RESUMEN

The potent costimulatory effect of CD137 has been implicated in several murine autoimmune disease models. CD137 costimulates and polarizes antigen-specific T cells toward a potent Th1/Tc1 response, and is essential for the development of experimental autoimmune encephalomyelitis (EAE), a murine model of Multiple Sclerosis (MS). This study aimed to investigate a role of CD137 in MS. Immunohistochemical and immunofluorescence staining of MS brain tissues was used to identify expression of CD137. CD137+ cells were identified in MS brain samples, with active lesions having the highest frequency of CD137+ cells. CD137 expression was found on several leukocyte subsets, including T cells, B cells and endothelial cells. In particular, CD137+ B cells were found in meningeal infiltrates. In vitro experiments showed that CD137 engagement on activated B cells increased early TNF and persistent IL-6 secretion with increased cell proliferation. These CD137+ B cells could interact with CD137L-expressing cells, secrete pro-inflammatory cytokines and accumulate in the meningeal infiltrate. This study demonstrates CD137 expression by activated B cells, enhancement of the inflammatory activity of B cells upon CD137 engagement, and provides evidence for a pathogenic role of CD137+ B cells in MS.

Asunto(s)

Ligando 4-1BB/metabolismo , Linfocitos B/inmunología , Interleucina-6/metabolismo , Meninges/metabolismo , Esclerosis Múltiple/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Femenino , Humanos , Activación de Linfocitos , Masculino , Meninges/patología , Persona de Mediana Edad , Transducción de Señal , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo

RESUMEN

Optogenetic stimulation of neural stem cells (NSCs) enables their activity-dependent photo-modulation. This provides a spatio-temporal tool for studying activity-dependent neurogenesis and for regulating the differentiation of the transplanted NSCs. Currently, this is mainly driven by viral transfection of channelrhodopsin-2 (ChR2) gene, which requires high irradiance and complex in vivo/vitro stimulation systems. Additionally, despite the extensive application of optogenetics in neuroscience, the transcriptome-level changes induced by optogenetic stimulation of NSCs have not been elucidated yet. Here, we made transformed NSCs (SFO-NSCs) stably expressing one of the step-function opsin (SFO)-variants of chimeric channelrhodopsins, ChRFR(C167A), which is more sensitive to blue light than native ChR2, via a non-viral transfection system using piggyBac transposon. We set up a simple low-irradiance optical stimulation (OS)-incubation system that induced c-fos mRNA expression, which is activity-dependent, in differentiating SFO-NSCs. More neuron-like SFO-NCSs, which had more elongated axons, were differentiated with daily OS than control cells without OS. This was accompanied by positive/negative changes in the transcriptome involved in axonal remodeling, synaptic plasticity, and microenvironment modulation with the up-regulation of several genes involved in the Ca2+-related functions. Our approach could be applied for stem cell transplantation studies in tissue with two strengths: lower carcinogenicity and less irradiance needed for tissue penetration.

Asunto(s)

Células-Madre Neurales/efectos de la radiación , Neurogénesis/efectos de la radiación , Optogenética , Señalización del Calcio , Línea Celular Transformada , Channelrhodopsins/biosíntesis , Channelrhodopsins/genética , Channelrhodopsins/efectos de la radiación , Elementos Transponibles de ADN , Regulación de la Expresión Génica/efectos de la radiación , Ontología de Genes , Genes Reporteros , Genes fos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/citología , Plasticidad Neuronal/efectos de la radiación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transcriptoma/efectos de la radiación , Regulación hacia Arriba/efectos de la radiación

RESUMEN

Optogenetics is an optical technique that exploits visible light for selective neuromodulation with spatio-temporal precision. Despite enormous effort, the effective stimulation of targeted neurons, which are located in deeper structures of the nervous system, by visible light, remains a technical challenge. Compared to visible light, near-infrared illumination offers a higher depth of tissue penetration owing to a lower degree of light attenuation. Herein, an overview of advances in developing new modalities for neural circuitry modulation utilizing upconversion-nanoparticle-mediated optogenetics is presented. These developments have led to minimally invasive optical stimulation and inhibition of neurons with substantially improved selectivity, sensitivity, and spatial resolution. The focus is to provide a comprehensive review of the mechanistic basis for evaluating upconversion parameters, which will be useful in designing, executing, and reporting optogenetic experiments.

Asunto(s)

Nanomedicina/métodos , Nanopartículas , Fenómenos Fisiológicos del Sistema Nervioso/genética , Optogenética/métodos , Animales , Humanos

RESUMEN

Cargo transport along axons, a physiological process mediated by motor proteins, is essential for neuronal function and survival. A current limitation in the study of axonal transport is the lack of a robust imaging technique with a high spatiotemporal resolution to visualize and quantify the movement of motor proteins in real-time and in different depth planes. Herein, we present a dynamic imaging technique that fully exploits the characteristics of upconversion nanoparticles. This technique can be used as a microscopic probe for the quantitative in situ tracking of retrograde transport neurons with single-particle resolution in multilayered cultures. This study may provide a powerful tool to reveal dynamic neuronal activity and intra-axonal transport function as well as any associated neurodegenerative diseases resulting from mutation or impairment in the axonal transport machinery.

Asunto(s)

Nanopartículas del Metal/química , Proteínas Motoras Moleculares/metabolismo , Neuronas/metabolismo , Animales , Axones/química , Axones/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Reprogramación Celular , Dineínas/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Rayos Infrarrojos , Ratones , Microscopía Fluorescente , Neuronas/citología , Transporte de Proteínas , Ratas

RESUMEN

BACKGROUND CONTEXT: Although general hypothermia is recognized as a clinically applicable neuroprotective intervention, acute moderate local hypothermia post contusive spinal cord injury (SCI) is being considered a more effective approach. Previously, we have investigated the feasibility and safety of inducing prolonged local hypothermia in the central nervous system of a rodent model. PURPOSE: Here, we aimed to verify the efficacy and neuroprotective effects of 5 and 8 hours of local moderate hypothermia (30±0.5°C) induced 2 hours after moderate thoracic contusive SCI in rats. STUDY DESIGN: Rats were induced with moderate SCI (12.5 mm) at its T8 section. Local hypothermia (30±0.5°C) was induced 2 hours after injury induction with an M-shaped copper tube with flow of cold water (12°C), from the T6 to the T10 region. Experiment groups were divided into 5-hour and 8-hour hypothermia treatment groups, respectively, whereas the normothermia control group underwent no hypothermia treatment. METHODS: The neuroprotective effects were assessed through objective weekly somatosensory evoked potential (SSEP) and motor behavior (basso, beattie and bresnahan Basso, Beattie and Bresnahan (BBB) scoring) monitoring. Histology on spinal cord was performed until at the end of day 56. All authors declared no conflict of interest. This work was supported by the Singapore Institute for Neurotechnology Seed Fund (R-175-000-121-733), National University of Singapore, Ministry of Education, Tier 1 (R-172-000-414-112.). RESULTS: Our results show significant SSEP amplitudes recovery in local hypothermia groups starting from day 14 post-injury onward for the 8-hour treatment group, which persisted up to days 28 and 42, whereas the 5-hour group showed significant improvement only at day 42. The functional improvement plateaued after day 42 as compared with control group of SCI with normothermia. This was supported by both 5-hour and 8-hour improvement in locomotion as measured by BBB scores. Local hypothermia also observed insignificant changes in its SSEP latency, as compared with the control. In addition, 5- and 8-hour hypothermia rats' spinal cord showed higher percentage of parenchyma preservation. CONCLUSIONS: Early local moderate hypothermia can be induced for extended periods of time post SCI in the rodent model. Such intervention improves functional electrophysiological outcome and motor behavior recovery for a long time, lasting until 8 weeks.

Asunto(s)

Contusiones/terapia , Hipotermia Inducida/métodos , Traumatismos de la Médula Espinal/terapia , Animales , Contusiones/fisiopatología , Potenciales Evocados Somatosensoriales , Femenino , Locomoción , Masculino , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/fisiopatología

RESUMEN

Reactive astrogliosis is a critical process in neuropathological conditions and neurotrauma. Although it has been suggested that it confers neuroprotective effects, the exact genomic mechanism has not been explored. The prevailing dogma of the role of astrogliosis in inhibition of axonal regeneration has been challenged by recent findings in rodent model's spinal cord injury, demonstrating its neuroprotection and axonal regeneration properties. We examined whether their neuroprotective and axonal regeneration potentials can be identify in human spinal cord reactive astrocytes in vitro. Here, reactive astrogliosis was induced with IL1ß. Within 24 hours of IL1ß induction, astrocytes acquired reactive characteristics. Transcriptome analysis of over 40000 transcripts of genes and analysis with PFSnet subnetwork revealed upregulation of chemokines and axonal permissive factors including FGF2, BDNF, and NGF. In addition, most genes regulating axonal inhibitory molecules, including ROBO1 and ROBO2 were downregulated. There was no increase in the gene expression of "Chondroitin Sulfate Proteoglycans" (CSPGs') clusters. This suggests that reactive astrocytes may not be the main CSPG contributory factor in glial scar. PFSnet analysis also indicated an upregulation of "Axonal Guidance Signaling" pathway. Our result suggests that human spinal cord reactive astrocytes is potentially neuroprotective at an early onset of reactive astrogliosis.

Asunto(s)

Astrocitos/metabolismo , Biomarcadores/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1beta/farmacología , Proteínas del Tejido Nervioso/metabolismo , Neuroprotección/genética , Médula Espinal/metabolismo , Astrocitos/citología , Astrocitos/efectos de los fármacos , Axones/metabolismo , Células Cultivadas , Feto/citología , Feto/efectos de los fármacos , Feto/metabolismo , Perfilación de la Expresión Génica , Humanos , Proteínas del Tejido Nervioso/genética , Médula Espinal/citología , Médula Espinal/efectos de los fármacos

RESUMEN

The cellular-level effects of low/high frequency oscillating magnetic field on excitable cells such as neurons are well established. In contrast, the effects of a homogeneous, static magnetic field (SMF) on Central Nervous System (CNS) glial cells are less investigated. Here, we have developed an in vitro SMF stimulation set-up to investigate the genomic effects of SMF exposure on oligodendrocyte differentiation and neurotrophic factors secretion. Human oligodendrocytes precursor cells (OPCs) were stimulated with moderate intensity SMF (0.3 T) for a period of two weeks (two hours/day). The differential gene expression of cell activity marker (c-fos), early OPC (Olig1, Olig2. Sox10), and mature oligodendrocyte markers (CNP, MBP) were quantified. The enhanced myelination capacity of the SMF stimulated oligodendrocytes was validated in a dorsal root ganglion microfluidics chamber platform. Additionally, the effects of SMF on the gene expression and secretion of neurotrophic factors- BDNF and NT3 was quantified. We also report that SMF stimulation increases the intracellular calcium influx in OPCs as well as the gene expression of L-type channel subunits-CaV1.2 and CaV1.3. Our findings emphasize the ability of glial cells such as OPCs to positively respond to moderate intensity SMF stimulation by exhibiting enhanced differentiation, functionality as well as neurotrophic factor release.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/metabolismo , Calcio/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Campos Magnéticos , Proteína Básica de Mielina/metabolismo , Oligodendroglía/metabolismo , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/genética , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Diferenciación Celular , Embrión de Mamíferos , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Técnicas Analíticas Microfluídicas , Proteína Básica de Mielina/genética , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurotrofina 3 , Factor de Transcripción 2 de los Oligodendrocitos/genética , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Oligodendroglía/citología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo

RESUMEN

Direct conversion through transdifferentiation is a promising cell reprogramming approach that induces a cell lineage conversion among adult cells without passing through an intermediate pluripotent phase. However, there is a need to critically evaluate the efficacy and safety of direct conversion to establish its feasibility as a clinically viable cell reprogramming technique. This review article aims to delineate some critical constraints of direct conversion as a cellular reprogramming approach. We report the most important challenges of lineage reprogramming through direct conversion and divide them into two major sections. The first section explores the obstacles that limit the efficiency of the direct conversion process. In this study, we discuss challenges such as lack of understanding of molecular mechanism and transcriptional control of direct conversion, low proliferative capacity of converted cells, and senescence and apoptosis as critical barriers of direct conversion. The second section focuses on addressing concerns of safety of directly converted cells. We describe issues of transgene load and epigenetic memory retention along with the constraints of currently available validation tools to characterize reprogrammed cells. Each issue mentioned above is evaluated in view of their origin, implications, progress made toward their resolution and scope for development of new strategies to address the constraints of the present technique.

Asunto(s)

Transdiferenciación Celular , Animales , Linaje de la Célula/genética , Reprogramación Celular/genética , Senescencia Celular/genética , Epigénesis Genética , Humanos , Modelos Biológicos

RESUMEN

Rapid progress in the field of stem cell therapy and cellular reprogramming provides convincing evidence of its feasibility in treating a wide range of pathologies through autologous cell replacement therapy. This review article describes in detail on three widely used approaches of somatic cell reprogramming: induced pluripotent stem cells, direct conversion and direct reprogramming, in the context of demyelination in the CNS. The potential limitations of each reprogramming technique are reviewed along with their distinct molecular approach to reprogramming. This is followed by an analysis on the scopes and challenges of its translational applications in deriving oligodendrocyte progenitor cells and oligodendrocytes for cell replacement treatment of demyelinating conditions in the CNS.

Asunto(s)

Transdiferenciación Celular , Técnicas de Reprogramación Celular/métodos , Enfermedades Desmielinizantes/terapia , Células Madre Pluripotentes Inducidas , Oligodendroglía , Trasplante de Células Madre/métodos , Animales , Autoinjertos , Humanos

RESUMEN

In this work, PEO-α-CD pseudorotaxane hydrogels were prepared. The gels were loaded with proteins, BSA and lysozyme, representing proteins with different molecular weights. The kinetics of protein release was studied. Factors such as PEO concentration, protein concentration and exposed surface area of the gels were investigated to understand their effects on the release of the encapsulated cargo. Erosion of the gel surface appeared to be the dominant factor for release of the proteins. Fitting the data to various models supported our hypothesis that the mechanism of release was primarily erosion-driven as the data was best described by zero order, power law and Hopfenberg model. The linear relationship between the amount of mass loss and time establishes the erosion of the polymer gel matrix to be the key factor for drug release.

Asunto(s)

Ciclodextrinas/química , Preparaciones de Acción Retardada/química , Hidrogeles/química , Rotaxanos/química , Sistemas de Liberación de Medicamentos , Modelos Teóricos , Peso Molecular , Muramidasa/química , Muramidasa/farmacología , Polietilenglicoles/química , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/farmacología