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1.
Cancers (Basel) ; 15(15)2023 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-37568574

RESUMO

This preliminary study seeks to determine the effect of R&P denervation on tumor growth and survival in immunocompetent rats bearing an aggressive and metastatic breast solid tumor. A novel microsurgical approach was applied "in situ", aiming to induce R&P denervation through the division of every single nerve fiber connecting the host with the primary tumor via its complete detachment and re-attachment, by resecting and reconnecting its supplying artery and vein (anastomosis). This preparation, known as microsurgical graft or flap, is radically denervated by definition, but also effectively delays or even impedes the return of innervation for a significant period of time, thus creating a critical and therapeutic time window. Mammary adenocarcinoma cells (HH-16.cl4) were injected into immunocompetent Sprague Dawley adult rats. When the tumors reached a certain volume, the subjects entered the study. The primary tumor, including a substantial amount of peritumoral tissue, was surgically isolated on a dominant artery and vein, which was resected and reconnected using a surgical microscope (orthotopic tumor auto-transplantation). Intending to simulate metastasis, two or three tumors were simultaneously implanted and only one was treated, using the surgical technique described herein. Primary tumor regression was observed in all of the microsurgically treated subjects, associated with a potent systemic anticancer effect and prolonged survival. In stark contrast, the subjects received a close to identical surgical operation; however, with the intact neurovascular connection, they did not achieve the therapeutic result. Animals bearing multiple tumors and receiving the same treatment in only one tumor exhibited regression in both the "primary" and remote- untreated tumors at a clinically significant percentage, with regression occurring in more than half of the treated subjects. A novel therapeutic approach is presented, which induces the permanent regression of primary and, notably, remote tumors, as well as, evidently, the naturally occurring metastatic lesions, at a high rate. This strategy is aligned with the impetus that comes from the current translational research data, focusing on the abrogation of the neuro-tumoral interaction as an alternative treatment strategy. More data regarding the clinical significance of this are expected to come up from a pilot clinical trial that is ongoing.

2.
Front Immunol ; 11: 575451, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33329540

RESUMO

CNS autoantigens conjugated to oxidized mannan (OM) induce antigen-specific T cell tolerance and protect mice against autoimmune encephalomyelitis (EAE). To investigate whether OM-peptides treat EAE initiated by human MHC class II molecules, we administered OM-conjugated murine myelin oligodendrocyte glycoprotein peptide 35-55 (OM-MOG) to humanized HLA-DR2b transgenic mice (DR2b.Ab°), which are susceptible to MOG-EAE. OM-MOG protected DR2b.Ab° mice against MOG-EAE by both prophylactic and therapeutic applications. OM-MOG reversed clinical symptoms, reduced spinal cord inflammation, demyelination, and neuronal damage in DR2b.Ab° mice, while preserving axons within lesions and inducing the expression of genes associated with myelin (Mbp) and neuron (Snap25) recovery in B6 mice. OM-MOG-induced tolerance was peptide-specific, not affecting PLP178-191-induced EAE or polyclonal T cell proliferation responses. OM-MOG-induced immune tolerance involved rapid induction of PD-L1- and IL-10-producing myeloid cells, increased expression of Chi3l3 (Ym1) in secondary lymphoid organs and characteristics of anergy in MOG-specific CD4+ T cells. The results show that OM-MOG treats MOG-EAE in a peptide-specific manner, across mouse/human MHC class II barriers, through induction of a peripheral type 2 myeloid cell response and T cell anergy, and suggest that OM-peptides might be useful for suppressing antigen-specific CD4+ T cell responses in the context of human autoimmune CNS demyelination.


Assuntos
Axônios/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Imunossupressores/farmacologia , Células Mieloides/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Adulto , Animais , Axônios/imunologia , Axônios/metabolismo , Axônios/patologia , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Regulação da Expressão Gênica , Grécia , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto Jovem
3.
Molecules ; 23(2)2018 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-29385090

RESUMO

In this report, amide-linked cyclic peptide analogues of the 87-99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP87-99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP72-85-induced EAE in Lewis rats. The Lys91 and Pro96 of MBP87-99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91-99)[Ala96]MBP87-99, cyclo(87-99)[Ala91,96]MBP87-99 and cyclo(87-99)[Arg91, Ala96]MBP87-99, but not wild-type linear MBP87-99, strongly inhibited MBP72-85-induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87-99)[Arg91, Ala96]MBP87-99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction.


Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Proteína Básica da Mielina , Fragmentos de Peptídeos , Peptídeos Cíclicos , Animais , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Proteína Básica da Mielina/síntese química , Proteína Básica da Mielina/química , Proteína Básica da Mielina/farmacologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Ratos , Ratos Endogâmicos Lew
4.
Methods Mol Biol ; 1651: 93-112, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28801902

RESUMO

Synthetic promoters have been developed in a number of different organisms and are capable of mediating specific and enhanced levels of gene expression. Typically, cis-regulatory regions from a few genes are randomly combined to generate a synthetic promoter library, and the sequences with the highest activity are selected for in target cell lines. Here we describe a novel approach that can be employed in the construction of synthetic promoters . Specifically, we use gene expression profiles obtained from microarray datasets to select the cis-regulatory elements that comprise the synthetic promoter library. By adopting this approach, we were able to construct several promoters that could specifically mediate gene expression in colorectal cancer cells. We develop a new selection criteria based on the observed transcriptome of target cells, the frequency that identified cis-regulatory sequences occur in identified gene modules, and the length of identified cis-regulatory regions. Our method allows for the generation of synthetic promoter libraries with increased level of specificity and facilitates the selection of promoters that are highly active only under predefined gene expression profiles.


Assuntos
Biblioteca Gênica , Genômica/métodos , Regiões Promotoras Genéticas , Transcriptoma , Animais , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Redes Reguladoras de Genes , Terapia Genética , Vetores Genéticos/genética , Humanos , Elementos Reguladores de Transcrição , Transfecção/métodos , beta-Galactosidase/genética
5.
Exp Neurol ; 267: 254-67, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25447934

RESUMO

Antigen presenting cells (APC) are critical for regulating immune responses. We tested mannan-peptide conjugates for targeting myelin peptides to APC to induce T cell tolerance and resistance to experimental autoimmune encephalomyelitis (EAE). Myelin peptides conjugated to mannan in oxidized (OM) or reduced (RM) forms protected mice against EAE in prophylactic and therapeutic protocols, with OM-conjugated peptides giving best results. Protection was peptide-specific and associated with reduced antigen-specific T cell proliferation, but not alterations in Th1, Th17 and Treg cell differentiation or T cell apoptosis compared to EAE controls. Bone marrow-derived dendritic cells (DC) loaded with OM-MOG showed up-regulated expression of co-stimulatory molecules, reduced PD-L1 expression and enhanced CD40-inducible IL-12 and IL-23 production compared to MOG DC, features consistent with immunogenic DC. OM-MOG induced active T cell tolerance because i.d. administration or passive transfer of OM-MOG DC suppressed ongoing EAE, while OM-MOG-vaccinated mice did not reduce the proliferation of transferred MOG-specific T cells. As in vivo, MOG-specific T cells cultured with OM-MOG DC showed reduced proliferation and equal Th1 and Th17 cell differentiation compared to those with MOG DC, but surprisingly cytokine production was unresponsive to CD40 engagement. Impaired effector T cell function was further evidenced in spinal cord sections from OM-MOG-vaccinated EAE mice, where markedly reduced numbers of CD3(+) T cells were present, restricted to leptomeninges and exceptional parenchymal lesions. Our results show that mannan-conjugated myelin peptides protect mice against EAE through the expansion of antigen-specific Th1 and Th17 cells with impaired proliferation responses and APC-induced co-stimulatory signals that are required for licensing them to become fully pathogenic T cells.


Assuntos
Encefalomielite Autoimune Experimental/terapia , Mananas/uso terapêutico , Proteína Básica da Mielina/uso terapêutico , Células Th1/fisiologia , Células Th17/fisiologia , Animais , Apoptose/fisiologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/imunologia , Antígeno Ki-67/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Peptídeos/uso terapêutico , Fatores de Tempo
6.
Molecules ; 19(11): 17968-84, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25375337

RESUMO

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization.


Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Epitopos/química , Glicoproteína Mielina-Oligodendrócito/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Encefalomielite Autoimune Experimental/metabolismo , Epitopos/metabolismo , Epitopos/farmacologia , Feminino , Humanos , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Proteínas da Mielina/química , Proteínas da Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Compostos de Tritil/química
7.
Brain ; 134(Pt 9): 2722-35, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21908876

RESUMO

Tumour necrosis factor mediates chronic inflammatory pathologies including those affecting the central nervous system, but non-selective tumour necrosis factor inhibitors exacerbate multiple sclerosis. In addition, TNF receptor SF1A, which encodes one of the tumour necrosis factor receptors, has recently been identified as a multiple sclerosis susceptibility locus in genome-wide association studies in large patient cohorts. These clinical data have emphasized the need for a better understanding of the beneficial effects of tumour necrosis factor during central nervous system inflammation. In this study, we present evidence that the soluble and transmembrane forms of tumour necrosis factor exert opposing deleterious and beneficial effects, respectively, in a multiple sclerosis model. We compared the effects, in experimental autoimmune encephalomyelitis, of selectively inhibiting soluble tumour necrosis factor, and of both soluble and transmembrane tumour necrosis factor. Blocking the action of soluble tumour necrosis factor, but not of soluble tumour necrosis factor and transmembrane tumour necrosis factor, protected mice against the clinical symptoms of experimental autoimmune encephalomyelitis. Therapeutic benefit was independent of changes in antigen-specific immune responses and focal inflammatory spinal cord lesions, but was associated with reduced overall central nervous system immunoreactivity, increased expression of neuroprotective molecules, and was dependent upon the activity of neuronal nuclear factor-κB, a downstream mediator of neuroprotective tumour necrosis factor/tumour necrosis factor receptor signalling, because mice lacking IκB kinase ß in glutamatergic neurons were not protected by soluble tumour necrosis factor blockade. Furthermore, blocking the action of soluble tumour necrosis factor, but not of soluble tumour necrosis factor and transmembrane tumour necrosis factor, protected neurons in astrocyte-neuron co-cultures against glucose deprivation, an in vitro neurodegeneration model relevant for multiple sclerosis, and this was dependent upon contact between the two cell types. Our results show that soluble tumour necrosis factor promotes central nervous system inflammation, while transmembrane tumour necrosis factor is neuroprotective, and suggest that selective inhibition of soluble tumour necrosis factor may provide a new way forward for the treatment of multiple sclerosis and possibly other inflammatory central nervous system disorders.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , NF-kappa B/imunologia , Neurônios/metabolismo , Fármacos Neuroprotetores/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Sistema Nervoso Central/anatomia & histologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Técnicas de Cocultura , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Etanercepte , Feminino , Glicoproteínas/imunologia , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Imunoglobulina G/uso terapêutico , Imunossupressores/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito , Neurônios/citologia , Fragmentos de Peptídeos/imunologia , Receptores do Fator de Necrose Tumoral/uso terapêutico , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores
9.
Genomics ; 96(2): 82-91, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20435134

RESUMO

The brain responds to injury and infection by activating innate defense and tissue repair mechanisms. Working upon the hypothesis that the brain defense response involves common genes and pathways across diverse pathologies, we analysed global gene expression in brain from mouse models representing three major central nervous system disorders, cerebral stroke, multiple sclerosis and Alzheimer's disease compared to normal brain using DNA microarray expression profiling. A comparison of dysregulated genes across disease models revealed common genes and pathways including key components of estrogen and TGF-beta signaling pathways that have been associated with neuroprotection as well as a neurodegeneration mediator, TRPM7. Further, for each disease model, we discovered collections of differentially expressed genes that provide novel insight into the individual pathology and its associated mechanisms. Our data provide a resource for exploring the complex molecular mechanisms that underlie brain neurodegeneration and a new approach for identifying generic and disease-specific targets for therapy.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Esclerose Múltipla/metabolismo , Transdução de Sinais/genética , Acidente Vascular Cerebral/metabolismo , Doença de Alzheimer/genética , Animais , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acidente Vascular Cerebral/genética , Biologia de Sistemas/métodos , Canais de Cátion TRPM/metabolismo
10.
Immunol Cell Biol ; 88(5): 586-95, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20212506

RESUMO

Tumor necrosis factor (TNF)- and TNF receptor I (TNFRI)-deficient mice are resistant to initiation and show delayed resolution of disease in paradigms of autoimmune disease, but the contribution of TNF/TNFRI signaling to T-cell activation and effector responses has not been determined. In this study, we investigated the role of TNFRI in T-cell receptor (TCR)-mediated T-cell activation in vitro and in vivo using CD3(+)-enriched primary T cells and mice deficient in TNFRI. Following TCR engagement, TNFRI knockout (KO) T cells showed significantly delayed proliferation, cell division, upregulation of interleukin 2 (IL-2) and IL-2 receptor alpha chain (CD25) mRNA and cell-surface expression of CD25 compared with wild-type (WT) cells. Thus, WT and TNFRI KO cells showed equivalent proliferation peaks at 48 and 72 h, respectively. TNFRI KO mice also developed a defective primary T-cell response to ovalbumin and an acute contact hypersensitivity response to oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one). However, TNFRI KO splenocytes that were stimulated by TCR engagement in vitro for 96 h produced significantly higher intracellular levels of interferon-gamma (IFN-gamma), IL-2 and TNF-alpha, but not IL-17, compared with WT cells, in correlation with their relatively higher proliferation rate at this time point. Further, TCR-stimulated CD3(+)-enriched TNFRI KO T cells showed similarly higher production and secretion of IFN-gamma and IL-2 compared with WT, suggesting that TNFRI-mediated cytokine regulation might involve a T-cell autonomous effect. Our results show a novel role for TNFRI as a positive T-cell costimulatory molecule that is important for timely T-cell activation and effector cytokine production and the development of primary immune responses in mice.


Assuntos
Citocinas/biossíntese , Ativação Linfocitária/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Western Blotting , Diferenciação Celular/imunologia , Proliferação de Células , Separação Celular , Citocinas/imunologia , Citometria de Fluxo , Imunoprecipitação , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/citologia , Linfócitos T/metabolismo , Tempo
11.
J Immunol ; 183(12): 7877-89, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20007573

RESUMO

Some aspects of CNS-directed autoimmunity in multiple sclerosis are modeled in mice by immunization with myelin Ags where tissue damage is driven by myelin-reactive Th1 and Th17 effector lymphocytes. Whether the CNS plays an active role in controlling such autoimmune diseases is unknown. We used mice in which IkappaB kinase beta was deleted from Ca(2+)/calmodulin-dependent kinase IIalpha-expressing neurons (nIKKbetaKO) to investigate the contribution of neuronal NF-kappaB to the development of myelin oligodendrocyte glycoprotein 35-55-induced experimental autoimmune encephalomyelitis. We show that nIKKbetaKO mice developed a severe, nonresolving disease with increased axon loss compared with controls and this was associated with significantly reduced CNS production of neuroprotective factors (vascular endothelial growth factor, CSF1-R, and FLIP) and increased production of proinflammatory cytokines (IL-6, TNF, IL-12, IL-17, and CD30L) and chemokines. The isolation of CNS-infiltrating monocytes revealed greater numbers of CD4(+) T cells, reduced numbers of NK1.1(+) cells, and a selective accumulation of Th1 cells in nIKKbetaKO CNS from early in the disease. Our results show that neurons play an important role in determining the quality and outcome of CNS immune responses, specifically that neuronal IkappaB kinase beta is required for neuroprotection, suppression of inflammation, limitation of Th1 lymphocyte accumulation, and enhancement of NK cell recruitment in experimental autoimmune encephalomyelitis-affected CNS and stress the importance of neuroprotective strategies for the treatment of multiple sclerosis.


Assuntos
Sistema Nervoso Central/enzimologia , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Quinase I-kappa B/fisiologia , Imunossupressores/metabolismo , Neurônios/enzimologia , Neurônios/imunologia , Animais , Axônios/imunologia , Axônios/patologia , Morte Celular/genética , Morte Celular/imunologia , Inibição de Migração Celular/genética , Inibição de Migração Celular/imunologia , Sistema Nervoso Central/patologia , Doença Crônica , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/genética , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Imunossupressores/farmacologia , Mediadores da Inflamação/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/patologia , Células Th1/imunologia , Células Th1/patologia
12.
Proc Natl Acad Sci U S A ; 105(16): 6185-90, 2008 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-18413601

RESUMO

CNS neurons use robust cytoprotective mechanisms to ensure survival and functioning under conditions of injury. These involve pathways induced by endogenous neuroprotective cytokines such as erythropoietin (EPO). Recently, in contrast to its well known deleterious roles, TNF has also been shown to exhibit neuroprotective properties. In the present study, we investigated the molecular mechanisms by which TNF receptor (TNFR)I mediates neuroprotection by comparing the gene expression profiles of lesioned cortex from WT and TNFRI KO mice after permanent middle cerebral artery occlusion. Several known neuroprotective molecules were identified as TNFRI targets, notably members of the Bcl-2 family, DNA repair machinery and cell cycle, developmental, and differentiation factors, neurotransmitters and growth factors, as well as their receptors, including EPO receptor (EPOR), VEGF, colony-stimulating factor receptor 1, insulin-like growth factor (IGF), and nerve growth factor (NGF). Further analysis showed that induction of EPOR and VEGF expression in primary cortical neurons after glucose deprivation (GD) largely depended on TNFRI and was further up-regulated by TNF. Also, EPO- and VEGF-induced neuroprotection against GD, oxygen-glucose deprivation, and NMDA excitotoxicity depended significantly on TNFRI presence. Finally, EPO prevented neuronal damage induced by kainic acid in WT but not TNFRI KO mice. Our results identify cross-talk between tissue protective cytokines, specifically that TNFRI is necessary for constitutive and GD-induced expression of EPOR and VEGF and for EPO-mediated neuroprotection.


Assuntos
Isquemia Encefálica/genética , Citoproteção/genética , Eritropoetina/genética , Agonistas de Aminoácidos Excitatórios/toxicidade , Neurônios/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Isquemia Encefálica/patologia , Morte Celular/genética , Perfilação da Expressão Gênica , Ácido Caínico/toxicidade , Masculino , Camundongos , Camundongos Knockout , N-Metilaspartato/toxicidade , Neurônios/efeitos dos fármacos , Neurônios/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptores da Eritropoetina/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/metabolismo
13.
Int Immunol ; 19(10): 1183-9, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17878261

RESUMO

c-FLIP(L) expression in T cells is required for mounting effective T cell responses and can also be critical for effector T cell differentiation, as has recently been shown by a number of in vivo studies in conditional knockout and transgenic mouse systems. Available data supports therefore a novel immunomodulatory role of this anti-apoptotic protein besides its traditionally proposed function in homeostatic maintenance of T cell populations. In this study, the responses to infection with Leishmania major of mice over-expressing FLIP(L) specifically in the T cell compartment (TgFLIP(L)) are assessed. Although previous studies have shown that FLIP(L) drives T cells towards a T(h)2 differentiation programme in various autoimmune and allergic paradigms, in this study, we show that TgFLIP(L) are able to overcome this T(h)2 bias in a dermal L. major infection model to mount a robust T(h)1 response to pathogen and effectively clear infection. Our results suggest that vaccination protocols designed to enhance FLIP(L) expression in T cells may be useful for the treatment of autoimmune diseases like multiple sclerosis, without necessarily compromising immune responses towards infectious agents.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Leishmania major , Leishmaniose Cutânea/imunologia , Células Th2/imunologia , Animais , Antígenos de Protozoários/imunologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Citocinas/metabolismo , Memória Imunológica , Camundongos , Camundongos Transgênicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Regulação para Cima
14.
J Neurosci ; 27(25): 6633-46, 2007 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-17581950

RESUMO

Knowledge of the molecular mechanisms that underlie neuron death after stroke is important to allow the development of effective neuroprotective strategies. In this study, we investigated the contribution of death receptor signaling pathways to neuronal death after ischemia using in vitro and in vivo models of ischemic injury and transgenic mice that are deficient in tumor necrosis factor receptor I (TNFRI KO) or show neuron-specific overexpression of the long isoform of cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein (FLIP(L)). Caspase 8 was activated in brain lesions after permanent middle cerebral artery occlusion (pMCAO) and in cortical neurons subjected to glucose deprivation (GD) and was necessary for GD-induced neuron death. Thus, neurons treated with zIETD-FMK peptide or overexpressing a dominant-negative caspase 8 mutant were fully protected against GD-induced death. The presence of the neuroprotective TNFRI was necessary for selectively sustaining p50/p65NF-kappaB activity and the expression of the p43 cleavage form of FLIP(L), FLIP(p43), an endogenous inhibitor of caspase 8, in pMCAO lesions and GD-treated neurons. Moreover, TNF pretreatment further upregulated p50/p65NF-kappaB activity and FLIP(p43) expression in neurons after GD. The knock-down of FLIP in wild-type (WT) neurons using a short hairpin RNA revealed that FLIP(L) is essential for TNF/TNFRI-mediated neuroprotection after GD. Furthermore, the overexpression of FLIP(L) was sufficient to rescue TNFRI KO neurons from GD-induced death and to enhance TNF neuroprotection in WT neurons, and neuron-specific expression of FLIP(L) in transgenic mice significantly reduced lesion volume after pMCAO. Our results identify a novel role for the TNFRI-NF-kappaB-FLIP(L) pathway in neuroprotection after ischemia and identify potential new targets for stroke therapy.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/fisiologia , Glucose/deficiência , Glucose/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Morte Celular/genética , Hipóxia Celular/genética , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/fisiologia
15.
J Neurosci ; 26(44): 11387-96, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17079667

RESUMO

Modification of arginine residues by citrullination is catalyzed by peptidylarginine deiminases (PADs), of which five are known, generating irreversible protein structural modifications. We have shown previously that enhanced citrullination of myelin basic protein contributed to destabilization of the myelin membrane in the CNS of multiple sclerosis (MS) patients. We now report increased citrullination of nucleosomal histones by PAD4 in normal-appearing white matter (NAWM) of MS patients and in animal models of demyelination. Histone citrullination was attributable to increased levels and activity of nuclear PAD4. PAD4 translocation into the nucleus was attributable to elevated tumor necrosis factor-alpha (TNF-alpha) protein. The elevated TNF-alpha in MS NAWM was not associated with CD3+ or CD8+ lymphocytes, nor was it associated with CD68+ microglia/macrophages. GFAP, a measure of astrocytosis, was the only cytological marker that was consistently elevated in the MS NAWM, suggesting that TNF-alpha may have been derived from astrocytes. In cell cultures of mouse and human oligodendroglial cell lines, PAD4 was predominantly cytosolic but TNF-alpha treatment induced its nuclear translocation. To address the involvement of TNF-alpha in targeting PAD4 to the nucleus, we found that transgenic mice overexpressing TNF-alpha also had increased levels of citrullinated histones and elevated nuclear PAD4 before demyelination. In conclusion, high citrullination of histones consequent to PAD4 nuclear translocation is part of the process that leads to irreversible changes in oligodendrocytes and may contribute to apoptosis of oligodendrocytes in MS.


Assuntos
Encéfalo/metabolismo , Citrulina/metabolismo , Modelos Animais de Doenças , Histonas/metabolismo , Hidrolases/metabolismo , Esclerose Múltipla/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Encéfalo/patologia , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Feminino , Histonas/genética , Humanos , Hidrolases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas
16.
J Immunol ; 173(11): 6619-26, 2004 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-15557152

RESUMO

Cellular FLIP (c-FLIP) is an endogenous inhibitor of death receptor-induced apoptosis through the caspase 8 pathway. It is an NF-kappaB-inducible protein thought to promote the survival of T cells upon activation, and its down-regulation has been implicated in activation-induced cell death. We have generated transgenic mice overexpressing human c-FLIP long form (c-FLIP(L)) specifically in T cells using the CD2 promoter (TgFLIP(L)). TgFLIP(L) mice exhibit increased IgG1 production upon stimulation by a T cell-dependent Ag and a markedly enhanced contact hypersensitivity response to allergen. In addition to showing augmented Th2-type responses, TgFLIP(L) mice are resistant to the development of myelin oligodendrocyte glycoprotein 35-55 peptide-induced experimental autoimmune encephalomyelitis, a Th1-driven autoimmune disease. In vitro analyses revealed that T cells of TgFLIP(L) mice proliferate normally, but produce higher levels of IL-2 and show preferential maturation of Th2 cytokine-producing cells in response to antigenic stimulation. After adoptive transfer, these (Th2) cells protected wild-type recipient mice from experimental autoimmune encephalomyelitis induction. Our results show that the constitutive overexpression of c-FLIP(L) in T cells is sufficient to drive Th2 polarization of effector T cell responses and indicate that it might function as a key regulator of Th cell differentiation.


Assuntos
Adjuvantes Imunológicos/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Subpopulações de Linfócitos T/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/genética , Transferência Adotiva , Animais , Anticorpos Monoclonais/farmacologia , Autoanticorpos/biossíntese , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Complexo CD3/imunologia , Morte Celular/genética , Morte Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Dermatite de Contato/imunologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/prevenção & controle , Glicoproteínas/imunologia , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/imunologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Subpopulações de Linfócitos T/imunologia , Células Th2/citologia , Células Th2/transplante , Receptor fas/fisiologia
17.
Proc Natl Acad Sci U S A ; 101(17): 6698-703, 2004 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-15096619

RESUMO

In multiple sclerosis, in which brain tissue becomes permeable to blood proteins, extravascular fibrin deposition correlates with sites of inflammatory demyelination and axonal damage. To examine the role of fibrin in neuroinflammatory demyelination, we depleted fibrin in two tumor necrosis factor transgenic mouse models of multiple sclerosis, transgenic lines TgK21 and Tg6074. In a genetic analysis, we crossed TgK21 mice into a fibrin-deficient background. TgK21fib(-/-) mice had decreased inflammation and expression of major histocompatibility complex class I antigens, reduced demyelination, and a lengthened lifespan compared with TgK21 mice. In a pharmacologic analysis, fibrin depletion, by using the snake venom ancrod, in Tg6074 mice also delayed the onset of inflammatory demyelination. Overall, these results indicate that fibrin regulates the inflammatory response in neuroinflammatory diseases. Design of therapeutic strategies based on fibrin depletion could potentially benefit the clinical course of demyelinating diseases such as multiple sclerosis.


Assuntos
Doenças Desmielinizantes , Fibrina/metabolismo , Inflamação/prevenção & controle , Esclerose Múltipla/fisiopatologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Linhagem Celular , Inflamação/metabolismo , Ativação de Macrófagos , Camundongos , Camundongos Transgênicos , Ativadores de Plasminogênio/metabolismo , Fator de Necrose Tumoral alfa/genética , Regulação para Cima
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