RESUMEN
BACKGROUND: Type 1 spinal muscular atrophy is a rare, progressive neuromuscular disease that is caused by low levels of functional survival of motor neuron (SMN) protein. Risdiplam is an orally administered, small molecule that modifies SMN2 pre-messenger RNA splicing and increases levels of functional SMN protein. METHODS: We report the results of part 1 of a two-part, phase 2-3, open-label study of risdiplam in infants 1 to 7 months of age who had type 1 spinal muscular atrophy, which is characterized by the infant not attaining the ability to sit without support. Primary outcomes were safety, pharmacokinetics, pharmacodynamics (including the blood SMN protein concentration), and the selection of the risdiplam dose for part 2 of the study. Exploratory outcomes included the ability to sit without support for at least 5 seconds. RESULTS: A total of 21 infants were enrolled. Four infants were in a low-dose cohort and were treated with a final dose at month 12 of 0.08 mg of risdiplam per kilogram of body weight per day, and 17 were in a high-dose cohort and were treated with a final dose at month 12 of 0.2 mg per kilogram per day. The baseline median SMN protein concentrations in blood were 1.31 ng per milliliter in the low-dose cohort and 2.54 ng per milliliter in the high-dose cohort; at 12 months, the median values increased to 3.05 ng per milliliter and 5.66 ng per milliliter, respectively, which represented a median of 3.0 times and 1.9 times the baseline values in the low-dose and high-dose cohorts, respectively. Serious adverse events included pneumonia, respiratory tract infection, and acute respiratory failure. At the time of this publication, 4 infants had died of respiratory complications. Seven infants in the high-dose cohort and no infants in the low-dose cohort were able to sit without support for at least 5 seconds. The higher dose of risdiplam (0.2 mg per kilogram per day) was selected for part 2 of the study. CONCLUSIONS: In infants with type 1 spinal muscular atrophy, treatment with oral risdiplam led to an increased expression of functional SMN protein in the blood. (Funded by F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT02913482.).
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Compuestos Azo/administración & dosificación , Fármacos Neuromusculares/administración & dosificación , Pirimidinas/administración & dosificación , Atrofias Musculares Espinales de la Infancia/tratamiento farmacológico , Proteína 1 para la Supervivencia de la Neurona Motora/sangre , Administración Oral , Compuestos Azo/efectos adversos , Compuestos Azo/farmacocinética , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Lactante , Masculino , Fármacos Neuromusculares/efectos adversos , Fármacos Neuromusculares/farmacocinética , Supervivencia sin Progresión , Pirimidinas/efectos adversos , Pirimidinas/farmacocinética , Empalme del ARN , Insuficiencia Respiratoria/etiología , Infecciones del Sistema Respiratorio/etiología , Atrofias Musculares Espinales de la Infancia/complicaciones , Atrofias Musculares Espinales de la Infancia/mortalidad , Proteína 1 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
OBJECTIVE: To characterize the natural history of spinal muscular atrophy (SMA) over 24 months using innovative measures such as wearable devices, and to provide evidence for the sensitivity of these measures to determine their suitability as endpoints in clinical trials. METHODS: Patients with Type 2 and 3 SMA (N = 81) with varied functional abilities (sitters, nonsitters, nonambulant, and ambulant) who were not receiving disease-modifying treatment were assessed over 24 months: motor function (Motor Function Measure [MFM]), upper limb strength (MyoGrip, MyoPinch), upper limb activity (ActiMyo® ), quantitative magnetic resonance imaging (fat fraction [FFT2 ] mapping and contractile cross-sectional area [C-CSA]), pulmonary function (forced vital capacity [FVC], peak cough flow, maximum expiratory pressure, maximum inspiratory pressure, and sniff nasal inspiratory pressure), and survival of motor neuron (SMN) protein levels. RESULTS: MFM32 scores declined significantly over 24 months, but not 12 months. Changes in upper limb activity could be detected over 6 months and continued to decrease significantly over 12 months, but not 24 months. Upper limb strength decreased significantly over 12 and 24 months. FVC declined significantly over 12 months, but not 24 months. FFT2 increased over 12 and 24 months, although not with statistical significance. A significant increase in C-CSA was observed at 12 but not 24 months. Blood SMN protein levels were stable over 12 and 24 months. INTERPRETATION: These data demonstrate that the MFM32, MyoGrip, MyoPinch, and ActiMyo® enable the detection of a significant decline in patients with Type 2 and 3 SMA over 12 or 24 months.
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Fuerza Muscular , Atrofia Muscular Espinal/diagnóstico por imagen , Atrofia Muscular Espinal/fisiopatología , Proteínas del Tejido Nervioso/sangre , Proteínas de Unión al ARN/sangre , Extremidad Superior/fisiopatología , Adolescente , Adulto , Niño , Preescolar , Evaluación de la Discapacidad , Progresión de la Enfermedad , Humanos , Estudios Longitudinales , Imagen por Resonancia Magnética , Actividad Motora , Atrofia Muscular Espinal/sangre , Pruebas de Función Respiratoria , Índice de Severidad de la Enfermedad , Factores de Tiempo , Adulto JovenRESUMEN
INTRODUCTION: To investigate the validity and reliability of the 32-item Motor Function Measure (MFM32) in individuals with neuromuscular disorders (NMD), including spinal muscular atrophy (SMA), aged 2-5 years, and in non-ambulant individuals with Types 2 or 3 SMA, aged 2-25 years. METHODS: Test-retest reliability (intraclass correlation coefficient [ICC]), internal consistency (Cronbach's alpha [α]), convergent validity (Spearman rank-order correlations), and known-groups validity (analysis of covariance comparing groups defined by the Clinical Global Impression of Severity [CGI-S] scale and Vignos grade) were calculated. The analysis was performed on a dataset provided by Hospices Civils De Lyon, extracted from the multinational MFM32 database. A total of 165 individuals were included in the analyses, of whom 84 were in the NMD group (aged 2-5 years) and 81 were in the SMA group (aged 2-25 years). RESULTS: Strong evidence of test-retest reliability (ICC: 2- to 5-years' population = 0.94-0.95; 2- to 25-years' population = 0.97), internal consistency (Cronbach's α: 2- to 5-years' population = 0.96; 2- to 25-years' population = 0.95), convergent validity (2- to 5-years' population: CGI-S rho = - 0.84, Vignos grade rho = - 0.79; 2- to 25-years' population: CGI-S rho = - 0.49), and known-groups validity (all P < 0.001) were demonstrated. CONCLUSIONS: These analyses provide supportive evidence of the validity and reliability of the MFM32 in younger individuals with NMDs, aged 2-5 years, and in non-ambulant individuals with Types 2 or 3 SMA, aged 2-25 years, supporting the use of the MFM32 across a wide age range.
RESUMEN
Spinal muscular atrophy (SMA) is a monogenic disorder caused by loss of function mutations in the survival motor neuron 1 gene, which results in a broad range of disease severity, from neonatal to adult onset. There is currently a concerted effort to define the natural history of the disease and develop outcome measures that accurately capture its complexity. As several therapeutic strategies are currently under investigation and both the FDA and EMA have recently approved the first medical treatment for SMA, there is a critical need to identify the right association of responsive outcome measures and biomarkers for individual patient follow-up. As an approved treatment becomes available, untreated patients will soon become rare, further intensifying the need for a rapid, prospective and longitudinal study of the natural history of SMA Type 2 and 3. Here we present the baseline assessments of 81 patients aged 2 to 30 years of which 19 are non-sitter SMA Type 2, 34 are sitter SMA Type 2, 9 non-ambulant SMA Type 3 and 19 ambulant SMA Type 3. Collecting these data at nine sites in France, Germany and Belgium established the feasibility of gathering consistent data from numerous and demanding assessments in a multicenter SMA study. Most assessments discriminated between the four groups well. This included the Motor Function Measure (MFM), pulmonary function testing, strength, electroneuromyography, muscle imaging and workspace volume. Additionally, all of the assessments showed good correlation with the MFM score. As the untreated patient population decreases, having reliable and valid multi-site data will be imperative for recruitment in clinical trials. The pending two-year study results will evaluate the sensitivity of the studied outcomes and biomarkers to disease progression. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02391831).
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Atrofias Musculares Espinales de la Infancia/epidemiología , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Estudios Longitudinales , Pulmón/fisiopatología , Masculino , Fuerza Muscular , Debilidad Muscular/complicaciones , Desempeño Psicomotor , Atrofias Musculares Espinales de la Infancia/complicaciones , Atrofias Musculares Espinales de la Infancia/fisiopatología , Adulto JovenRESUMEN
AIMS: Sphingosine-1-phosphate receptor (S1PR) modulating therapies are currently in the clinic or undergoing investigation for multiple sclerosis (MS) treatment. However, the expression of S1PRs is still unclear in the central nervous system under normal conditions and during neuroinflammation. METHODS: Using immunohistochemistry we examined tissues from both grey and white matter MS lesions for sphingosine-1-phosphate receptor 1 (S1P1 ) and 5 (S1P5 ) expression. Tissues from Alzheimer's disease (AD) cases were also examined. RESULTS: S1P1 expression was restricted to astrocytes and endothelial cells in control tissues and a decrease in endothelial cell expression was found in white matter MS lesions. In grey matter MS lesions, astrocyte expression was lost in active lesions, while in quiescent lesions it was restored to normal expression levels. CNPase colocalization studies demonstrated S1P5 expression on myelin and both were reduced in demyelinated lesions. In AD tissues we found no difference in S1P1 expression. CONCLUSION: These data demonstrate a differential modulation of S1PRs in MS lesions, which may have an impact on S1PR-directed therapies.
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Encéfalo/metabolismo , Esclerosis Múltiple/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Astrocitos/metabolismo , Encéfalo/patología , Células Endoteliales/metabolismo , Femenino , Sustancia Gris/metabolismo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/patología , Receptores de Esfingosina-1-Fosfato , Sustancia Blanca/metabolismoRESUMEN
Lymphocyte trafficking is critically regulated by the Sphingosine 1-phosphate receptor-1 (S1P(1)), a G protein-coupled receptor that has been highlighted as a promising therapeutic target in autoimmunity. Fingolimod (FTY720, Gilenya) is a S1P(1) receptor agonist that has recently been approved for the treatment of multiple sclerosis (MS). Here, we report the discovery of NIBR-0213, a potent and selective S1P(1) antagonist that induces long-lasting reduction of peripheral blood lymphocyte counts after oral dosing. NIBR-0213 showed comparable therapeutic efficacy to fingolimod in experimental autoimmune encephalomyelitis (EAE), a model of human MS. These data provide convincing evidence that S1P(1) antagonists are effective in EAE. In addition, the profile of NIBR-0213 makes it an attractive candidate to further study the consequences of S1P(1) receptor antagonism and to differentiate the effects from those of S1P(1) agonists.
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Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Dipéptidos/farmacología , Dipéptidos/uso terapéutico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Administración Oral , Compuestos de Anilina/administración & dosificación , Compuestos de Anilina/química , Animales , Células CHO , Cricetinae , Cricetulus , Dipéptidos/administración & dosificación , Dipéptidos/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Encefalomielitis Autoinmune Experimental/patología , Femenino , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Recuento de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Estructura Molecular , Ratas , Ratas Endogámicas Lew , Ratas Wistar , Receptores de Esfingosina-1-Fosfato , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
FTY720 (fingolimod) has demonstrated efficacy in multiple sclerosis (MS). We evaluated the effects of FTY720 on progressive disability, viral load, and antibody responses in mice infected with Theiler's murine encephalomyocarditis virus (TMEV). FTY720 and phosphorylated FTY720 (FTY720-P) were detected in the brain after intraperitoneal injection of the drug. Bioactivity of FTY720 was confirmed by reduced numbers of mononuclear cells in the spleen and blood after treatment. No significant differences were found in disability progression, viral load, and serum antibody responses between the FTY720-treated versus the PBS-treated mice. There was less production of IgG within the CNS in the FTY-treated group on some measures.
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Modelos Animales de Enfermedad , Inmunosupresores/uso terapéutico , Esclerosis Múltiple/patología , Esclerosis Múltiple/virología , Glicoles de Propileno/uso terapéutico , Esfingosina/análogos & derivados , Theilovirus/efectos de los fármacos , Animales , Infecciones por Cardiovirus/tratamiento farmacológico , Infecciones por Cardiovirus/inmunología , Infecciones por Cardiovirus/patología , Femenino , Clorhidrato de Fingolimod , Inmunosupresores/farmacología , Inyecciones Intraperitoneales , Ratones , Esclerosis Múltiple/tratamiento farmacológico , Glicoles de Propileno/farmacología , Esfingosina/farmacología , Esfingosina/uso terapéutico , Theilovirus/inmunología , Carga Viral/efectos de los fármacos , Carga Viral/inmunologíaRESUMEN
BACKGROUND: Angiogenesis is a common finding in chronic inflammatory diseases; however, its role in multiple sclerosis (MS) is unclear. Central nervous system lesions from both MS and experimental autoimmune encephalomyelitis (EAE), the animal model of MS, contain T cells, macrophages and activated glia, which can produce pro-angiogenic factors. Previous EAE studies have demonstrated an increase in blood vessels, but differences between the different phases of disease have not been reported. Therefore we examined angiogenic promoting factors in MS and EAE lesions to determine if there were changes in blood vessel density at different stages of EAE. METHODS: In this series of experiments we used a combination of vascular casting, VEGF ELISA and immunohistochemistry to examine angiogenesis in experimental autoimmune encephalomyelitis (EAE). Using immunohistochemistry we also examined chronic active MS lesions for angiogenic factors. RESULTS: Vascular casting and histological examination of the spinal cord and brain of rats with EAE demonstrated that the density of patent blood vessels increased in the lumbar spinal cord during the relapse phase of the disease (p < 0.05). We found an increased expression of VEGF by inflammatory cells and a decrease in the recently described angiogenesis inhibitor meteorin. Examination of chronic active human MS tissues demonstrated glial expression of VEGF and glial and blood vessel expression of the pro-angiogenic receptor VEGFR2. There was a decreased expression of VEGFR1 in the lesions compared to normal white matter. CONCLUSIONS: These findings reveal that angiogenesis is intimately involved in the progression of EAE and may have a role in MS.
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Inductores de la Angiogénesis/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Esclerosis Múltiple/patología , Neovascularización Patológica , Adulto , Anciano , Animales , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Cobayas , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/metabolismo , Ratas , Ratas Endogámicas Lew , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Complement factor C3 is the central component of the complement system and a key inflammatory protein activated in Alzheimer's disease (AD). Previous studies demonstrated that inhibition of C3 by overexpression of soluble complement receptor-related protein y in an AD mouse model led to reduced microgliosis, increased amyloid beta (Abeta) plaque burden, and neurodegeneration. To further address the role of C3 in AD pathology, we generated a complement C3-deficient amyloid precursor protein (APP) transgenic AD mouse model (APP;C3(-/-)). Brains were analyzed at 8, 12, and 17 months of age by immunohistochemical and biochemical methods and compared with age-matched APP transgenic mice. At younger ages (8-12 months), no significant neuropathological differences were observed between the two transgenic lines. In contrast, at 17 months of age, APP;C3(-/-) mice showed significant changes of up to twofold increased total Abeta and fibrillar amyloid plaque burden in midfrontal cortex and hippocampus, which correlated with (1) significantly increased Tris-buffered saline (TBS)-insoluble Abeta(42) levels and reduced TBS-soluble Abeta(42) and Abeta(40) levels in brain homogenates, (2) a trend for increased Abeta levels in the plasma, (3) a significant loss of neuronal-specific nuclear protein-positive neurons in the hippocampus, and (4) differential activation of microglia toward a more alternative phenotype (e.g., significantly increased CD45-positive microglia, increased brain levels of interleukins 4 and 10, and reduced levels of CD68, F4/80, inducible nitric oxide synthase, and tumor necrosis factor). Our results suggest a beneficial role for complement C3 in plaque clearance and neuronal health as well as in modulation of the microglia phenotype.
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Precursor de Proteína beta-Amiloide/genética , Complemento C3/deficiencia , Microglía/metabolismo , Degeneración Nerviosa/metabolismo , Fenotipo , Placa Amiloide/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Complemento C3/genética , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/patología , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Placa Amiloide/genética , Placa Amiloide/patologíaRESUMEN
BACKGROUND: Activation of microglia is a part of the inflammatory response in neurodegenerative diseases but its role in the pathophysiology of these diseases is still unclear. The osteopetrotic (op/op) mouse lacks colony-stimulating factor-1 (CSF-1) and thus has a deficiency in microglia and macrophages. Prior reports have demonstrated that op/op mice deposit amyloid beta (Abeta) plaques, similar to those found in Alzheimer's disease. The purpose of these studies was to confirm this and to determine if the lack of CSF-1 affects the development of dopaminergic neurons and the expression of CD200, a known microglial inhibitory protein. METHOD: We examined the central nervous system of op/op mice at 30 days, 60 days and 7 months of age and wildtype littermates at 30 days using immunohistochemistry and histochemistry. RESULTS: We found a decrease in the number of microglia in 1 month-old op/op mice compared to wildtype (WT) littermates as measured by CD11b, CD45, CD32/16, CD68, CD204 and F4/80 immunoreactivity. Abeta plaques were not detected, while the number of dopaminergic neurons appeared normal. The expression of CD200 appeared to be normal, but there appeared to be a lower expression in the substantia nigra. CONCLUSION: In contrast to a prior report we did not detect Abeta deposition in the central nervous system of op/op mice at 30 days, 60 days or 7 months of age and there was a normal number of dopaminergic neurons. This indicates that op/op mice may be useful to examine the effects of microglia on neurodegenerative disease progression by breeding them to different transgenic mouse models. In addition, the lack of CSF-1 does not appear to affect CD200 expression by neurons but we did note a decrease in the substantia nigra of op/op and WT mice, suggesting that this may be a mechanism by which microglia control may be attenuated in this specific area during Parkinson's disease.
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Dopamina/metabolismo , Hipocampo/patología , Microglía/patología , Neuronas/patología , Osteopetrosis/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Dopamina/genética , Hipocampo/metabolismo , Ratones , Ratones Mutantes , Microglía/metabolismo , Neuronas/metabolismo , Osteopetrosis/genética , Osteopetrosis/metabolismoRESUMEN
Active and passive Abeta immunotherapy in Alzheimer's disease (AD)-like mouse models lowers cerebral amyloid-beta protein (Abeta) levels, especially if given early in the disease process, and improves cognitive deficits. In 2002, a Phase IIa clinical trial was halted due to meningoencephalitis in approximately 6% of the AD patients. It is hypothesized that the immunogen, full-length Abeta1-42, may have led to an autoimmune response. Currently, we are developing novel Abeta peptide immunogens for active immunization in amyloid precursor protein transgenic mice (APP Tg) to target Abeta B cell epitopes (within Abeta1-15) and avoid Abeta-specific T cell epitopes (Abeta16-42) so as to generate a safe and effective AD vaccine. Intranasal immunization with dendrimeric Abeta1-15 (16 copies of Abeta1-15 on a lysine core) or a tandem repeat of Abeta1-15 joined by 2 lysines and conjugated to an RGD motif with a mutated form of an E. coli-derived adjuvant generated robust Abeta titers in both wildtype and APP Tg mice. The Abeta antibodies recognized a B cell epitope within Abeta1-7, were mostly T-helper 2 associated immunoglobulin isotypes, bound human AD and APP Tg plaques, and detected Abeta oligomers. Splenic T cells reacted to the immunogens but not full-length Abeta. Six months of intranasal immunization (from 6-to-12 months of age) of J20 mice with each immunogen lowered insoluble Abeta42 by 50%, reduced plaque burden and gliosis, and increased Abeta in plasma. Interestingly, Abeta antibody generation was influenced by route of immunization. Transcutaneous immunization with dbeta1-15, but not full-length Abeta, led to high Abeta titers. In summary, our short Abeta immunogens induced robust titers of predominantly Th2 antibodies that were able to clear cerebral Abeta in the absence of Abeta-specific T cell reactivity, indicating the potential for a safer vaccine. We remain optimistic about the potential of such a vaccine for prevention and treatment of AD.
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Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/prevención & control , Vacunas contra el Alzheimer/uso terapéutico , Péptidos beta-Amiloides/inmunología , Fragmentos de Péptidos/uso terapéutico , Péptidos beta-Amiloides/química , Animales , Modelos Animales de Enfermedad , Humanos , Fragmentos de Péptidos/inmunologíaRESUMEN
Immunization of humans and APP-tg mice with full-length beta-amyloid (Abeta) results in reduced cerebral Abeta levels. However, due to adverse events in the AN1792 trial, alternative vaccines are required. We investigated dendrimeric Abeta1-15 (dAbeta1-15), which is composed of 16 copies of Abeta1-15 peptide on a branched lysine core and thus, includes an Abeta-specific B cell epitope but lacks the reported T cell epitope. Immunization by subcutaneous, transcutaneous, and intranasal routes of B6D2F1 wildtype mice led to anti-Abeta antibody production. Antibody isotypes were mainly IgG1 for subcutaneous or transcutaneous immunization and IgG2b for intranasal immunization, suggestive of a Th2-biased response. All Abeta antibodies preferentially recognized an epitope in Abeta1-7. Intranasal immunization of J20 APP-tg mice resulted in a robust humoral immune response with a corresponding significant reduction in cerebral plaque burden. Splenocyte proliferation against Abeta peptide was minimal indicating the lack of an Abeta-specific cellular immune response. Anti-Abeta antibodies bound monomeric, oligomeric, and fibrillar Abeta. Our data suggest that dAbeta1-15 may be an effective and potentially safer immunogen for Alzheimer's disease (AD) vaccination.
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Péptidos beta-Amiloides/inmunología , Precursor de Proteína beta-Amiloide/genética , Fragmentos de Péptidos/inmunología , Vacunas/inmunología , Administración Cutánea , Administración Intranasal , Péptidos beta-Amiloides/administración & dosificación , Análisis de Varianza , Animales , Anticuerpos/sangre , Especificidad de Anticuerpos , Proliferación Celular , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas de Escherichia coli/inmunología , Hipocampo/metabolismo , Humanos , Inmunoglobulina G , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/administración & dosificación , Vacunas/administración & dosificaciónRESUMEN
BACKGROUND: Immunotherapy for Alzheimer's disease (AD) is emerging as a potential treatment. However, a clinical trial (AN1792) was halted after adverse effects occurred in a small subset of subjects, which may have been caused by a T cell-mediated immunological response. In general, aging limits the humoral immune response, therefore, immunogens and vaccination regimes are required that induce a strong antibody response with less potential for an adverse immune response. METHOD: In the current study, we immunized both wildtype and J20 APP-tg mice with a priming injection of Abeta1-40/42, followed by multiple intranasal boosts with the novel immunogen dAbeta1-15 (16 copies of Abeta1-15 on a lysine tree), Abeta1-15 peptide or Abeta1-40/42 full length peptide. RESULTS: J20 APP-tg mice primed with Abeta1-40/42 subcutaneously and subsequently boosted intranasally with Abeta1-15 peptide did not generate a cellular or humoral immune response. In contrast, J20 APP-tg mice boosted intranasally with dAbeta1-15 or full length Abeta1-40/42 produced high levels of anti-Abeta antibodies. Splenocyte proliferation was minimal in mice immunized with dAbeta1-15. Wildtype littermates of the J20 APP-tg mice produced higher amounts of anti-Abeta antibodies compared to APP-tg mice but also had low T cell proliferation. The anti-Abeta antibodies were mainly composed of IgG2b and directed to an epitope within the Abeta1-7 region, regardless of the immunogen. Examination of the brain showed a significant reduction in Abeta plaque burden in the J20 APP-tg mice producing antibodies compared to controls. Biochemically, Abeta40 or Abeta42 were also reduced in brain homogenates and elevated in plasma but the changes did not reach significance. CONCLUSION: Our results demonstrate that priming with full length Abeta40/42 followed by boosting with dAbeta1-15 but not Abeta1-15 peptide led to a robust humoral immune response with a minimal T cell response in J20 APP-tg mice. In addition, Abeta plaque burden was reduced in mice producing anti-Abeta antibodies. Interestingly, wildtype mice produced higher levels of anti-Abeta antibodies, indicating that immune tolerance may be present in J20 APP-tg mice. Together, these data suggest that dAbeta1-15 but not Abeta1-15 peptide may be useful as a boosting immunogen in an AD vaccination regime.
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Amyloid-beta (Abeta) immunotherapy lowers cerebral Abeta and improves cognition in mouse models of Alzheimer's disease (AD). A clinical trial using active immunization with Abeta1-42 was suspended after approximately 6% of patients developed meningoencephalitis, possibly because of a T-cell reaction against Abeta. Nevertheless, beneficial effects were reported in antibody responders. Consequently, alternatives are required for a safer vaccine. The Abeta1-15 sequence contains the antibody epitope(s) but lacks the T-cell reactive sites of full-length Abeta1-42. Therefore, we tested four alternative peptide immunogens encompassing either a tandem repeat of two lysine-linked Abeta1-15 sequences (2xAbeta1-15) or the Abeta1-15 sequence synthesized to a cross-species active T1 T-helper-cell epitope (T1-Abeta1-15) and each with the addition of a three-amino-acid RGD (Arg-Gly-Asp) motif (R-2xAbeta1-15; T1-R-Abeta1-15). High anti-Abeta antibody titers were observed in wild-type mice after intranasal immunization with R-2xAbeta1-15 or 2xAbeta1-15 plus mutant Escherichia coli heat-labile enterotoxin LT(R192G) adjuvant. Moderate antibody levels were induced after immunization with T1-R-Abeta1-15 or T1-Abeta1-15 plus LT(R192G). Restimulation of splenocytes with the corresponding immunogens resulted in moderate proliferative responses, whereas proliferation was absent after restimulation with full-length Abeta or Abeta1-15. Immunization of human amyloid precursor protein, familial AD (hAPP(FAD)) mice with R-2xAbeta1-15 or 2xAbeta1-15 resulted in high anti-Abeta titers of noninflammatory T-helper 2 isotypes (IgG1 and IgG2b), a lack of splenocyte proliferation against full-length Abeta, significantly reduced Abeta plaque load, and lower cerebral Abeta levels. In addition, 2xAbeta1-15-immunized hAPP(FAD) animals showed improved acquisition of memory compared with vehicle controls in a reference-memory Morris water-maze behavior test that approximately correlated with anti-Abeta titers. Thus, our novel immunogens show promise for future AD vaccines.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Discapacidades para el Aprendizaje/terapia , Fragmentos de Péptidos/inmunología , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Análisis de Varianza , Animales , Anticuerpos/sangre , Especificidad de Anticuerpos , Toxinas Bacterianas/inmunología , Conducta Animal , Fenómenos Biofísicos , Biofisica , Química Encefálica , Proliferación Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Enterotoxinas/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Mapeo Epitopo/métodos , Proteínas de Escherichia coli/inmunología , Inmunización Secundaria , Inmunohistoquímica/métodos , Discapacidades para el Aprendizaje/etiología , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/administración & dosificación , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factores de Tiempo , VacunasRESUMEN
Alzheimer disease (AD), the most common form of dementia, is without an effective cure or preventive treatment. Recently, amyloid-beta protein (Abeta) has become a major therapeutic target. Many efforts are underway to either reduce the production of Abeta or enhance its clearance. In 1999, Schenk and colleagues first showed that active immunization with full-length Abeta lowered cerebral Abeta levels in transgenic mice. These findings have been confirmed and extended in various transgenic mouse models of AD using both active and passive Abeta immunization. Cognitive improvement also has been reported in association with active and passive Abeta vaccination in AD-like mouse models, even in the absence of significant reductions in cerebral Abeta loads. In 2004, the authors reported that active immunization with full-length Abeta in aged nonhuman primates, Caribbean vervets, reduced cerebral Abeta levels and gliosis. Proposed mechanisms of Abeta clearance by immunotherapy include disruption of Abeta aggregates, Abeta phagocytosis by microglia, neutralization of Abeta oligomers at the synapse, and increased efflux of Abeta from brain to blood. A phase IIa clinical trial was halted in 2002 because of the appearance of meningoencephalitis in approximately 6% of the AD patients. Although the exact cause of these adverse events is unknown, the immunogen, full-length Abeta1-42, may have been recognized as a self-antigen leading to an autoimmune response in some patients. Limited cognitive stabilization and apparent plaque clearance have been reported in subsets of patients who generated antibody titers. Currently, a passive immunization trial with a recombinant humanized monoclonal Abeta antibody is underway in humans. In the meantime, the authors are developing novel Abeta peptide immunogens for active immunization to target Abeta B cell epitope(s) and avoid Abeta-specific T-cell reactions in order to generate a safe and effective AD vaccine. The authors remain optimistic about the potential of such a vaccine for the prevention and treatment of AD.
Asunto(s)
Enfermedad de Alzheimer/prevención & control , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/inmunología , Inmunización , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/psicología , Péptidos beta-Amiloides/metabolismo , Animales , Chlorocebus aethiops , Humanos , Inmunización Pasiva , Ratones , Ratones TransgénicosRESUMEN
Activated microglia and reactive astrocytes invade and surround cerebral beta amyloid (Abeta) plaques in Alzheimer's disease (AD), but the role of microglia in plaque development is still unclear. In this study, minocycline was administered for 3 months, prior to and early in Abeta plaque formation in amyloid precursor protein transgenic mice (APP-tg). When minocycline was given to younger mice, there was a small but significant increase in Abeta deposition in the hippocampus, concurrent with improved cognitive performance relative to vehicle treated mice. If APP-tg mice received minocycline after Abeta deposition had begun, microglial activation was suppressed but this did not affect Abeta deposition or improve cognitive performance. In vitro studies demonstrated that minocycline suppressed microglial production of IL-1beta, IL-6, TNF, and NGF. Thus, minocycline has different effects on Abeta plaque deposition and microglia activation depending on the age of administration. Our data suggest that this may be due to the effects of minocycline on microglial function. Therefore, anti-inflammatory therapies to suppress microglial activation or function may reduce cytokine production but enhance Abeta plaque formation early in AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Precursor de Proteína beta-Amiloide/metabolismo , Gliosis/tratamiento farmacológico , Microglía/metabolismo , Minociclina/farmacología , Placa Amiloide/efectos de los fármacos , Factores de Edad , Envejecimiento/fisiología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Precursor de Proteína beta-Amiloide/genética , Animales , Animales Recién Nacidos , Antibacterianos/farmacología , Antiinflamatorios/efectos adversos , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/fisiopatología , Línea Celular Tumoral , Células Cultivadas , Cognición/efectos de los fármacos , Cognición/fisiología , Citocinas/biosíntesis , Citocinas/efectos de los fármacos , Modelos Animales de Enfermedad , Encefalitis/genética , Encefalitis/metabolismo , Encefalitis/fisiopatología , Gliosis/fisiopatología , Gliosis/prevención & control , Humanos , Ratones , Ratones Transgénicos , Microglía/efectos de los fármacos , Placa Amiloide/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Abeta vaccination or passive transfer of human-specific anti-Abeta antibodies are approaches under investigation to prevent and/or treat Alzheimer's disease (AD). Successful active Abeta vaccination requires a strong and safe adjuvant to induce anti-Abeta antibody formation. We compared the adjuvants monophosphoryl lipid A (MPL)/trehalose dicorynomycolate (TDM), cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin LT(R192G) for their ability to induce a humoral and cellular immune reaction, using fibrillar Abeta1-40/42 as a common immunogen in wildtype B6D2F1 mice. Subcutaneous (s.c.) administration with MPL/TDM resulted in anti-Abeta antibodies levels up to four times higher compared to s.c. LT(R192G). Using MPL/TDM, the anti-Abeta antibodies induced were mainly IgG2b, IgG1 and lower levels of IgG2a and IgM, with a moderate splenocyte proliferation and IFN-gamma production in vitro upon stimulation with Abeta1-40/42. LT(R192G), previously shown by us to induce robust titers of anti-Abeta antibodies, generated predominantly IgG2b and IgG1 anti-Abeta antibodies with very low splenocyte proliferation and IFN-gamma production. Weekly intranasal (i.n.) administration over 11 weeks of Abeta40/42 with CTB induced only moderate levels of antibodies. All immunogens generated antibodies that recognized mainly the Abeta1-7 epitope and specifically detected amyloid plaques on AD brain sections. In conclusion, MPL/TDM, in addition to LT(R192G), is an effective adjuvant when combined with Abeta40/42 and may aid in the design of Abeta immunotherapy.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Enfermedad de Alzheimer/prevención & control , Vacunas contra el Alzheimer/administración & dosificación , Péptidos beta-Amiloides/inmunología , Enfermedad de Alzheimer/inmunología , Animales , Formación de Anticuerpos/efectos de los fármacos , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/inmunología , Toxina del Cólera/administración & dosificación , Toxina del Cólera/inmunología , Enterotoxinas/administración & dosificación , Enterotoxinas/inmunología , Escherichia coli/inmunología , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/inmunología , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunoterapia , Lípido A/administración & dosificación , Lípido A/análogos & derivados , Lípido A/inmunología , Masculino , RatonesRESUMEN
Active amyloid beta (A beta) vaccination has been shown to be effective in clearing cerebral A beta and improving cognitive function in mouse models of Alzheimer's disease. However, an A beta vaccine clinical trial was suspended after meningoencephalitis was detected in a subset of subjects. Passive immunization has been suggested to be a safer alternative to active A beta immunization but there are reports of increased risk of microhemorrhages associated with its administration in aged beta-amyloid precursor protein transgenic mice bearing abundant vascular amyloid deposition. In addition, the cost may be prohibitive for large-scale clinical use. Therefore, we are designing novel A beta immunogens that encompass the B cell epitope of A beta but lack the T cell-reactive sites. These immunogens induced the production of A beta-specific antibodies in the absence of an A beta-specific cellular immune response in wild-type mice and are being tested in beta-amyloid precursor protein transgenic mice. These data together with published reports from several other groups suggest that a safe, active A beta vaccine is a tenable goal.
Asunto(s)
Enfermedad de Alzheimer/prevención & control , Vacunas contra el Alzheimer/uso terapéutico , Péptidos beta-Amiloides/inmunología , Vacunas contra el Alzheimer/efectos adversos , Animales , Formación de Anticuerpos , Ensayos Clínicos como Asunto , Epítopos/inmunología , Humanos , Inmunoterapia , Ratones , Ratones Transgénicos , Secuencias Repetidas en TándemRESUMEN
The cerebral accumulation of beta-amyloid (Abeta) is a pathological hallmark of Alzheimer's disease (AD). Abeta vaccination or anti-Abeta specific antibodies may be a possible therapeutic option for AD. Previously, we demonstrated variation in the humoral response between B6D2F1 and C57BL/6 during short term (14 weeks) Abeta immunization. In the present study, we determined the humoral and cellular immune responses in these same mouse strains to a longer period of Abeta vaccination and further refined the major B cell epitope to Ass1-7. B6D2F1 mice generated a greater humoral and Th1 immune response versus C57BL/6 mice. Immunization with 25 microg Abeta produced a greater T cell response in B6D2F1 mice compared to 50 or 100 microg Abeta but resulted in comparable humoral immunity. Thus, Abeta vaccination is affected by the genetic background and amount of Abeta peptide used as immunogen. These data may help explain some differences observed in Abeta immunization studies in mice of various genetic backgrounds and aid in the design of Abeta vaccines.
Asunto(s)
Vacunas contra el Alzheimer/inmunología , Péptidos beta-Amiloides/inmunología , Formación de Anticuerpos , Inmunidad Celular , Péptidos beta-Amiloides/administración & dosificación , Animales , Anticuerpos/sangre , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Inmunoglobulina G/sangre , Interferón gamma/análisis , Interleucina-2/análisis , Interleucina-4/análisis , Interleucina-5/análisis , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Especificidad de la Especie , Factores de Tiempo , VacunaciónRESUMEN
Amyloid beta (A beta) immunization of amyloid precursor protein (APP)-transgenic (tg) mice with human A beta induces humoral immunity, however, the immune response to endogenous rodent A beta is unknown. Fourteen-month J20 APP-tg mice and non-tg littermates were immunized subcutaneously followed by chronic intranasal boosting with human or rodent A beta peptide and adjuvant LT(R192G). Rodent A beta-immunized APP-tg mice had anti-rodent A beta antibody levels of 257.8 micrograms/ml and those immunized with human A beta had anti-human A beta antibodies of 120.8 micrograms/ml. Non-tg littermates had anti-rodent and anti-human A beta antibody concentrations of 98.8 and 231.1 microgram/ml, respectively. Inter-species cross-reactivity was minimal. Anti-human A beta antibodies were predominately IgG1 and IgG2b, while anti-rodent A beta antibodies were equally IgG1, IgG2a, and IgG2b. Anti-human A beta antibodies recognized an epitope within human A beta1-9. Anti-rodent A beta antibodies did not stain Alzheimer's disease (AD) plaques but bound some plaques in APP-tg mice. Splenocytes proliferated modestly to their respective antigen and secreted low levels of IL-2 and IFN-gamma. Therefore, immunizing APP-tg and non-tg mice with rodent A beta resulted in a species-specific humoral response with modest T cell reactivity.