1 levels in paired serum and cerebrospinal fluid (CSF) samples of 75 MS patients, 15 healthy subjects, and patients with other neurological diseases. Paired synovial fluid and serum samples of patients with juvenile idiopathic arthritis, and paired sputum and serum samples of asthma patients were also studied. Mean serum caspase-1 concentrations did not differ between groups. Caspase-1 was detected in the CSF of patients with acute, but not stable, MS [7.5 +/- (SEM) 0.9 pg/ml; test's sensitivity, 56% and specificity, 100%]. Its levels correlated with pleocytosis. The highest mean caspase-1 levels were found in the arthritic synovial fluids (945.5 +/- 126.6 pg/ml, which correlated with erythrocyte sedimentation rate), and in the sputum samples (370.1 +/- 71.0 pg/ml, which correlated with the number of macrophages in the sputum). On condition that caspase-1 is determined in the fluids pertaining to the disease-specific inflammatory sites, its level is a reliable marker of ongoing immune-inflammatory response. The enzyme measurement in CSF can also help define state-trait in MS.
Assuntos
Artrite Juvenil/enzimologia , Asma/enzimologia , Líquidos Corporais/enzimologia , Caspase 1/sangue , Esclerose Múltipla/enzimologia , Artrite Juvenil/sangue , Artrite Juvenil/líquido cefalorraquidiano , Biomarcadores/análise , Caspase 1/líquido cefalorraquidiano , Células Cultivadas , Humanos , Esclerose Múltipla/sangue , Esclerose Múltipla/líquido cefalorraquidiano , Escarro/enzimologia , Líquido Sinovial/enzimologiaRESUMO
We describe a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique for the quantification of the messenger RNA of human and murine tumor necrosis factor alpha (TNFalpha) and related receptors. This protocol can be adapted for blood, peripheral blood lymphocytes, or other tissues. We propose a dot-blot technique which, if properly set up, is fast and quantitatively reliable. We describe two different detection protocols employing either radioactive or, alternatively for laboratories that cannot or do not want to use radioactivity, fluorescent-labeled probes. We also describe our calculations for relative quantification, based on the use of a positive control sample that becomes the reference value used to compare experimental samples. These protocols have as their aim to provide a flexible tool that can be employed in several different human and murine experimental settings by laboratories with different equipment.
Assuntos
Bioquímica/métodos , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator de Necrose Tumoral alfa/biossíntese , Animais , Autorradiografia , Fluoresceína/metabolismo , Corantes Fluorescentes/farmacologia , Humanos , Camundongos , Hibridização de Ácido Nucleico , RNA/metabolismoRESUMO
MVs are known vehicles of horizontal communication among cells, currently under scrutiny as powerful biomarkers in several pathological processes. The potential advantage of MVs relies on the assumption that their content reflects processes ongoing in pathologically relevant cell types. We have described that MVs of myeloid origin in the CSF are a marker of microglia/macrophage activation. Myeloid cells have different activation types, resulting in diverse functional phenotypes. Knowledge on the activation type of myeloid cells during disease would be of paramount importance for the understanding of ongoing pathogenic processes. We show here that macrophages activated in vitro in different ways all release increased amounts of MVs compared with NS cells. Moreover, we show that macrophage-derived MVs contain a repertoire of mRNAs that is not the result of casual sampling from the parental cells, as it is characterized by distinct mRNA enrichments and species. Nevertheless, mRNA content of MVs clearly allows identification in vivo of the activated phenotype of the cell of origin, indicating carryover of functional macrophage traits. We propose that detection of mRNAs in myeloid MVs permits identification of myeloid cell activation type during disease, allowing for further stratification of pathological processes.
Assuntos
Micropartículas Derivadas de Células/imunologia , Ativação de Macrófagos , Macrófagos Peritoneais/imunologia , RNA Mensageiro/imunologia , Animais , Feminino , CamundongosRESUMO
During multiple sclerosis (MS) inflammatory attacks, and in subsequent clinical recovery phases, immune cells contribute to neuronal and oligodendroglial cell survival and tissue repair by secreting growth factors. Animal studies showed that growth factors also play a substantial role in regulating synaptic plasticity, and namely in long-term potentiation (LTP). LTP could drive clinical recovery in relapsing patients by restoring the excitability of denervated neurons. We recently reported that maintenance of synaptic plasticity reserve is crucial to contrast clinical deterioration in MS and that the platelet-derived growth factor (PDGF) may play a key role in its regulation. We also reported that a Hebbian form of LTP-like cortical plasticity, explored by paired associative stimulation (PAS), correlates with clinical recovery from a relapse in MS. Here, we explored the role of PDGF in clinical recovery and in adaptive neuroplasticity in relapsing-remitting MS (RR-MS) patients. We found a correlation between the cerebrospinal fluid (CSF) PDGF concentrations and the extent of clinical recovery after a relapse, as full recovery was more likely observed in patients with high PDGF concentrations and poor recovery in subjects with low PDGF levels. Consistently with the idea that PDGF-driven synaptic plasticity contributes to attenuate the clinical consequences of tissue damage in RR-MS, we also found a striking correlation between CSF levels of PDGF and the amplitude of LTP-like cortical plasticity explored by PAS. CSF levels of fibroblast growth factor, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor did not correlate with clinical recovery nor with measures of synaptic transmission and plasticity.
Assuntos
Potenciação de Longa Duração/fisiologia , Esclerose Múltipla Recidivante-Remitente/fisiopatologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Adulto , Convalescença , Eletromiografia , Potencial Evocado Motor , Feminino , Fatores de Crescimento de Fibroblastos/líquido cefalorraquidiano , Fator Estimulador de Colônias de Granulócitos/líquido cefalorraquidiano , Fator Estimulador de Colônias de Granulócitos e Macrófagos/líquido cefalorraquidiano , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Córtex Motor/fisiopatologia , Esclerose Múltipla Recidivante-Remitente/metabolismo , Fator de Crescimento Derivado de Plaquetas/líquido cefalorraquidiano , Índice de Gravidade de Doença , Estimulação Magnética Transcraniana , Adulto JovemRESUMO
The immune system shapes synaptic transmission and plasticity in experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis (MS). These synaptic adaptations are believed to drive recovery of function after brain lesions, and also learning and memory deficits and excitotoxic neurodegeneration; whether inflammation influences synaptic plasticity in MS patients is less clear. In a cohort of 59 patients with MS, we found that continuous theta-burst transcranial magnetic stimulation did not induce the expected long-term depression (LTD)-like synaptic phenomenon, but caused persisting enhancement of brain cortical excitability. The amplitude of this long-term potentiation (LTP)-like synaptic phenomenon correlated with the concentration of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) in the cerebrospinal fluid. In MS and EAE, the brain and spinal cord are typically enriched of CD3(+) T lymphocyte infiltrates, which are, along with activated microglia and astroglia, a major cause of inflammation. Here, we found a correlation between the presence of infiltrating T lymphocytes in the hippocampus of EAE mice and synaptic plasticity alterations. We observed that T lymphocytes from EAE, but not from control mice, release IL-1ß and promote LTP appearance over LTD, thereby mimicking the facilitated LTP induction observed in the cortex of MS patients. EAE-specific T lymphocytes were able to suppress GABAergic transmission in an IL-1ß-dependent manner, providing a possible synaptic mechanism able to lower the threshold of LTP induction in MS brains. Moreover, in vivo blockade of IL-1ß signaling resulted in inflammation and synaptopathy recovery in EAE hippocampus. These data provide novel insights into the pathophysiology of MS.
Assuntos
Interleucina-1beta/fisiologia , Potenciação de Longa Duração/fisiologia , Esclerose Múltipla Recidivante-Remitente/fisiopatologia , Adolescente , Adulto , Animais , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Gliose/etiologia , Gliose/patologia , Hipocampo/fisiopatologia , Humanos , Infusões Intraventriculares , Proteína Antagonista do Receptor de Interleucina 1/administração & dosagem , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Distribuição Aleatória , Subpopulações de Linfócitos T/metabolismo , Ritmo Teta , Estimulação Magnética Transcraniana , Adulto Jovem , Ácido gama-Aminobutírico/fisiologiaRESUMO
The aim of this study is to investigate the expression of P2X7R, IL-1beta and the ATP activity modulating ecto-apyrase CD39 on peripheral blood monocytes of MS patients and to observe the possible effects of Glatiramer Acetate (GA) on such expression. Twelve RR treatment-free MS patients were selected and peripheral blood monocytes were obtained. The expression of P2X7R, IL-1beta and CD39 on monocytes was investigated by qrt-PCR. The in vitro effects of GA on the expression of monocytes stimulated with BzATP (a potent P2X7R agonist)-were evaluated. Ten healthy donors (HDs) were similarly studied. Finally, 5 MS patients were given GA therapy and the monocytes obtained before treatment, after 3 and 12 months of GA treatment were similarly investigated. No differences were found in P2X7R, IL-1beta and CD39 expression between patients and controls. In MS Bz-ATP stimulated monocytes, GA pre-conditioning clearly downregulated P2X7R (p=0.003) but IL-1beta expression also showed a decreasing trend (p=0.07). Conversely, CD39 showed an increasing trend (p=0.07). Similar evidence was found in HDs. GA in vivo treatment induced a reduction in the expression that was clear for P2X7R and CD39 (p<0.05) but only not significant for IL-1beta after 12 months of treatment. Monocytes from both MS and control subjects express P2X7R, IL-1beta and CD39, and GA seems to interfere with such expression.
Assuntos
Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/imunologia , Peptídeos/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/metabolismo , Adulto , Antígenos CD/biossíntese , Antígenos CD/metabolismo , Apirase/biossíntese , Apirase/metabolismo , Feminino , Acetato de Glatiramer , Humanos , Interleucina-1beta/biossíntese , Interleucina-1beta/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Esclerose Múltipla Recidivante-Remitente/metabolismo , Cultura Primária de Células , Receptores Purinérgicos P2X7/biossínteseRESUMO
FoxP3⺠Treg cells are believed to play a role in the occurrence of autoimmunity and in the determination of clinical recurrences. Contradictory reports are, however, available describing frequency and function of Treg cells during autoimmune diseases. We examined, by both polychromatic flow cytometry, and real-time RT-PCR, several Treg markers in peripheral blood mononuclear cells from patients with multiple sclerosis (MS), an autoimmune disease affecting the central nervous system. We found that Tregs, as defined by CD25, CD39, FoxP3, CTLA4, and GITR expression, were significantly decreased in stable MS patients as compared to healthy donors, but, surprisingly, restored to normal levels during an acute clinical attack. We conclude that Treg cells are not involved in causing clinical relapses, but rather react to inflammation in the attempt to restore homeostasis.