RESUMEN
Sanfilippo syndrome (MPS III) is an autosomal recessive inherited disorder causing dementia in children, following an essentially normal early developmental period. First symptoms typically include delayed language development, hyperactivity and/or insomnia from 2 years of age, followed by unremitting and overt loss of previously acquired skills. There are no approved treatments, and the median age of death is 18 years. Treatments under clinical trial demonstrate therapeutic benefit when applied pre-symptomatically in children diagnosed early through known familial inheritance risk. Newborn screening for Sanfilippo syndrome would enable pre-symptomatic diagnosis and optimal therapeutic benefit, however, many fold more patients with Sanfilippo syndrome are expected to be identified in the population than present with childhood dementia. Therefore, the capacity to stratify which Sanfilippo infants will need treatment in toddlerhood is necessary. While diagnostic methods have been developed, and continue to be refined, currently there are no tools or laboratory-based biomarkers available to provide pre-symptomatic prognosis. There is also a lack of progression and neurocognitive response-to-treatment biomarkers; disease stage and rate of progression are currently determined by age at symptom onset, loss of cerebral grey matter volume by magnetic resonance imaging and developmental quotient score for age. Robust blood-based biomarkers are an urgent unmet need. In this review, we discuss the development of biomarker assays for Sanfilippo based on the neuropathological pathways known to change leading into symptom onset and progression, and their performance as biomarkers in other neurodegenerative diseases. We propose that neural-derived exosomes extracted from blood may provide an ideal liquid biopsy to detect reductions in synaptic protein availability, and mitochondrial function. Furthermore, given the prominent role of neuroinflammation in symptom expression, glial fibrillary acidic protein detection in plasma/serum, alongside measurement of active brain atrophy by neurofilament light chain, warrant increased investigation for prognostic, progression and neurocognitive response-to-treatment biomarker potential in Sanfilippo syndrome and potentially other childhood dementias.
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Demencia , Mucopolisacaridosis III , Niño , Lactante , Recién Nacido , Humanos , Adolescente , Mucopolisacaridosis III/diagnóstico , Mucopolisacaridosis III/patología , Biomarcadores , PronósticoRESUMEN
BACKGROUND AND PURPOSE: The aim was to evaluate urinary neopterin, a marker of pro-inflammatory state, as a potential biomarker of disease prognosis and progression in amyotrophic lateral sclerosis (ALS); and to compare its utility to urinary neurotrophin receptor p75 extracellular domain (p75ECD ). METHODS: This was an observational study including 21 healthy controls and 46 people with ALS, 29 of whom were sampled longitudinally. Neopterin and p75ECD were measured using enzyme-linked immunoassays. Baseline and longitudinal changes in clinical measures, neopterin and urinary p75ECD were examined, and prognostic utility was explored by survival analysis. RESULTS: At baseline, urinary neopterin was higher in ALS compared to controls (181.7 ± 78.9 µmol/mol creatinine vs. 120.4 ± 60.8 µmol/mol creatinine, p = 0.002, Welch's t test) and correlated with the Revised ALS Functional Rating Scale (r = -0.36, p = 0.01). Combining previously published urinary p75ECD results from 22 ALS patients with a further 24 ALS patients, baseline urinary p75ECD was also higher compared to healthy controls (6.0 ± 2.7 vs. 3.2 ± 1.0 ng/mg creatinine, p < 0.0001) and correlated with the Revised ALS Functional Rating Scale (r = -0.36, p = 0.01). Urinary neopterin and p75ECD correlated with each other at baseline (r = 0.38, p = 0.009). In longitudinal analysis, urinary neopterin increased on average (±SE) by 6.8 ± 1.1 µmol/mol creatinine per month (p < 0.0001) and p75ECD by 0.19 ± 0.02 ng/mg creatinine per month (p < 0.0001) from diagnosis in 29 ALS patients. CONCLUSION: Urinary neopterin holds promise as marker of disease progression in ALS and is worthy of future evaluation for its potential to predict response to anti-inflammatory therapies.
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Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/diagnóstico , Biomarcadores , Progresión de la Enfermedad , Humanos , Neopterin , PronósticoRESUMEN
The p75 neurotrophin receptor (p75(NTR); also known as NGFR) can mediate neuronal apoptosis in disease or following trauma, and facilitate survival through interactions with Trk receptors. Here we tested the ability of a p75(NTR)-derived trophic cell-permeable peptide, c29, to inhibit p75(NTR)-mediated motor neuron death. Acute c29 application to axotomized motor neuron axons decreased cell death, and systemic c29 treatment of SOD1(G93A) mice, a common model of amyotrophic lateral sclerosis, resulted in increased spinal motor neuron survival mid-disease as well as delayed disease onset. Coincident with this, c29 treatment of these mice reduced the production of p75(NTR) cleavage products. Although c29 treatment inhibited mature- and pro-nerve-growth-factor-induced death of cultured motor neurons, and these ligands induced the cleavage of p75(NTR) in motor-neuron-like NSC-34 cells, there was no direct effect of c29 on p75(NTR) cleavage. Rather, c29 promoted motor neuron survival in vitro by enhancing the activation of TrkB-dependent signaling pathways, provided that low levels of brain-derived neurotrophic factor (BDNF) were present, an effect that was replicated in vivo in SOD1(G93A) mice. We conclude that the c29 peptide facilitates BDNF-dependent survival of motor neurons in vitro and in vivo.
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Muerte Celular/fisiología , Péptidos de Penetración Celular/metabolismo , Neuronas Motoras/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Animales , Apoptosis/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Supervivencia Celular/fisiología , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal/fisiología , Médula Espinal/metabolismo , Médula Espinal/fisiología , Superóxido Dismutasa/metabolismoRESUMEN
Multifunctional SiO2 microtubes for targeted drug delivery are produced with precise control over shape and size by combining lithography and electrochemical etching. The hollow core is loaded with a lipophilic anticancer drug generating nanopills and an antibody is conjugated to the external surface for cancer cell targeting. Results demonstrate selective killing of neuroblastoma cells that express the cognate receptor.
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Antineoplásicos/química , Camptotecina/administración & dosificación , Sistemas de Liberación de Medicamentos , Nanomedicina/métodos , Nanopartículas/química , Dióxido de Silicio/química , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Electroquímica , Humanos , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microtúbulos/química , Neoplasias/metabolismo , Proteínas del Tejido Nervioso/química , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Receptores de Factor de Crecimiento Nervioso/química , Propiedades de SuperficieRESUMEN
Healthcare facilities produce millions of tons of waste annually, with a significant portion consisting of diagnostic plasticware. Here, we introduce a new detection platform that completely replaces traditional assay plates with a piece of membrane, offering a much greener and more sustainable alternative. The membrane, integrated within the portable vortex fluidic device (P-VFD), enables rapid detection of a clinically relevant protein biomarker, urinary p75ECD. This biomarker is utilized to evaluate the prognosis, disease severity, and progression of amyotrophic lateral sclerosis (ALS). This assay has a limit-of-detection (LOD) of 4.03 pg, which is comparable to the plate-based assay (2.24 pg) and has been optimized through a full factorial design of experiments (DOE). P-VFD has great potential in quantifying p75ECD in human biofluids and can significantly reduce the assay time to 5 min compared to the current plate-based p75ECD ELISA assay (3 days), with at least a 4.4-fold reduction in the usage of the detection antibody.
RESUMEN
Amyotrophic lateral sclerosis is a relentless neurodegenerative disease that is mostly fatal within 3-5 years and is diagnosed on evidence of progressive upper and lower motor neuron degeneration. Around 15% of those with amyotrophic lateral sclerosis also have frontotemporal degeneration, and gene mutations account for â¼10%. Amyotrophic lateral sclerosis is a variable heterogeneous disease, and it is becoming increasingly clear that numerous different disease processes culminate in the final degeneration of motor neurons. There is a profound need to clearly articulate and measure pathological process that occurs. Such information is needed to tailor treatments to individuals with amyotrophic lateral sclerosis according to an individual's pathological fingerprint. For new candidate therapies, there is also a need for methods to select patients according to expected treatment outcomes and measure the success, or not, of treatments. Biomarkers are essential tools to fulfil these needs, and urine is a rich source for candidate biofluid biomarkers. This review will describe promising candidate urinary biomarkers of amyotrophic lateral sclerosis and other possible urinary candidates in future areas of investigation as well as the limitations of urinary biomarkers.
RESUMEN
Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a progressive neurological disorder, characterised by the death of upper and lower motor neurons. The aetiology of ALS remains unknown, and treatment options are limited. Endogenous retroviruses (ERVs), specifically human endogenous retrovirus type K (HERV-K), have been proposed to be involved in the propagation of neurodegeneration in ALS. ERVs are genomic remnants of ancient viral infection events, with most being inactive and not retaining the capacity to encode a fully infectious virus. However, some ERVs retain the ability to be activated and transcribed, and ERV transcripts have been found to be elevated within the brain tissue of MND patients. A hallmark of ALS pathology is altered localisation of the transactive response (TAR) DNA binding protein 43 kDa (TDP-43), which is normally found within the nucleus of neuronal and glial cells and is involved in RNA regulation. In ALS, TDP-43 aggregates within the cytoplasm and facilitates neurodegeneration. The involvement of ERVs in ALS pathology is thought to occur through TDP-43 and neuroinflammatory mediators. In this review, the proposed involvement of TDP-43, HERV-K and immune regulators on the onset and progression of ALS will be discussed. Furthermore, the evidence supporting a therapy based on targeting ERVs in ALS will be reviewed.
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Esclerosis Amiotrófica Lateral , Retrovirus Endógenos , Infecciones por VIH , Enfermedad de la Neurona Motora , Humanos , Esclerosis Amiotrófica Lateral/genética , Enfermedad de la Neurona Motora/patología , Neuronas Motoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Infecciones por VIH/patologíaRESUMEN
Inflammatory conditions are critically influenced by neuroimmune crosstalk. Cytokines and neurotrophic factors shape the responses of both nervous and immune systems. Although much progress has been made, most findings to date are based on expression of recombinant (tagged) proteins. The examination of receptor interactions by immunoprecipitation (IP) at endogenous levels provides further insight into the more subtle regulations of immune responses. Here, we present a comprehensive workflow and an optimized IP protocol that provide step-by-step instructions to investigate neurotrophin receptor p75NTR at endogenous, low abundance levels: from lysate preparation and confirmation of receptor expression to antibody validation and successful detection of protein-protein interactions. We employ human melanoma cell line A375 to validate specific antibodies and IP conditions, and apply these methods to explore p75NTR interactions in human leukemic plasmacytoid dendritic cell line PMDC05 detecting 14-3-3ϵ:p75NTR interaction in this cell type. With p75NTR as an exemplary protein, our approach provides a strategy to detect specific interaction partners even under endogenous, low abundance expression conditions.
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Anticuerpos/inmunología , Hibridomas/inmunología , Inmunoprecipitación/métodos , Proteínas del Tejido Nervioso/inmunología , Receptores de Factor de Crecimiento Nervioso/inmunología , Flujo de Trabajo , Proteínas 14-3-3/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Células Dendríticas/inmunología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Espectrometría de Masas , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/metabolismoRESUMEN
The degeneration of cholinergic basal forebrain (cBF) neurons in Alzheimer's disease (AD) leads to the cognitive impairment associated with this condition. cBF neurons express the p75 neurotrophin receptor (p75NTR), which mediates cell death, and the extracellular domain of p75NTR can bind to amyloid beta (Aß) and promote its degradation. Here, we investigated the contribution of cBF neuronal p75NTR to the progression of AD by removing p75NTR from cholinergic neurons in the APP/PS1 familial AD mouse strain. Conditional loss of p75NTR slowed cognitive decline and reduced both Aß accumulation into plaques and gliosis. Expression of the amyloid protein precursor and its cleavage enzymes ADAM10 and BACE1 were unchanged. There was also no upregulation of p75NTR in non-cholinergic cell types. This indicates that a direct interaction between cBF-expressed p75NTR and Aß does not contribute significantly to the regulation of Aß load. Rather, loss of p75NTR from cBF neurons, which results in increased cholinergic innervation of the cortex, appears to regulate alternative, more dominant, Aß clearance mechanisms.
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Envejecimiento/metabolismo , Péptidos beta-Amiloides/metabolismo , Prosencéfalo Basal/metabolismo , Disfunción Cognitiva/metabolismo , Neuronas/metabolismo , Presenilina-1/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Alelos , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Genotipo , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Background: Neuroinflammation and human endogenous retroviruses (HERV) are thought to have a role in the pathophysiology of amyotrophic lateral sclerosis (ALS). Therapy directed against endogenous retroviruses has demonstrated positive effects during in vitro and biomarker studies. Consequently, the present study was undertaken to assess the safety and tolerability of long-term antiretroviral therapy (ART), Triumeq (abacavir, lamivudine, and dolutegravir) exposure in patients with ALS, and efficacy against biomarkers of disease progression. Methods: Patients were observed during a 10-week lead-in period before receiving Triumeq treatment for 24 weeks at four specialist ALS centers. The primary outcomes were safety and tolerability. Secondary outcomes included HERV-K expression levels, urinary p75ECD levels, neurophysiological parameters, and clinical indicators. The ENCALS prediction model was applied to provide an estimate of the cohort survival. The trial was registered (NCT02868580). Findings: 40 patients with ALS received Triumeq and 35 (88%) completed treatment. There were no drug-related serious adverse events; one patient was withdrawn from the study due to a drug-associated increase in liver enzymes. A favorable response on HERV-K expression levels was observed, accompanied by a decline in ALSFRS-R progression rate of 21.8% (95% CI -4.8%-48.6%) and the amount of urinary p75ECD measured. One patient died five months after stopping treatment, while five were expected to have died during the treatment period (interquartile range 2-8). Interpretation: Long-term Triumeq exposure was safe and well tolerated in this cohort. There was suggestive indication for a possible biological response in some pharmacodynamic and clinical biomarkers. A larger international phase 3 trial will be deployed to assess the effect of Triumeq on overall survival and disease progression. Funding: Funding was provided by the FightMND Foundation; MND Research Institute of Australia; MND Association, United Kingdom, and GSK. ViiV Healthcare provided the Triumeq.
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Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Didesoxinucleósidos/farmacología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Lamivudine/farmacología , Adolescente , Adulto , Anciano , Esclerosis Amiotrófica Lateral/fisiopatología , Biomarcadores/análisis , Estudios de Cohortes , Didesoxinucleósidos/efectos adversos , Progresión de la Enfermedad , Combinación de Medicamentos , Femenino , Compuestos Heterocíclicos con 3 Anillos/efectos adversos , Humanos , Lamivudine/efectos adversos , Masculino , Persona de Mediana Edad , Oxazinas , Piperazinas , Piridonas , Adulto JovenRESUMEN
This study addressed the suitability of the NSC-34 cell line as a motor neuron-like model for investigating neurotrophin receptor trafficking and associated subcellular processes. Initially, culture conditions were optimized for the use of NSC-34 cells in confocal microscopy. Cell surface markers, as well as markers associated with the regulated endosomal pathway thought to be associated with neurotrophin receptor transport, were identified. The study revealed the presence of a number of molecules previously not described in the literature, including the tropomyosin-like receptor kinase C (TrkC), sortilin, the vesicular acetylcholine transporter (VAChT), and the lipid raft-associated ganglioside GT1b. The presence of both sortilin and Gt1b was of special interest, insofar as these markers have been implicated in direct relationships with the p75NTR receptor. Evidence is provided for neurotrophin-dependent internalization of p75NTR and TrkB. Both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) increased the rate of internalization of p75NTR, with internalization dynamics comparable to those described for other cell lines. Thus, these studies not only describe components of the regulatory process governing the trafficking of this important receptor but also clearly demonstrate the value of NSC-34 cells as a suitable motor neuron model for the study of internalization and trafficking of cell surface molecules.
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Línea Celular , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Anticuerpos/inmunología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Diferenciación Celular , Línea Celular/citología , Línea Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Citosol/metabolismo , Endocitosis/efectos de los fármacos , Gangliósidos/inmunología , Proteínas de la Membrana/metabolismo , Modelos Neurológicos , Neuronas Motoras/metabolismo , Factor de Crecimiento Nervioso/farmacología , Transporte de Proteínas , Receptor trkB/inmunología , Receptor trkB/metabolismo , Receptores de Factor de Crecimiento Nervioso/efectos de los fármacos , Receptores de Factor de Crecimiento Nervioso/inmunologíaRESUMEN
To comprehensively assess whether p75ECD in urine could be a candidate biomarker for ALS evaluation. Urine samples were collected from 101 ALS patients, 108 patients with other neurological disease (OND) and 97 healthy controls. 61 ALS patients were followed up with clinical data including ALSFRS-r every 6 to 12 months, 23 ALS patients died and 17 ALS patients lost touch during follow up period. Enzyme-linked immunoassay was employed to determine urine p75ECD concentration. The ALSFRS-r was employed to assess the severity of ALS. The concentration of p75ECD in ALS was significantly higher than that of OND and CTRL (p < 0.001). Additionally, urine p75ECD concentrations in ALS-definite grade patients were significantly higher than that in ALS-probable grade and ALS-possible grade patients (p < 0.001). Higher urine p75ECD concentrations were correlated with increased clinical stage (p = 0.0309); urine p75ECD concentrations and ALSFRS-r were negatively correlated (p = 0.022); and urine p75ECD concentration in the fast-progressing ALS group was significantly higher than that in slow-progression (p = 0.0026). Our finding indicates that urine p75ECD concentration provides additional evidence for patients with clinically suspected ALS, and can be employed to evaluate ALS-severity.
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Esclerosis Amiotrófica Lateral/metabolismo , Biomarcadores/orina , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/orina , Receptores de Factor de Crecimiento Nervioso/química , Adulto , Anciano , Esclerosis Amiotrófica Lateral/mortalidad , Esclerosis Amiotrófica Lateral/orina , Pueblo Asiatico , Biomarcadores/química , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dominios Proteicos , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVE: To evaluate urinary neurotrophin receptor p75 extracellular domain (p75ECD) levels as disease progression and prognostic biomarkers in amyotrophic lateral sclerosis (ALS). METHODS: The population in this study comprised 45 healthy controls and 54 people with ALS, 31 of whom were sampled longitudinally. Urinary p75ECD was measured using an enzyme-linked immunoassay and validation included intra-assay and inter-assay coefficients of variation, effect of circadian rhythm, and stability over time at room temperature, 4°C, and repeated freeze-thaw cycles. Longitudinal changes in urinary p75ECD were examined by mixed model analysis, and the prognostic value of baseline p75ECD was explored by survival analysis. RESULTS: Confirming our previous findings, p75ECD was higher in patients with ALS (5.6 ± 2.2 ng/mg creatinine) compared to controls (3.6 ± 1.4 ng/mg creatinine, p < 0.0001). Assay reproducibility was high, with p75ECD showing stability across repeated freeze-thaw cycles, at room temperature and 4°C for 2 days, and no diurnal variation. Urinary p75ECD correlated with the revised ALS Functional Rating Scale at first evaluation (r = -0.44, p = 0.008) and across all study visits (r = -0.36, p < 0.0001). p75ECD also increased as disease progressed at an average rate of 0.19 ng/mg creatinine per month (p < 0.0001). In multivariate prognostic analysis, bulbar onset (hazard ratio [HR] 3.0, p = 0.0035), rate of disease progression from onset to baseline (HR 4.4, p < 0.0001), and baseline p75ECD (HR 1.3, p = 0.0004) were predictors of survival. CONCLUSIONS: The assay for urinary p75ECD is analytically robust and shows promise as an ALS biomarker with prognostic, disease progression, and potential pharmacodynamic application. Baseline urinary p75ECD provides prognostic information and is currently the only biological fluid-based biomarker of disease progression.
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Esclerosis Amiotrófica Lateral , Biomarcadores/orina , Progresión de la Enfermedad , Proteínas del Tejido Nervioso/orina , Adulto , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/fisiopatología , Esclerosis Amiotrófica Lateral/orina , Estudios de Cohortes , Creatinina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Receptores de Factor de Crecimiento Nervioso , Reproducibilidad de los ResultadosRESUMEN
In this study, p75NTREXONIII knockout mice were used as immune-naive hosts to produce functional antibodies to human p75NTR. Three monoclonal antibodies were produced and named MLR1, MLR2 and MLR3, and isotyped as IgG1, IgG2a and IgG2a, respectively. MLR1 and MLR2 bound to human p75NTR with higher affinity than the well-characterized ME20.4 in ELISA and also recognized p75NTR present on neurons in both rat and mouse. MLR1 and MLR2 bound to nerves known to express p75NTR following injection into Balb/C mice but not p75NTREXONIII knockout mice, indicating the antibodies are directed against the ligand binding extracellular region absent in knockout mice. Both MLR1 and MLR2 partially blocked NGF induced cell death in a mouse cell-line that expresses p75NTR but not TrKA. Importantly, intracerebroventricular injections indicated MLR2 was internalized within the cell bodies of mouse basal forebrain neurons, further demonstrating that this antibody is biologically active.
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Anticuerpos Monoclonales/fisiología , Especificidad de Anticuerpos , Ratones Noqueados/inmunología , Receptor de Factor de Crecimiento Nervioso/genética , Receptor de Factor de Crecimiento Nervioso/inmunología , Animales , Reacciones Antígeno-Anticuerpo , Vasos Sanguíneos/metabolismo , Western Blotting/métodos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo/métodos , Humanos , Inmunohistoquímica/métodos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Sprague-DawleyRESUMEN
Human adult dental pulp stem cells (DPSC) are a heterogeneous stem cell population, which are able to differentiate down neural, chondrocyte, osteocyte and adipocyte lineages. We studied the expression pattern of p75 neurotrophin receptors (p75NTR), a marker of neural stem cells, within human DPSC populations from eight donors. p75NTR are expressed at low levels (<10%) in DPSC. Importantly, p75(+) DPSC represent higher expression levels of SOX1 (neural precursor cell marker), SOX2 (cell pluripotency marker) and nestin (neural stem cell marker) in comparison to p75(-) DPSC. Our results suggest that p75(+) hDPSC may denote a subpopulation with greater neurogenic potential.
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Pulpa Dental/citología , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Células Madre/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Citometría de Flujo , Humanos , Factores de Transcripción SOXB1/metabolismoRESUMEN
Full length TrkC (TrkC-FL) is a receptor tyrosine kinase whose mRNA can be spliced to a truncated TrkC.T1 isoform lacking the kinase domain. Neurotrophin-3 (NT-3) activates TrkC-FL to maintain motor neuron health and function and TrkC.T1 to produce neurotoxic TNF-α; hence resulting in opposing pathways. In mouse and human ALS spinal cord, the reduction of miR-128 that destabilizes TrkC.T1 mRNA results in up-regulated TrkC.T1 and TNF-α in astrocytes. We exploited conformational differences to develop an agonistic mAb 2B7 that selectively activates TrkC-FL, to circumvent TrkC.T1 activation. In mouse ALS, 2B7 activates spinal cord TrkC-FL signals, improves spinal cord motor neuron phenotype and function, and significantly prolongs life-span. Our results elucidate biological paradoxes of receptor isoforms and their role in disease progression, validate the concept of selectively targeting conformational epitopes in naturally occurring isoforms, and may guide the development of pro-neuroprotective (TrkC-FL) and anti-neurotoxic (TrkC.T1) therapeutic strategies.
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Esclerosis Amiotrófica Lateral/fisiopatología , Receptor trkC/fisiología , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/inmunología , Astrocitos/fisiología , Modelos Animales de Enfermedad , Humanos , Ratones , MicroARNs/fisiología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Factores de Crecimiento Nervioso/fisiología , Fármacos Neuroprotectores/uso terapéutico , Conformación Proteica , Isoformas de Proteínas/fisiología , Ratas , Receptor trkC/efectos de los fármacos , Receptor trkC/inmunología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Mice with high numbers of the Cu/Zn superoxide dismutase-1 G93A transgene (SOD1(G93A) G1H) have become the most commonly used animal model to study amyotrophic lateral sclerosis. This study investigated changes in size, numbers, and cell stress/death markers of motor neuron numbers in G1H mice that re-express the common p75 neurotrophin receptor (p75NTR). SOD1(G93A) G1H mice and age-matched C57BL/6J controls at 60, 80, 100, 120 days and end stage/140 days were analyzed for p75NTR, choline acetyltransferase (ChAT), activating transcription factor 3 (ATF3), and cleaved caspase-3. In addition, motor neuron counts and soma sizes were recorded. Motor neurons re-expressing p75NTR in SOD1(G93A) G1H mice were first observed at 80 days, and this continued to 140 days, peaking at 100-120 days at â¼5%. The soma area of motor neurons re-expressing p75NTR was always 600-800 µm(2) , suggesting that these are alpha motor neurons, which was confirmed after examination of somas post injection of a retrogradely transported antibody to p75NTR in 110-day-old SOD1(G93A) G1H mice. In motor neurons not re-expressing p75NTR, the frequency of small soma 200-400 µm2 motor neurons increased, whereas the larger 600-900 µm2 motor neurons decreased with progression, indicating that large motor neurons were dying off and shrinking in the process. There was minimal coexpression of p75NTR with ATF3, a marker for cell stress, but 85% coexpressed the apoptotic marker cleaved caspase-3. These findings indicate that in SOD1(G93A) G1H mice, p75NTR re-expression is detectable from 80 days in a small population of large motor neurons that represent 5% of the total motor neurons. Furthermore, p75NTR re-expression occurs in larger alpha motor neurons that express cleaved caspsase-3 and are destined to die.
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Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Receptores de Factor de Crecimiento Nervioso/metabolismo , Factor de Transcripción Activador 3/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Caspasa 3/metabolismo , Recuento de Células , Tamaño de la Célula , Colina O-Acetiltransferasa/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Vértebras Lumbares , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuritas/metabolismo , Neuritas/patología , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa/genéticaRESUMEN
The ability to selectively kill cancerous cell populations while leaving healthy cells unaffected is a key goal in anticancer therapeutics. The use of nanoporous silica-based materials as drug-delivery vehicles has recently proven successful, yet production of these materials requires costly and toxic chemicals. Here we use diatom microalgae-derived nanoporous biosilica to deliver chemotherapeutic drugs to cancer cells. The diatom Thalassiosira pseudonana is genetically engineered to display an IgG-binding domain of protein G on the biosilica surface, enabling attachment of cell-targeting antibodies. Neuroblastoma and B-lymphoma cells are selectively targeted and killed by biosilica displaying specific antibodies sorbed with drug-loaded nanoparticles. Treatment with the same biosilica leads to tumour growth regression in a subcutaneous mouse xenograft model of neuroblastoma. These data indicate that genetically engineered biosilica frustules may be used as versatile 'backpacks' for the targeted delivery of poorly water-soluble anticancer drugs to tumour sites.
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Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Camptotecina/administración & dosificación , Camptotecina/uso terapéutico , Diatomeas/metabolismo , Animales , Anticuerpos , Línea Celular Tumoral , Clonación Molecular , Diatomeas/genética , Sistemas de Liberación de Medicamentos , Regulación de la Expresión Génica , Ingeniería Genética , Inmunoglobulina G , Liposomas , Linfoma de Células B/tratamiento farmacológico , Ratones , Micelas , Nanopartículas , Neoplasias Experimentales/tratamiento farmacológico , Neuroblastoma/tratamiento farmacológico , Tamaño de la Partícula , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Dióxido de Silicio/metabolismo , Trasplante HeterólogoRESUMEN
Objective biomarkers for amyotrophic lateral sclerosis would facilitate the discovery of new treatments. The common neurotrophin receptor p75 is up regulated and the extracellular domain cleaved from injured neurons and peripheral glia in amyotrophic lateral sclerosis. We have tested the hypothesis that urinary levels of extracellular neurotrophin receptor p75 serve as a biomarker for both human motor amyotrophic lateral sclerosis and the SOD1(G93A) mouse model of the disease. The extracellular domain of neurotrophin receptor p75 was identified in the urine of amyotrophic lateral sclerosis patients by an immuno-precipitation/western blot procedure and confirmed by mass spectrometry. An ELISA was established to measure urinary extracellular neurotrophin receptor p75. The mean value for urinary extracellular neurotrophin receptor p75 from 28 amyotrophic lateral sclerosis patients measured by ELISA was 7.9±0.5 ng/mg creatinine and this was significantly higher (p<0.001) than 12 controls (2.6±0.2 ng/mg creatinine) and 19 patients with other neurological disease (Parkinson's disease and Multiple Sclerosis; 4.1±0.2 ng/mg creatinine). Pilot data of disease progression rates in 14 MND patients indicates that p75NTR(ECD) levels were significantly higher (pâ=â0.0041) in 7 rapidly progressing patients as compared to 7 with slowly progressing disease. Extracellular neurotrophin receptor p75 was also readily detected in SOD1(G93A) mice by immuno-precipitation/western blot before the onset of clinical symptoms. These findings indicate a significant relation between urinary extracellular neurotrophin receptor p75 levels and disease progression and suggests that it may be a useful marker of disease activity and progression in amyotrophic lateral sclerosis.
Asunto(s)
Esclerosis Amiotrófica Lateral/diagnóstico , Biomarcadores/orina , Receptor de Factor de Crecimiento Nervioso/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Western Blotting/métodos , Creatinina , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoprecipitación/métodos , Ratones , Ratones Endogámicos C57BL , Receptor de Factor de Crecimiento Nervioso/genética , Superóxido Dismutasa/genéticaRESUMEN
Microglial activation is a central event in neurodegeneration. Novel technologies are sought for that specifically manipulate microglial function in order to delineate their role in onset and progression of neuropathologies. We investigated for the first time whether non-viral gene delivery based on polyethyleneglycol-polyethyleneimine conjugated to the monoclonal anti-CD11b antibody OX42 ("OX42-immunogene") could be used to specifically target microglia. We first conducted immunofluorescence studies with the OX42 antibody and identified its microglial integrin receptor CD11b as a potential target for receptor-mediated gene transfer based on its cellular specificity in mixed glia culture and in vivo and found that the OX42 antibody is rapidly internalized and trafficked to acidic organelles in absence of activation of the respiratory burst. We then performed transfection experiments with the OX42-immunogene in vitro and in rat brain showing that the OX42-immunogene although internalized was degraded intracellularly and did not cause substantial gene expression in microglia. Investigation of specific barriers to microglial gene transfer revealed that aggregated OX42-immunogene polyplexes stimulated the respiratory burst that likely involved Fcγ-receptors. Transfections in the presence of the endosomolytic agent chloroquine improved transfection efficiency indicating that endosomal escape may be limited. This study identifies CD11b as an entry point for antibody-mediated gene transfer into microglia and takes important steps toward the further development of OX42-immunogenes.