RESUMEN
OBJECTIVES: Drug treatment for children with epilepsy should, ideally, be governed by evidence from adequate and well-controlled clinical studies. However, these studies are difficult to conduct, and so direct evidence supporting the informed use of specific drugs is often lacking. The Research Roundtable for Epilepsy (RRE) met in 2020 to align on an approach to therapy development for focal seizures in children age 1 month <2 years of age. METHODS: The RRE reviewed the regulatory landscape, epidemiology, seizure semiology, antiseizure medicine pharmacology, and safety issues applicable to this population. RESULTS: After reviewing evidence, the conclusion was that pediatric efficacy trials would be impracticable to conduct but a waiver of the regulatory requirement to conduct any study would lead to an absence of information to guide dosing in a critical population. Review of available data and discussion of RRE attendees led to the conclusion that the requirements for extrapolation of efficacy from older children down to infants from age 1 month to <2 years old appeared to be met. After the RRE, the US Food and Drug Administration (FDA) approved brivaracetam for use in children with focal epilepsy above the age of 1 month in August 2021 and lacosamide in October 2021, both based on the principle of extrapolation from data in older children. SIGNIFICANCE: These recommendations should result in more rapid accessibility of antiseizure medications for infants.
Asunto(s)
Epilepsias Parciales , Epilepsia , Adolescente , Anticonvulsivantes/uso terapéutico , Niño , Epilepsias Parciales/tratamiento farmacológico , Epilepsia/tratamiento farmacológico , Humanos , Lactante , Lacosamida/uso terapéutico , Convulsiones/tratamiento farmacológicoRESUMEN
OBJECTIVE: To evaluate the efficacy and safety of ganaxolone as adjunctive therapy in adults with uncontrolled partial-onset seizures despite taking up to three concomitant antiepileptic drugs (AEDs). METHODS: Adults aged 18-69 years and refractory to conventional AEDs were enrolled in a multicenter, double-blind, placebo-controlled trial. After an 8-week baseline period, patients were randomized 2:1 to ganaxolone 1,500 mg/day or placebo for a 10-week treatment period (2-week forced titration and 8-week maintenance) followed by either tapering or entry into an open-label extension study. The primary endpoint was mean weekly seizure frequency. Secondary endpoints included the proportion of patients experiencing ≥50% reduction in seizure frequency (responder rate), percent change in mean weekly seizure frequency, seizure-free days, and quality of life. Safety and tolerability assessments included adverse events (AEs), treatment discontinuation, and clinical laboratory evaluations. Efficacy analyses were performed on the intent-to-treat population. RESULTS: Of 147 randomized patients (98 ganaxolone, 49 placebo), 131 completed the study; 95% of participants titrated up to 1,500 mg/day and 78% maintained this dose. From baseline to endpoint, mean weekly seizure frequency decreased with ganaxolone (6.5-5.2) versus placebo (9.2-10.8), representing an 11.4% decrease versus placebo (p = 0.0489, analysis of covariance [ANCOVA]). Mean percent change from baseline was -17.6% with ganaxolone versus 2.0% with placebo (p = 0.0144, Kruskal-Wallis test). Responder rates were 24% with ganaxolone versus 15% with placebo (p = 0.19). Discontinuation due to adverse events was similar with ganaxolone (7.1%) and placebo (6.1%). Common adverse events were mild to moderate in severity and included dizziness (16.3% vs. 8.2%), fatigue (16.3% vs. 8.2%), and somnolence (13.3% vs. 2.0%). SIGNIFICANCE: Ganaxolone 1,500 mg/day reduced partial-onset seizure frequency and was generally safe and well tolerated in this phase 2 study. These results support continued development of ganaxolone for adult patients with refractory partial-onset seizures.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Pregnanolona/análogos & derivados , Convulsiones/tratamiento farmacológico , Adolescente , Adulto , Anciano , Análisis de Varianza , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pregnanolona/uso terapéutico , Resultado del Tratamiento , Adulto JovenRESUMEN
Inborn errors of immunity (IEI) are a group of diseases in humans that typically present as increased susceptibility to infections, autoimmunity, hyperinflammation, allergy, and in some cases malignancy. Among newly identified genes linked to IEIs include 3 independent reports of 9 individuals from 7 independent kindreds with severe primary immunodeficiency disease (PID) and autoimmunity due to loss-of-function mutations in the NCKAP1L gene encoding Hematopoietic protein 1 (HEM1). HEM1 is a hematopoietic cell specific component of the WASp family verprolin homologous (WAVE) regulatory complex (WRC), which acts downstream of multiple immune receptors to stimulate actin nucleation and polymerization of filamentous actin (F-actin). The polymerization and branching of F-actin is critical for creating force-generating cytoskeletal structures which drive most active cellular processes including migration, adhesion, immune synapse formation, and phagocytosis. Branched actin networks at the cell cortex have also been implicated in acting as a barrier to regulate inappropriate vesicle (e.g. cytokine) secretion and spontaneous antigen receptor crosslinking. Given the importance of the actin cytoskeleton in most or all hematopoietic cells, it is not surprising that HEM1 deficient children present with a complex clinical picture that involves overlapping features of immunodeficiency and autoimmunity. In this review, we will provide an overview of what is known about the molecular and cellular functions of HEM1 and the WRC in immune and other cells. We will describe the common clinicopathological features and immunophenotypes of HEM1 deficiency in humans and provide detailed comparative descriptions of what has been learned about Hem1 disruption using constitutive and immune cell-specific mouse knockout models. Finally, we discuss future perspectives and important areas for investigation regarding HEM1 and the WRC.
Asunto(s)
Síndromes de Inmunodeficiencia , Humanos , Animales , Ratones , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunologíaRESUMEN
Malaria parasite infection weakens colonization resistance against Salmonella enterica serovar (S.) Typhimurium. S. Typhimurium is a member of the Enterobacterales, a taxon that increases in abundance when the colonic microbiota is disrupted or when the colonic mucosa is inflamed. However, here, we show that infection of mice with Plasmodium yoelii enhances S. Typhimurium colonization by weakening host control in the upper GI tract. P. yoelii-infected mice had elevated gastric pH. Stimulation of gastric acid secretion during P. yoelii infection restored stomach acidity and colonization resistance, demonstrating that parasite-induced hypochlorhydria increases gastric survival of S. Typhimurium. Furthermore, blockade of P. yoelii-induced TNF-α signaling was sufficient to prevent elevation of gastric pH and enhance S. Typhimurium colonization during concurrent infection. Collectively, these data suggest that abundance in the fecal microbiota of facultative anaerobes, such as S. Typhimurium, can be increased by suppressing antibacterial defenses in the upper GI tract, such as gastric acid.
Asunto(s)
Microbioma Gastrointestinal , Malaria , Animales , Heces/microbiología , Intestino Delgado , Ratones , Salmonella typhimurium/fisiologíaRESUMEN
Strong evidence supports that CNS-specific CD4(+) T cells are central to the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Using a model of spontaneous EAE, we demonstrated that myelin basic protein (MBP)-specific CD4(+) T cells up-regulate activation markers in the CNS-draining cervical lymph nodes at a time when there is no T cell activation anywhere else, including the CNS, and before the appearance of clinical signs. In spontaneous EAE, the number of MBP-specific T cell numbers does not build up gradually in the CNS; instead, a swift migration of IFN-gamma-producing T cells into the CNS takes place approximately 24 h before the onset of neurological signs of EAE. Surgical excision of the cervical lymph nodes in healthy pre-EAE transgenic mice delayed the onset of EAE and resulted in a less severe disease. In EAE induced by immunization with MBP/CFA, a similar activation of T cells in the draining lymph nodes of the injection site precedes the disease. Taken together, our results suggest that peripheral activation of T cells in draining lymph nodes is an early event in the development of EAE, which paves the way for the initial burst of IFN-gamma-producing CD4(+) T cell into the CNS.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Movimiento Celular/inmunología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Proteína Básica de Mielina/inmunología , Animales , Biomarcadores/líquido cefalorraquídeo , Linfocitos T CD4-Positivos/metabolismo , Movimiento Celular/genética , Células Cultivadas , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/cirugía , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Escisión del Ganglio Linfático , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ganglios Linfáticos/cirugía , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factores de Tiempo , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.
Asunto(s)
Antozoos/metabolismo , Fluorescencia , Proteínas Luminiscentes/metabolismo , Animales , Color , Dinoflagelados/fisiología , SimbiosisRESUMEN
Amyloid plaques are a hallmark of Alzheimer disease, but their importance in its pathogenesis is controversial. By neuronal labeling and transcranial two-photon imaging, we show in a transgenic mouse model of Alzheimer disease that dendrites passing through or near fibrillar amyloid deposits undergo spine loss and shaft atrophy, and nearby axons develop large varicosities, together leading to neurite breakage and large-scale, permanent disruption of neuronal connections. Thus, fibrillar amyloid deposition is more detrimental to neuronal circuitry than previously thought, underscoring the importance of prevention and early clearance of plaques.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Corteza Cerebral/patología , Ovillos Neurofibrilares/metabolismo , Neuronas/fisiología , Placa Amiloide/metabolismo , Sinapsis/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Diagnóstico por Imagen/métodos , Técnicas In Vitro , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Neuritas/metabolismo , Presenilina-1 , Sinapsis/patología , Factores de TiempoRESUMEN
Dystrophic neurites are associated with fibrillar amyloid deposition in Alzheimer's disease (AD), but the frequency and types of changes in synaptic structures near amyloid deposits have not been well characterized. Using high-resolution confocal microscopy to image lipophilic dye-labeled dendrites and thioflavin-S-labeled amyloid plaques, we systematically analyzed the structural changes of dendrites associated with amyloid deposition in both a transgenic mouse model of AD (PSAPP) and in human postmortem brain. We found that in PSAPP mice, dendritic branches passing through or within 40 mum from amyloid deposits displayed various dendritic abnormalities such as loss of dendritic spines, shaft atrophy, bending, abrupt branch endings, varicosity formation, and sprouting. Similar structural alterations of dendrites were seen in postmortem human AD tissue, with spine loss as the most common abnormality in both PSAPP mice and human AD brains. These results demonstrate that fibrillar amyloid deposits and their surrounding microenvironment are toxic to dendrites and likely contribute to significant disruption of neuronal circuits in AD.
Asunto(s)
Enfermedad de Alzheimer/patología , Dendritas/patología , Placa Amiloide/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Atrofia , Tamaño de la Célula , Dendritas/ultraestructura , Espinas Dendríticas/patología , Humanos , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Transgénicos , Microscopía Confocal , Neuronas/patología , Presenilina-1/genética , Sinapsis/patología , Sinapsis/fisiologíaRESUMEN
To gain a better understanding of the natural function of fluorescent proteins, we have undertaken quantitative analyses of these proteins in a single species of coral, Montastraea cavernosa, residing around Turneffe atoll, on the Belizean Barrier Reef. We identified at least 10 members of a fluorescent protein family in this species, which consist of 4 distinct spectral classes. As much as a 10-fold change in the overall expression of fluorescent proteins was observed from specimen to specimen, suggesting that fluorescent proteins are dynamically regulated in response to environmental or physiological conditions. We found that the expression of some proteins was inversely correlated with depth, and that groups of proteins were coordinately expressed. There was no relationship between the expression of fluorescent proteins and the natural coloration of the Montastraea cavernosa specimens in this study. These findings have implications for current hypotheses regarding the properties and natural function of fluorescent proteins.
Asunto(s)
Antozoos/fisiología , Ambiente , Regulación de la Expresión Génica/fisiología , Proteínas Luminiscentes/fisiología , Animales , Antozoos/química , Antozoos/genética , Secuencia de Bases , Cartilla de ADN/química , Perfilación de la Expresión Génica/veterinaria , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Filogenia , Pigmentación , ARN Ribosómico 18S/análisis , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Sensibilidad y Especificidad , Estadística como Asunto , Tubulina (Proteína)/análisis , Tubulina (Proteína)/biosíntesisRESUMEN
BACKGROUND: Microglia are known as resident immune cells in the brain. ß-amyloid (Aß) plaques in the brain of Alzheimer's disease (AD) are surrounded by microglia, but whether and how microglia affect the formation and maintenance of plaques remains controversial. METHODS: We depleted microglia by injecting diphtheria toxin (DT) in CX 3 CR1 CreER/+ :R26 DTR/+ (CX 3 CR1-iDTR) mice crossed with APPswe/PSEN1dE9 (APP/PS1) mice. Intravital time-lapse imaging was performed to examine changes in the number and size of Congo Red-labeled amyloid plaques over 1-2 weeks. We also examined spine density and shaft diameter of dendrites passing through plaques in a PSAPP mouse model of AD (PS1 M146L line 6.2 × Tg2576) crossed with Thy1 YFP H-line mice. RESULTS: We found that DT administration to CX 3 CR1-iDTR mice efficiently ablated microglia within one week and that microglia repopulated in the second week after DT administration. Microglia depletion didn't affect the number of amyloid plaques, but led to ~13% increase in the size of Aß plaques within one week. Moreover, microglia repopulation was associated with the stabilization of plaque size during the second week. In addition, we found dendritic spine loss and shaft atrophy in the distal parts of dendrites passing through plaques. CONCLUSION: Our results demonstrate the important role of microglia in limiting the growth of Aß plaques and plaque-associated disruption of neuronal connection.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Microglía/metabolismo , Placa Amiloide/metabolismo , Enfermedad de Alzheimer/inmunología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones Transgénicos , Placa Amiloide/inmunología , Imagen de Lapso de Tiempo/métodosRESUMEN
Gas-bubble disease occurs in aquatic species that are exposed to water that is supersaturated with gases. In February 2007, municipal water supersaturated with gas was inadvertently pumped into the vivarium's aquatic housing systems and affected approximately 450 adult female Xenopus laevis. The inflow of supersaturated water was stopped immediately, the holding tanks aggressively aerated, and all experimental manipulations and feeding ceased. Within the first 6 h after the event, morbidity approached 90%, and mortality reached 3.5%. Acutely affected frogs showed clinical signs of gas-bubble disease: buoyancy problems, micro- and macroscopic bubbles in the foot webbing, hyperemia in foot webbing and leg skin, and loss of the mucous slime coat. All of the frogs that died or were euthanized had areas of mesenteric infarction, which resulted in intestinal epithelial necrosis and degeneration of the muscular tunic. Over the subsequent 2 wk, as gas saturation levels returned to normal, the clinical symptoms resolved completely in the remaining frogs. However, 3 mo later, 85% of them failed to lay eggs or produce oocytes, and the remaining 15% produced oocytes of low number and poor quality, yielding cytosolic extracts with poor to no enzymatic activity. Histology of the egg mass from a single 2- to 3-y-old frog at 3 mo after disease resolution revealed irregularly shaped oocytes, few large mature oocytes, and numerous small, degenerating oocytes. At 6 mo after the incident, the remaining frogs continued to fail to produce eggs of sufficient quantity or quality after hormonal priming. The researchers consequently opted to cull the remainder of the colony and repopulate with new frogs.
Asunto(s)
Embolia Aérea/veterinaria , Hiperoxia/veterinaria , Infarto/veterinaria , Mesenterio/irrigación sanguínea , Enfermedades Peritoneales/veterinaria , Xenopus laevis/sangre , Enfermedad Aguda , Animales , Femenino , Infarto/mortalidad , Estrés Oxidativo , Enfermedades Peritoneales/mortalidad , Abastecimiento de AguaRESUMEN
Preclinical and clinical research supports a role for neuroactive steroids in the pathophysiology of posttraumatic stress disorder (PTSD). We investigated ganaxolone (a synthetic 3ß-methylated derivative of allopregnanolone, a GABAergic neuroactive steroid) for treatment of PTSD in a proof-of-concept, multisite, double-blind, placebo-controlled trial. Veteran and non-veteran participants (n = 112) were randomized to ganaxolone or placebo at biweekly escalating doses of 200, 400, and 600 mg twice daily for 6 weeks. During an open-label 6-week extension phase, the initial ganaxolone group continued ganaxolone, while the placebo group crossed over to ganaxolone. Eighty-six and 59 participants, respectively, completed the placebo-controlled and open-label phases. A modified intent-to-treat mixed model repeated measures analysis revealed no significant differences between the effects of ganaxolone and placebo on Clinician Administered PTSD Symptom (CAPS) scores, global well-being, negative mood, or sleep. Dropout rates did not differ between groups, and ganaxolone was generally well tolerated. Trough blood levels of ganaxolone at the end of the double-blind phase were, however, lower than the anticipated therapeutic level of ganaxolone in >35% of participants on active drug. Pharmacokinetic profiling of the ganaxolone dose regimen used in the trial and adverse event sensitivity analyses suggest that under-dosing may have contributed to the failure of ganaxolone to out-perform placebo. Future investigations of ganaxolone may benefit from higher dosing, rigorous monitoring of dosing adherence, a longer length of placebo-controlled testing, and targeting of treatment to PTSD subpopulations with demonstrably dysregulated pre-treatment neuroactive steroid levels. Clinicaltrials.gov identifier: NCT01339689.
Asunto(s)
Pregnanolona/análogos & derivados , Trastornos por Estrés Postraumático/tratamiento farmacológico , Trastornos por Estrés Postraumático/epidemiología , Adulto , Estudios Cruzados , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pregnanolona/uso terapéutico , Prueba de Estudio Conceptual , Trastornos por Estrés Postraumático/diagnóstico , Resultado del TratamientoRESUMEN
The diagnosis of prion diseases in humans is challenging due to a lack of specific and sensitive non-invasive tests. Many forms of human prion disease including variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome, and 10% of sporadic CJD cases are associated with amyloid deposition. Several positron emission tomography (PET) ligands have recently been developed to directly image beta-amyloid associated with Alzheimer disease. One of them, methoxy-X04, is a fluorescent derivative of Congo red with high binding affinity toward amyloid fibrils and good blood-brain barrier permeability. Using methoxy-X04, we investigated whether amyloid-targeting ligands can be also employed for direct imaging of amyloid deposits associated with some prion diseases. Such a method could potentially become a novel diagnostic approach for these conditions. Studies were performed on MB mice infected with the 87V mouse-adapted scrapie strain. Labeling of PrP amyloid plaques in brains of presymptomatic and symptomatic mice was demonstrated using in vivo transcranial two-photon microscopy after systemic administration of methoxy-X04. During real-time imaging, PrP amyloid deposits could be clearly distinguished 15 min after intravenous administration of methoxy-X04. The ligand showed rapid clearance from brain areas that did not contain amyloid deposits. PrP amyloid deposits could also be detected by direct application of methoxy-X04 on cerebellar sections from GSS patients. These results suggest that methoxy-X04 or similar derivatives could be used as PET imaging agents to improve the diagnosis of human prion diseases associated with amyloid deposition.
Asunto(s)
Alquenos , Péptidos beta-Amiloides/análisis , Derivados del Benceno , Placa Amiloide/patología , Enfermedades por Prión/diagnóstico por imagen , Enfermedades por Prión/patología , Tomografía Computarizada de Emisión/métodos , Alquenos/metabolismo , Alquenos/farmacocinética , Animales , Derivados del Benceno/metabolismo , Derivados del Benceno/farmacocinética , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Humanos , Tasa de Depuración Metabólica/efectos de los fármacos , Tasa de Depuración Metabólica/fisiología , Ratones , Placa Amiloide/metabolismo , Valor Predictivo de las Pruebas , Enfermedades por Prión/metabolismo , Reproducibilidad de los Resultados , Scrapie/diagnóstico por imagen , Scrapie/metabolismo , Scrapie/patología , EstilbenosRESUMEN
PURPOSE: A pilot study of the safety, tolerability, dose range and potential efficacy of ganaxolone for the treatment of refractory epilepsy in pediatric and adolescent subjects. METHODS: We report the results of a nonrandomized, nonblinded, open-label, dose-escalation trial of ganaxolone in pediatric subjects (5-15 years) suffering from refractory epilepsy. Subjects received an oral suspension of ganaxolone in a 1:1 complex with beta-cyclodextrin in a dose escalation (1 mg/kg, b.i.d. to 12 mg/kg t.i.d.) schedule over 16 days. This was followed by a maintenance period for 8 weeks. Subjects that showed significant response were eligible for a compassionate use extension period. RESULTS: Fifteen subjects enrolled, eight completed the trial and three continued in the open-label compassionate-use extension period. All subject exhibited refractory partial or generalized epilepsy. In an intent-to-treat analysis, four (25%) were considered substantial responders (>or=50% reduction in seizure frequency), two (13%) were considered moderate responders (between 25 and 50% reduction in seizure frequency) and the remainder were considered nonresponders (<24% reduction). Three subjects entered the extension phase, one remained essentially seizure-free for over 3.5 years of ganaxolone administration. Ganaxolone was tolerated well. A total of 17 adverse events were reported in 10 patients, all were considered mild to moderate in severity. Somnolence was the most frequently (nine) reported adverse event. CONCLUSIONS: This pilot study is consistent with other clinical studies indicating that ganaxolone has anticonvulsant activity in humans. The results of this study encourage the further study of ganaxolone as an antiepileptic therapy.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Epilepsia/tratamiento farmacológico , Pregnanolona/análogos & derivados , Adolescente , Factores de Edad , Anticonvulsivantes/efectos adversos , Anticonvulsivantes/farmacología , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Epilepsia/prevención & control , Femenino , Humanos , Masculino , Proyectos Piloto , Pregnanolona/efectos adversos , Pregnanolona/farmacología , Pregnanolona/uso terapéutico , Resultado del TratamientoRESUMEN
Particle-mediated ballistic delivery of fluorescent dyes has been recently used to label neuronal populations in a rapid and efficient fashion. Here we describe detailed protocols for this technique as well as recent improvements in its implementation. This technique allows rapid labeling of entire neurons in a Golgi-like manner after membranes of individual neurons are contacted by particles coated with lipophilic dyes. Neurons can be labeled by dyes of different colors at controlled densities to facilitate the study of structural interactions between cells. Furthermore, in conjunction with other histochemical labeling methods, the technique can be used to study changes in neuronal structures associated with pathologic processes in animal models or postmortem human brain. In addition to lipophilic dyes, water-soluble molecules such as calcium indicators can also be delivered efficiently with this technique. The method of ballistic delivery of indicators thus provides new avenues to probe the structure and function of the nervous system.
Asunto(s)
Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/análisis , Neuronas/química , Coloración y Etiquetado/métodos , Animales , Ratones , Coloración y Etiquetado/instrumentaciónRESUMEN
A 10-day winter sampling campaign was conducted in downtown Toronto for particulate matter (PM) air pollution in the fine (<2.5 microm) size range. An aerosol laser ablation mass spectrometer (LAMS), a tapered-element oscillating microbalance (TEOM), and an aerodynamic particle sizer (APS) were operated in parallel to characterize the PM on-line. In this study, the LAMS observed differences in the chemical composition between three separate episodes with higher PM2.5 mass and APS counts. LAMS results showed that in one instance of elevated PM, organic amines were present in the particulates. Temporal analyses of this episode revealed chemical transformations as the amines, characterized by m/z peaks 58(C3HeN)+, 86(C5H2N)+, and nitrates, increased in number concentration while Ca and hydrocarbon particle classes concurrently decreased. On another day, sulfates were found to have increased significantly. The third event was only 4 h in duration and exhibited an increase in the number of submicron-sized K/hydrocarbons and sulfate-containing particles. In this last event, the hydrocarbons and a K to Fe ratio enrichment indicated there was likely a contribution from a combustion source. This work offers some of the first insights into single particle size and chemistry in a cold winter climate.