RESUMO
Multiple Sclerosis (MS) has been reported to be associated with intestinal inflammation and gut dysbiosis. To elucidate the underlying biology of MS-linked gut inflammation, we investigated gut infiltration of immune cells during the development of spontaneous experimental autoimmune encephalomyelitis (EAE) in humanized transgenic (Tg) mice expressing HLA-DR2a and human T cell receptor (TCR) specific for myelin basic protein peptide (MBP87-99)/HLA-DR2a complexes. Strikingly, we noted the simultaneous development of EAE and colitis, suggesting a link between autoimmune diseases of the central nervous system (CNS) and intestinal inflammation. Examination of the colon in these mice revealed the infiltration of MBP-specific Th17 cells as well as recruitment of neutrophils. Furthermore, we observed that fecal Lipocalin-2 (Lcn-2), a biomarker of intestinal inflammation, was significantly elevated and predominantly produced by the gut-infiltrating neutrophils. We then extended our findings to MS patients and demonstrate that their fecal Lcn-2 levels are significantly elevated compared to healthy donors (HDs). The elevation of fecal Lcn-2 levels correlated with reduced bacterial diversity and increased levels of other intestinal inflammation markers including neutrophil elastase and calprotectin. Of interest, bacteria thought to be beneficial for inflammatory bowel disease (IBD) such as Anaerobutyricum, Blautia, and Roseburia, were reduced in fecal Lcn-2-high MS patients. We also observed a decreasing trend in serum acetate (a short-chain fatty acid) levels in MS Lcn-2-high patients compared to HDs. Furthermore, a decrease in the relative abundance of Blautia massiliensis was significantly associated with a reduction of acetate in the serum of MS patients. This study suggests that gut infiltration of Th17 cells and recruitment of neutrophils are associated with the development of gut dysbiosis and intestinal inflammation, and that fecal Lcn-2 level is a sensitive biological indicator for gut dysbiosis in multiple sclerosis.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Humanos , Camundongos , Animais , Disbiose/complicações , Lipocalina-2 , Biomarcadores Ambientais , Camundongos Transgênicos , Inflamação/complicaçõesRESUMO
OBJECTIVE: Monthly scanning with triple-dose gadopentetate dimeglumine has been shown to be associated with progressive increases in bone T1 hyperintensity, hypophosphatemia, and leukopenia. This study was performed to retrospectively investigate the potential associations among these phenomena. METHODS: This retrospective analysis involved patients who had received monthly triple-dose gadopentetate dimeglumine for up to 2 years as part of treatment for multiple sclerosis. Monthly magnetic resonance imaging scans of the brain (n = 67) were segmented to evaluate the signal intensity in the cranial marrow. Potential associations among the marrow T1 hyperintensity, serum phosphate concentration, and white blood cell count were examined. RESULTS: Patients in the no leukopenia group showed a statistically significant mean monthly increase in the bone marrow signal-to-noise ratio of 0.0430/month. Patients in the leukopenia group showed a mean monthly increase in the bone marrow signal-to-noise ratio of 0.0398/month, but this was not statistically significant. Patients in the hypophosphatemia group were significantly less likely to develop leukopenia than patients who had never developed hypophosphatemia. CONCLUSIONS: Although monthly administration of triple-dose gadopentetate dimeglumine over 13 months has been associated with progressive increases in leukopenia, hypophosphatemia, and T1 signal intensity of bone, this study showed an inverse relationship between leukopenia and hypophosphatemia.
Assuntos
Hipofosfatemia , Leucopenia , Compostos Organometálicos , Medula Óssea , Núcleos Cerebelares , Meios de Contraste , Gadolínio , Gadolínio DTPA , Humanos , Hipofosfatemia/induzido quimicamente , Leucopenia/induzido quimicamente , Imageamento por Ressonância Magnética , Estudos Retrospectivos , CrânioRESUMO
BACKGROUND: Relapses of multiple sclerosis decrease during pregnancy, when the hormone estriol is increased. Estriol treatment is anti-inflammatory and neuroprotective in preclinical studies. In a small single-arm study of people with multiple sclerosis estriol reduced gadolinium-enhancing lesions and was favourably immunomodulatory. We assessed whether estriol treatment reduces multiple sclerosis relapses in women. METHODS: We did a randomised, double-blind, placebo-controlled phase 2 trial at 16 academic neurology centres in the USA, between June 28, 2007, and Jan 9, 2014. Women aged 18-50 years with relapsing-remitting multiple sclerosis were randomly assigned (1:1) with a random permuted block design to either daily oral estriol (8 mg) or placebo, each in combination with injectable glatiramer acetate 20 mg daily. Patients and all study personnel, except for pharmacists and statisticians, were masked to treatment assignment. The primary endpoint was annualised relapse rate after 24 months, with a significance level of p=0.10. Relapses were confirmed by an increase in Expanded Disability Status Scale score assessed by an independent physician. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00451204. FINDINGS: We enrolled 164 patients: 83 were allocated to the estriol group and 81 were allocated to the placebo group. The annualised confirmed relapse rate was 0.25 relapses per year (95% CI 0.17-0.37) in the estriol group versus 0.37 relapses per year (0.25-0.53) in the placebo group (adjusted rate ratio 0.63, 95% CI 0.37-1.05; p=0.077). The proportion of patients with serious adverse events did not differ substantially between the estriol group and the placebo group (eight [10%] of 82 patients vs ten [13%] of 76 patients). Irregular menses were more common in the estriol group than in the placebo group (19 [23%] vs three [4%], p=0.0005), but vaginal infections were less common (one [1%] vs eight [11%], p=0.0117). There were no differences in breast fibrocystic disease, uterine fibroids, or endometrial lining thickness as assessed by clinical examination, mammogram, uterine ultrasound, or endometrial lining biopsy. INTERPRETATION: Estriol plus glatiramer acetate met our criteria for reducing relapse rates, and treatment was well tolerated over 24 months. These results warrant further investigation in a phase 3 trial. FUNDING: National Institutes of Health, National Multiple Sclerosis Society, Conrad N Hilton Foundation, Jack H Skirball Foundation, Sherak Family Foundation, and the California Community Foundation.
Assuntos
Estriol/administração & dosagem , Acetato de Glatiramer/administração & dosagem , Esclerose Múltipla Recidivante-Remitente/diagnóstico , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Adjuvantes Imunológicos/administração & dosagem , Adulto , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
PURPOSE OF REVIEW: Recent studies indicate a role for immune dysregulation in the pathogenesis of multiple sclerosis, an inflammatory demyelinating and degenerative disease of the central nervous system. This review addresses the current mechanisms of immune dysregulation in the development of multiple sclerosis, including the impact of environmental risk factors on immunity in both multiple sclerosis and its animal models. RECENT FINDINGS: CD4 T-helper (Th) cells have long been implicated as the main drivers of pathogenesis of multiple sclerosis. However, current studies indicate that multiple sclerosis is largely a heterogeneous disease process, which involves both innate and adaptive immune-mediated inflammatory mechanisms that ultimately contribute to demyelination and neurodegeneration. Therefore, B cells, CD8 T cells, and microglia/macrophages can also play an important role in the immunopathogenesis of multiple sclerosis apart from proinflammatory CD4 Th1/Th17 cell subsets. Furthermore, increasing evidence indicates that environmental risk factors, such as Vitamin D deficiency, Epstein-Barr virus, smoking, Western diet, and the commensal microbiota, influence the development of multiple sclerosis through interactions with genetic variants of multiple sclerosis, thus leading to the dysregulation of immune responses. SUMMARY: A better understanding of immune-mediated mechanisms in the pathogenesis of multiple sclerosis and the contribution of environmental risk factors toward the development of multiple sclerosis will help further improve therapeutic approaches to prevent disease progression.
Assuntos
Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Sistema Imunitário/patologia , Esclerose Múltipla , Animais , Citocinas/metabolismo , Humanos , Sistema Imunitário/fisiopatologia , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologiaRESUMO
B cells are necessary to maintain disease activity in relapsing multiple sclerosis (MS) and produce matrix metallopeptidase-9 (MMP-9), which disrupts the blood-brain barrier. MMP-9 protein expression was increased and expression of microRNA-320a (miR-320a), which targets MMP-9 mRNA, was significantly decreased in B lymphocytes of MS patients during a disease relapse compared to remission. Functional significance of these findings was demonstrated by transfecting human B lymphocytes with miR-320a inhibitor, which led to increased MMP-9 expression and secretion. In summary, expression of miR-320a is decreased in B cells of MS patients and may contribute to increased blood-brain barrier permeability and neurological disability.
Assuntos
Linfócitos B/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/metabolismo , Esclerose Múltipla/patologia , Adolescente , Adulto , Feminino , Perfilação da Expressão Gênica , Humanos , Interleucina-6/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , MicroRNAs/genética , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Transfecção , Adulto JovemRESUMO
Natalizumab is a humanized monoclonal antibody against the leukocyte adhesion molecule very late antigen (VLA)-4, and is currently an approved therapy for patients with relapsing-remitting multiple sclerosis (RRMS). However, it is unknown whether natalizumab is beneficial for progressive forms of MS. Therefore, we assessed the effects of anti-VLA-4 monoclonal antibody (mAb) therapy in a progressive experimental autoimmune encephalomyelitis (EAE) mouse model. Notably, we found that early therapy could significantly reduce the severity of progressive EAE, while treatment initiated at an advanced stage was less efficient. Furthermore, we observed the accumulation of a novel subset of GM-CSF-producing CD11b+CD4+ T cells in the CNS throughout disease progression. Importantly, early therapeutic anti-VLA-4 mAb treatment suppressed the accumulation of these GM-CSF-producing CD11b+CD4+ T cells in the CNS along with activated microglia/macrophages populations, and also conferred a protective effect against inflammation-mediated neurodegeneration, including demyelination and axonal loss. Collectively, our data suggest that early treatment with anti-VLA-4 mAb can provide neuroprotection against progressive CNS autoimmune disease by preventing the accumulation of pathogenic GM-CSF-producing CD11b+CD4+ T cells in the CNS.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno CD11b/metabolismo , Linfócitos T CD4-Positivos/imunologia , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Integrina alfa4beta1/imunologia , Animais , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Citometria de Fluxo , Técnicas Imunoenzimáticas , Integrina alfa4beta1/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Oligodendroglia are cells of the central nervous system (CNS) that form myelin sheath, which insulates neuronal axons. Neuropathologies of the CNS include dysmyelination of axons in multiple sclerosis and CNS trauma. Cell replacement is a promising but largely untested therapy for dysmyelination. Shiverer mouse, a genetic mutant that does not synthesize full-length myelin basic protein (MBP), a critical prerequisite protein in CNS myelin sheath formation, provides an unequivocal model for determining the potential of stem cells to become oligodendroglia. We demonstrate that adult wild-type mouse bone marrow stem cells can express MBP and ensheath axons when transplanted into Shiverer brain.
Assuntos
Axônios/metabolismo , Encéfalo/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Proteína Básica da Mielina/biossíntese , Bainha de Mielina/metabolismo , Animais , Axônios/patologia , Encéfalo/patologia , Encéfalo/cirurgia , Células Cultivadas , Células-Tronco Hematopoéticas/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes Neurológicos , Bainha de Mielina/patologiaRESUMO
OBJECTIVE: Viral infections have been implicated in the pathogenesis of multiple sclerosis (MS). Plasmacytoid dendritic cells (pDCs) are present in peripheral blood, cerebrospinal fluid, and brain lesions of MS patients. pDCs sense viral DNA via Toll-like receptor 9 (TLR9), which has to be cleaved from the N-terminal to become functional (TLR9 processing). pDCs activated with TLR9 agonists promote T-helper type 1 (Th1)/T-helper type 17 (Th17) responses. In the animal model of MS, TLR9 agonists can induce disease. We hypothesized that pDCs are inhibited by disease-modifying therapy such as interferon (IFN)-ß, consequently decreasing the frequency of MS attacks. METHODS: We separated pDCs from healthy subjects and patients diagnosed with relapsing-remitting MS and clinically isolated syndrome. Cytokine secretion by pDCs activated with TLR9 agonists was measured by enzyme-linked immunosorbent assay and multianalyte profiling. TLR9 gene and protein expression was studied by DNA microarrays and western blot. RESULTS: In untreated patients, pDCs activated with TLR9 agonists produced increased levels of IFN-α, a Th1-promoting cytokine, as compared to healthy subjects. In IFN-ß-treated patients, activated pDCs had decreased ability to produce both IFN-α and the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α as compared to untreated patients. pDCs separated from IFN-ß-treated patients had significantly reduced levels of the processed TLR9 protein but normal levels of the full-length TLR9 protein and TLR9 gene expression as compared to untreated patients. INTERPRETATION: This finding represents a novel immunomodulatory mechanism of IFN-ß: inhibition of TLR9 processing. This results in decreased activation of pDCs by viral pathogens and, thus, may affect the frequency of MS exacerbations.
Assuntos
Células Dendríticas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Interferon beta/farmacologia , Esclerose Múltipla/sangue , Receptor Toll-Like 9/metabolismo , Adolescente , Adulto , Células Cultivadas , Células Dendríticas/virologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/metabolismo , Interferon beta/uso terapêutico , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/genética , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is neuroprotective in animal models of neurodegenerative diseases. However, BDNF has a short half-life and its efficacy in the central nervous system (CNS), when delivered peripherally, is limited due to the blood-brain barrier (BBB). We have developed a means of delivering BDNF into the CNS using genetically engineered bone marrow stem cells (BMSCs) as a vehicle, and have explored the clinical effects of BDNF on outcomes in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). BDNF-engineered-BMSCs were transplanted (i.v.) into irradiated 2-week-old SJL/J female mice. Eight weeks after transplantation, mice were immunized with a peptide of proteolipid protein (PLP(139-151)). Mice, which had received BDNFengineered BMSCs, showed a significant delay in EAE onset and a reduction in overall clinical severity compared to mice receiving BMSC transfected with an empty vector lacking the BDNF gene. In addition, pathological examination showed that BDNF delivery reduced demyelination and increased remyelination. Inhibition of pro-inflammatory cytokines TNF-alpha and IFN-gamma and enhanced expression of the antiinflammatory cytokines IL-4, IL-10, and IL-11 were found in the CNS tissues of the BDNF transplanted group. These results support the use of BMSCs as vehicles to deliver BDNF into the CNS of EAE animals. This is a potentially novel therapeutic approach that might be used to deliver BDNF gene or genes for other therapeutic proteins into the CNS in MS or in other diseases of the CNS in which accessibility of therapeutic proteins is limited due to the BBB.
Assuntos
Transplante de Medula Óssea/métodos , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/uso terapêutico , Encefalomielite Autoimune Experimental/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Vetores Genéticos/farmacologia , Vetores Genéticos/uso terapêutico , Camundongos , Proteína Proteolipídica de Mielina/imunologia , Bainha de Mielina/imunologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Fragmentos de Peptídeos/imunologia , Resultado do TratamentoRESUMO
Interferon-beta (IFN-beta), an approved treatment of multiple sclerosis (MS), produces only partial clinical responses. IFN-beta therapy has been limited by its short serum half-life and limited ability to cross the blood brain barrier. We have developed a means of delivering the IFN-beta gene both systemically and into the central nervous system (CNS) using bone marrow stem cells (BMSCs) as a vehicle and examined the therapeutic efficacy of this approach in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. A retroviral expression vector (pLXSN-IFNbeta) was used to stably transfect virus producer PA317 cells to generate retrovirus containing the IFN-beta gene which then was used to transduce BMSCs. IFN-beta engineered BMSCs were transplanted (i.v.) into mice that then were immunized with proteolipoprotein (PLP) to initiate EAE. IFN-beta-engineered BMSCs transplanted mice showed a significant inhibition of EAE onset, and the overall clinical severity was less compared to control groups. IFN-beta delivery strongly reduced infiltration of mononuclear cells possibly by inhibiting cell adhesion molecules. Reduced demyelination and increased remyelination were also observed in the IFN-beta treated group. Furthermore, inhibition of the pro-inflammatory cytokines TNF-alpha, IFN-gamma and IL-12 and enhanced expression of the anti-inflammatory cytokines IL-10, IL-4 and TGF-beta was observed in CNS tissue. In addition, mice receiving IFN-beta had reduced apoptosis and increases in growth promoting factors including BDNF, CNTF, PDGF and VEGF. These results suggest that BMSCs can be used as vehicles to deliver the IFN-beta into the CNS. This is a potentially novel therapeutic approach which might be used in MS and other diseases of the CNS in which drug access is limited.
Assuntos
Células da Medula Óssea/fisiologia , Transplante de Medula Óssea/métodos , Encefalomielite Autoimune Experimental/prevenção & controle , Interferon beta/uso terapêutico , Animais , Antígenos CD34/metabolismo , Células da Medula Óssea/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Técnicas de Transferência de Genes , Marcação In Situ das Extremidades Cortadas , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferon beta/biossíntese , Interferon beta/genética , Camundongos , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Proteína Proteolipídica de Mielina , Fragmentos de Peptídeos , Prevenção Secundária , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
BACKGROUND: Glatiramer acetate therapy is thought to be effective for multiple sclerosis (MS) by promoting T(H)2 cytokine deviation, possibly in the brain, but the exact mechanism and site of action are incompletely understood. Determining the site of action and effect of glatiramer on cell trafficking is of major importance in designing rational combination therapy clinical trials. OBJECTIVE: To determine whether glatiramer therapy will also act in the peripheral blood through bystander modulation of chemokine receptor (CKR) expression and cytokine production on T lymphocytes. DESIGN: Before-and-after trial. SETTING: A university MS specialty center. PATIENTS: Ten patients with relapsing-remitting MS. INTERVENTIONS: Treatment with glatiramer for 12 months and serial phlebotomy. MAIN OUTCOME MEASURES: Cytokine production, CKR expression, and cell migration. RESULTS: The glatiramer-reactive T cells were T(H)2 cytokine biased, consistent with previous studies. We found a significant reduction in the expression of the T(H)1 inflammation associated with the CKRs CXCR3, CXCR6, and CCR5 on glatiramer- and myelin-reactive T cells generated from patients with MS receiving glatiramer therapy vs baseline. Conversely, expression of the lymph node-homing CKR, CCR7, was markedly enhanced on the glatiramer-reactive T cells derived from patients with MS undergoing glatiramer therapy. There was a reduction in the percentage of CD4+ glatiramer-reactive T cells and an increase in the number of CD8+ glatiramer-reactive T cells. CONCLUSIONS: Glatiramer may suppress autoreactive CD4+ effector memory T cells and enhance CD8+ regulatory responses, and bystander modulation of CKRs may occur in the periphery.
Assuntos
Efeito Espectador/efeitos dos fármacos , Efeito Espectador/imunologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Receptores de Quimiocinas/biossíntese , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Acetato de Glatiramer , Humanos , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia , Linfócitos T/metabolismoRESUMO
A major challenge in the treatment of neurological disorders is the effective delivery of molecules into the CNS and, more so, to the lesion site. The blood-brain barrier restricts the delivery of therapeutic molecules into the CNS when injected intravenously. This difficulty is further compounded by the short half-life of certain therapeutic agents. Organ-targeted protein delivery could circumvent these difficulties, provided that the corresponding cDNA can be delivered and expressed in the target tissue effectively and safely. Recent studies from a number of laboratories indicate that a subset of adult bone marrow cells home to the CNS among other organs and can be engineered to deliver and express therapeutic proteins into the CNS. This article, which is focused on work from the authors' research, reviews the opportunities and difficulties presented by this approach.
Assuntos
Células da Medula Óssea/fisiologia , Transplante de Medula Óssea , Sistema Nervoso Central/fisiologia , Organismos Geneticamente Modificados , Animais , Lesões Encefálicas/terapia , Camundongos , RatosRESUMO
Bone marrow, or cells selected from bone marrow, were reported recently to give rise to cells with a neural phenotype after in vitro treatment with neural-inducing factors or after delivery into the brain. However, we showed previously that untreated bone marrow cells express products of the neural myelin basic protein gene, and we demonstrate here that a subset of ex vivo bone marrow cells expresses the neurogenic transcription factor Pax-6 as well as neuronal genes encoding neurofilament H, NeuN (neuronal nuclear protein), HuC/HuD (Hu-antigen C/Hu-antigen D), and GAD65 (glutamic acid decarboxylase 65), as well as the oligodendroglial gene encoding CNPase (2',3' cyclic nucleotide 3'-phosphohydrolase). In contrast, astroglial glial fibrillary acidic protein (GFAP) was not detected. These cells also were CD34+, a marker of hematopoietic stem cells. Cultures of these highly proliferative CD34+ cells, derived from adult mouse bone marrow, uniformly displayed a phenotype comparable with that of hematopoietic progenitor cells (CD45+, CD34+, Sca-1+, AA4.1+, cKit+, GATA-2+, and LMO-2+). The neuronal and oligodendroglial genes expressed in ex vivo bone marrow also were expressed in all cultured CD34+ cells, and GFAP was not observed. After CD34+ cell transplantation into adult brain, neuronal or oligodendroglial markers segregated into distinct nonoverlapping cell populations, whereas astroglial GFAP appeared, in the absence of other neural markers, in a separate set of implanted cells. Thus, neuronal and oligodendroglial gene products are present in a subset of bone marrow cells, and the expression of these genes can be regulated in brain. The fact that these CD34+ cells also express transcription factors (Rex-1 and Oct-4) that are found in early development elicits the hypothesis that they may be pluripotent embryonic-like stem cells.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Neurônios/metabolismo , Fatores de Transcrição , Animais , Antígenos CD34/biossíntese , Western Blotting , Células da Medula Óssea/metabolismo , Encéfalo/metabolismo , Divisão Celular , Transplante de Células , Proteínas de Ligação a DNA/biossíntese , Proteínas ELAV , Proteína Semelhante a ELAV 3 , Proteína Semelhante a ELAV 4 , Proteínas do Olho , Proteína Glial Fibrilar Ácida/biossíntese , Glutamato Descarboxilase/biossíntese , Proteínas de Homeodomínio/biossíntese , Imuno-Histoquímica , Interleucina-3/metabolismo , Interleucina-6/metabolismo , Isoenzimas/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/biossíntese , Proteínas de Neurofilamentos/biossíntese , Fator 3 de Transcrição de Octâmero , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Fenótipo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/biossíntese , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Células-Tronco/metabolismo , Fatores de TempoRESUMO
Experimental and MRI evidence suggest that glatiramer acetate's (Copaxone) therapeutic effect in multiple sclerosis (MS) could be mediated by anti-inflammatory GA-reactive Th2 cells that enter the brain, cross-react with myelin antigens, and produce bystander suppression. Furthermore, a neuroprotective effect, possibly mediated by neurotrophic factors such as BDNF, has been suggested based on experimental evidence in animal models, and the observation that inflammatory cells can elaborate BDNF. Therefore, we examined BDNF production in 73 GA, 13 MBP, and 22 TT-reactive short-term T-cell lines from 12 MS patients treated with GA. Ten of 73 GA-TCL (14%), 1 of the MBP-TCL (3%), and 2 of the TT-TCL (9%) produced BDNF levels two standard deviations above the mean levels produced by resting TCL. RT-PCR analysis confirmed BDNF expression in some GA- and MBP-reactive TCL. The mean BDNF level produced by GA-TCL was significantly higher than that for MBP-TCL, or TT-TCL when lines originating from the same patients were compared (P=0.033). All 10 high BDNF-producing GA-reactive TCL were Th2-biased as determined by the IL-5/IFN-gamma levels ratio. A positive correlation was observed between BDNF and IL-5 (Th2 indicator) (P=0.006) but not with IFN-gamma Th1 indicator) levels in GA-TCL derived from MS patients during but not pre-treatment. We conclude that while BDNF production by T cells is not antigen-specific, GA-reactive TCL are more likely to produce BDNF, and to be Th2-biased.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Peptídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Adulto , Idoso , Fator Neurotrófico Derivado do Encéfalo/genética , Feminino , Acetato de Glatiramer , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Peptídeos/uso terapêutico , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
One favored mechanism of action of glatiramer acetate (GA) in multiple sclerosis (MS) involves the induction of GA-reactive Th2 cells that are believed to enter the central nervous system and mediate bystander suppression in response to cross-reactive myelin antigens. To test this hypothesis, we examined the proliferative response and cytokine release from peripheral blood mononuclear cells (PBMCs) of 12 MS patients treated with GA, in response to 16 myelin peptides that were previously described as immunodominant or encephalitogenic and a tetanus peptide as a control antigen. Interferon-gamma (IFN-gamma) and IL-5 (markers of Th1 and Th2 responses, respectively) were assayed by enzyme-linked immunosorbent assay (ELISA). GA-stimulated PBMCs from 9 of 12 patients (75%) proliferated to one or more myelin peptides. Among the 16 peptides tested, GA-stimulated PBMCs from the majority of the patients proliferated in response to MOG(21-44). PBMCs from two thirds of the patients produced IL-5 in response to myelin peptides, while half of them produced IFN-gamma. Th1/Th0/Th2 cytokine phenotypes demonstrated that responses from 10 of 12 patients were either Th0- or Th2-biased. Responses from two patients were Th1-biased. Conversely, some myelin-specific T-cell lines (TCLs) responded to GA by proliferation (3 of 21 TCLs), IL-5 release (11 of 21 TCLs), and IFN-gamma release (3 of 21 TCLs). These results indicate that GA-reactive TCLs can respond to a spectrum of myelin peptides in a Th2-biased fashion, which is consistent with the concept of bystander suppression. Furthermore, some myelin-specific TCLs are able to recognize GA, with a tendency to produce more IL-5 than IFN-gamma, which would suggest a systemic modulatory effect of the drug.
Assuntos
Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Proteínas da Mielina/imunologia , Peptídeos/imunologia , Células Th2/imunologia , Células Th2/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/imunologia , Adjuvantes Imunológicos/sangue , Adjuvantes Imunológicos/farmacologia , Adulto , Idoso , Sequência de Aminoácidos , Linhagem Celular , Citocinas/biossíntese , Feminino , Acetato de Glatiramer , Humanos , Imunofenotipagem , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Proteína Básica da Mielina/imunologia , Proteína Proteolipídica de Mielina/imunologia , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/imunologia , Peptídeos/sangue , Peptídeos/farmacologiaRESUMO
Glatiramer acetate (GA) (Copaxone(R)) is a worldwide-approved drug for the treatment of relapsing multiple sclerosis (MS), an autoimmune disease of the CNS. The drug is a synthetic copolymer with an amino acid composition based on the structure of myelin basic protein, one of the autoantigens implicated in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE). Developed initially as a "tool" to study EAE, the drug unexpectedly inhibited disease and was subsequently developed for the treatment of MS. The drug has been shown in controlled clinical trials to significantly reduce relapse rate and progression of disability in MS with long-term efficacy, remarkable safety, and tolerability. Efficacy as measured by magnetic resonance imaging parallels its clinical benefits as manifested by a reduction in gadolinium-enhancing lesions and brain atrophy. The mechanism of action of the drug in humans is believed to involve the induction of glatiramer-reactive regulatory cells, including CD4+ and CD8+ T-cells. Glatiramer-reactive Th2 cells are believed to enter the brain and, through cross-reactivity with myelin antigens, produce bystander suppression, antiinflammatory effects, and neuroprotection.
Assuntos
Imunossupressores/farmacologia , Esclerose Múltipla/tratamento farmacológico , Peptídeos/farmacologia , Administração Oral , Ensaios Clínicos como Assunto , Acetato de Glatiramer , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Esclerose Múltipla/patologia , Peptídeos/administração & dosagem , Peptídeos/efeitos adversosRESUMO
The interferon-beta (IFNbeta) gene is not inducible in neuronal cells in response to measles virus (MV) due to lack of nuclear factor kappa B (NF-kappaB) activation. NF-kappaB is normally sequestered in the cytoplasm by an inhibitor (IkappaBalpha). Previously, the authors demonstrated that the failure to activate neuronal NF-kappaB by MV was due to the inability to phosphorylate and degrade its inhibitor, IkappaBalpha. Here the authors demonstrate that transient transfection of a brain cDNA library into neuronal cells restores the ability of MV to activate NF-kappaB. In addition, tumor necrosis factor-alpha (TNFalpha), but not interleukin-1 (IL-1) or lipopolysaccharide (LPS), stimulation resulted in IkappaBalpha phosphorylation and degradation in two neuronal cell lines. These results indicate that failure of MV to activate neuronal NF-kappaB is due to a signaling defect and that MV utilizes an NF-kappaB signaling pathway distinct from that of TNFalpha, but may overlap with that for IL-1 and LPS.
Assuntos
Vírus do Sarampo , NF-kappa B/metabolismo , Neuroglia/virologia , Neurônios/virologia , Linhagem Celular , Biblioteca Gênica , Teste de Complementação Genética , Humanos , Proteínas I-kappa B/metabolismo , Interferon beta/farmacologia , Interleucina-1 , Lipopolissacarídeos , Inibidor de NF-kappaB alfa , Neuroglia/metabolismo , Neurônios/metabolismo , Fosforilação , Transdução de Sinais , Fator de Necrose Tumoral alfaRESUMO
The peripheral delivery of interferon-beta (IFN-beta) for the treatment of central nervous system (CNS) diseases is only partially effective because of the blood-brain barrier (BBB). To circumvent this problem, we evaluated the feasibility of genetically altering bone marrow cells ex vivo and using them as vehicles to transfer the IFN-beta cDNA into the mouse CNS. An IFN-beta retroviral expression vector (pLXSN-IFNbeta) was used to stably transfect PA317 cells. The supernatant from these producer cells, which expressed IFN-beta mRNA and protein, were used to infect bone marrow cells. When transplanted into irradiated mice, IFN-beta-engineered marrow cells accessed the CNS and expressed IFN-beta mRNA and protein. Marrow cells transduced with a control neomycin vector entered the brain and expressed the neomycin but not the IFN-beta gene. In the CNS, IFN-beta delivered by marrow cells induced the mRNA expression of 2',5'-oligoadenylate synthetase (2',5'-OAS), indicating biologic activity. Our findings demonstrating that bone marrow cells can serve as a delivery system for IFN-beta cDNA into the CNS could have implications for the treatment of neurologic disorders, such as multiple sclerosis (MS), viral encephalitis, and brain tumors.
Assuntos
Transplante de Medula Óssea , Encéfalo/metabolismo , Vetores Genéticos/farmacocinética , Interferon beta/genética , 2',5'-Oligoadenilato Sintetase/biossíntese , 2',5'-Oligoadenilato Sintetase/genética , Animais , Barreira Hematoencefálica , DNA Complementar/genética , Indução Enzimática , Estudos de Viabilidade , Genes Reporter , Genes Sintéticos , Terapia Genética , Interferon beta/biossíntese , Canamicina Quinase/biossíntese , Canamicina Quinase/genética , Camundongos , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Organismos Livres de Patógenos Específicos , TransfecçãoRESUMO
MS is an immunologically mediated disease, as determined by observation of the response to immunotherapy and the existence of an animal model, experimental autoimmune encephalitis. Interferon (IFN) beta-1b, IFN beta-1a, and glatiramer acetate, the therapies used for relapsing or remitting MS, have mechanisms of action that address the immunologic pathophysiology of MS. The IFNs bind to cell surface-specific receptors, initiating a cascade of signaling pathways that end with the secretion of antiviral, antiproliferative, and immunomodulatory gene products. Glatiramer acetate, a synthetic molecule, inhibits the activation of myelin basic protein-reactive T cells and induces a T-cell repertoire characterized by anti-inflammatory effects. Although the two classes of drugs have some overlapping mechanisms of action, the IFNs rapidly block blood-brain barrier leakage and gadolinium (Gd) enhancement within 2 weeks, whereas glatiramer acetate produces less rapid resolution of Gd-enhanced MRI activity. IFN beta has no direct effects in the CNS, but glatiramer acetate-specific T cells are believed to have access to the CNS, where they can exert anti-inflammatory and possibly neuroprotective effects.