RESUMO
PURPOSE: To provide an initial assessment of the safety of a recombinant adeno-associated virus vector expressing RPE65 (rAAV2-CB-hRPE65) in adults and children with retinal degeneration caused by RPE65 mutations. DESIGN: Nonrandomized, multicenter clinical trial. PARTICIPANTS: Eight adults and 4 children, 6 to 39 years of age, with Leber congenital amaurosis (LCA) or severe early-childhood-onset retinal degeneration (SECORD). METHODS: Patients received a subretinal injection of rAAV2-CB-hRPE65 in the poorer-seeing eye, at either of 2 dose levels, and were followed up for 2 years after treatment. MAIN OUTCOME MEASURES: The primary safety measures were ocular and nonocular adverse events. Exploratory efficacy measures included changes in best-corrected visual acuity (BCVA), static perimetry central 30° visual field hill of vision (V30) and total visual field hill of vision (VTOT), kinetic perimetry visual field area, and responses to a quality-of-life questionnaire. RESULTS: All patients tolerated subretinal injections and there were no treatment-related serious adverse events. Common adverse events were those associated with the surgical procedure and included subconjunctival hemorrhage in 8 patients and ocular hyperemia in 5 patients. In the treated eye, BCVA increased in 5 patients, V30 increased in 6 patients, VTOT increased in 5 patients, and kinetic visual field area improved in 3 patients. One subject showed a decrease in BCVA and 2 patients showed a decrease in kinetic visual field area. CONCLUSIONS: Treatment with rAAV2-CB-hRPE65 was not associated with serious adverse events, and improvement in 1 or more measures of visual function was observed in 9 of 12 patients. The greatest improvements in visual acuity were observed in younger patients with better baseline visual acuity. Evaluation of more patients and a longer duration of follow-up will be needed to determine the rate of uncommon or rare side effects or safety concerns.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Amaurose Congênita de Leber/terapia , Degeneração Retiniana/terapia , Adulto , Criança , Eletrorretinografia , Feminino , Vetores Genéticos , Humanos , Injeções Intraoculares , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/fisiopatologia , Masculino , Qualidade de Vida , Degeneração Retiniana/etiologia , Acuidade Visual/fisiologia , Campos Visuais/fisiologia , Adulto Jovem , cis-trans-Isomerases/genéticaRESUMO
Approximately 80% of trafficked individuals access health care during their victimization. The emergency department is a frontline of care. This study examines current screening practices of Western Washington emergency department nurses to determine nurse and facility readiness for improved identification. Interviews were conducted with nurses to understand their current screening practices for identifying potential victims of sexual exploitation; the acceptability of existing screening questions for use in their settings; and their opinions about the utility of a standardized screening tool for identifying victims and improving care. There is an absence of formal protocols and screening tools, limited clarity regarding roles and responsibilities in the identification and care of trafficked persons among the health care team, and a desire to provide improved care quality to patients. Standardized processes and screening may lead to more efficient and effective identification of, care for, and linkage to vital support services and resources.
Assuntos
Vítimas de Crime , Serviço Hospitalar de Emergência , Humanos , Washington , Hospitais , Programas de RastreamentoRESUMO
A comparison of the preparation ability of two root canal instrumentation systems in oval-shaped canals using micro-computed tomography was undertaken. Thirty extracted, single-rooted, human mandibular premolars with radiographically similar canal morphology were selected, allocated to two groups (N = 15) and prepared with TRUShape or Vortex Blue (VB). Each sample was subjected to three scans (20 µm resolution): pre-preparation and after preparation to sizes #30 and #40. Three-dimensional data sets were evaluated for canal volume, surface area and surface treatment. Matched axial slices in apical, middle and coronal root thirds were evaluated for cross-sectional area, roundness and transportation. Preparation with both instruments increased canal volumes and surface areas similarly and significantly (P < 0.001) with no significant difference between groups. TRUShape significantly enhanced surface treatment at both apical sizes (P < 0.05). Transportation exceeded 100 µm in only eight out of 90 cross sections. Both instruments performed similarly during preparation. TRUShape, however, significantly enhanced surface treatment.
Assuntos
Cavidade Pulpar , Preparo de Canal Radicular , Dente Pré-Molar , Desenho de Equipamento , Humanos , Dente Molar , Níquel , Titânio , Microtomografia por Raio-XRESUMO
A localized, intermittent muscle-cooling protocol was implemented to determine cooling garment efficacy in reducing upper extremity muscular fatigue and tremor in novice ( n = 10) and experienced surgeons ( n = 9). Subjects wore a muscle-cooling garment while performing multiple trials of a forearm exercise and paired suturing task to induce muscular fatigue and exercise-induced tremor. A reduction in tremor amplitude and an extension in time to fatigue were expected with muscle cooling as compared with control trials. Each subject completed an intervention session (5°C cooling condition) and a control session (32°C or thermal neutral condition). A paired samples t test indicated that tremor amplitude was significantly reduced ( t [8] = 1.89458; p < 0.05) in experienced surgeons in two dimensions (up and down, and back and forth). Tremor amplitude was reduced in novice surgeons but the effect was not significant. Time to fatigue and suture time improved in both cohorts with muscle cooling, but the effect did not reach significance. Results from the pilot work suggest muscle cooling as an intervention for reduction of fatigue and tremor is very promising, warranting further investigation. Surgical specialties that require prolonged procedures might benefit more from this intervention.
RESUMO
We describe a technique to position patients with severe kyphosis so the head is horizontal. The legs are lifted into the Trendelenburg position, and the patient is supported with straps similar to those of a parachute. Although the patient's legs are high above the head and jugular distension has been observed, we have not noticed posterior pressure in the 2 cases we describe. This technique is similar to that described by others but has the additional security of the parachute-like straps.
Assuntos
Catarata/etiologia , Cifose/etiologia , Aparelhos Ortopédicos , Facoemulsificação/métodos , Postura , Idoso , Desenho de Equipamento , Humanos , Implante de Lente Intraocular , Masculino , Facoemulsificação/instrumentaçãoRESUMO
In artificial phospholipid bilayers, dual measurements of laurdan steady-state anisotropy and emission spectra can be used to identify the presence of liquid ordered phases. Human erythrocytes were used as a model to test whether similar measurements could be applied to biological samples. Specifically, laurdan anisotropy and emission spectra were obtained from native erythrocytes before and after treatment with calcium ionophore and from the microvesicles (known to be enriched in liquid ordered domains) shed from the cells during calcium entry. Spectral and anisotropy data were consistent with an increased order and reduced fluidity of erythrocyte membrane lipids upon ionophore treatment. Microvesicle membranes appeared more ordered than native erythrocytes and similar to ionophore-treated cells based on laurdan emission. In contrast, the anisotropy value was lower in microvesicles compared to ionophore-treated cells, suggesting greater probe mobility. Parallel measurements of diphenylhexatriene anisotropy corroborated the laurdan data. These results were consistent with the liquid ordered property of microvesicle membranes based on comparisons to behavior in artificial membranes. Two-photon microscopy was used to examine the distribution of laurdan fluorescence along the surface of erythrocyte membranes before and after ionophore treatment. A dual spatial analysis of laurdan anisotropy, as revealed by the distribution of laurdan emission spectra, and intensity excited by polarized light suggested that the plasma membranes of ionophore-treated erythrocytes may also exhibit elevated numbers of liquid ordered domains.
Assuntos
2-Naftilamina/análogos & derivados , Membrana Celular/metabolismo , Eritrócitos/metabolismo , Corantes Fluorescentes , Lauratos , Coloração e Rotulagem , Difenilexatrieno , Polarização de Fluorescência , Humanos , Microdomínios da Membrana/metabolismoRESUMO
Steroidogenic acute regulatory (StAR) protein facilitates import of cholesterol into adrenal and gonadal mitochondria where cholesterol is converted to pregnenolone, initiating steroidogenesis. StAR acts exclusively on the outer mitochondrial membrane (OMM) by unknown mechanisms. To identify StAR domains involved in membrane association, we reacted N-62 StAR with small unilamellar vesicles (SUVs) composed of lipids resembling the OMM. Solvent-exposed domains were digested with trypsin, Asp-N, or pepsin at different pH levels, and StAR peptides protected from proteolysis were identified by mass spectrometry. At pH 4 SUVs completely protected residues 259-282; at pH 6.5 this region was partially digested into 254-272, 254-273, and 254-274. Computer-graphic modeling of N-62 StAR indicated these peptides correspond to the C-terminal alpha4 helix and that residues Leu(275), Thr(263), and Arg(272) in alpha4 form stabilizing interactions with Gln(128), Asp(150), and Asp(106) in adjacent loops. CD spectroscopy of a 37-mer model of alpha4 (residues 247-287) indicated a random coil in aqueous buffer, but in 40% methanol the peptide was alpha-helical and achieved maximal alpha-helicity at pH 5.0 in the presence of SUVs. Reacting the 37-mer with diethyl pyrocarbamate incorporated into SUVs increased the number of modified residues. Thus the C-terminal alpha4 helix is critically involved in the membrane association of StAR with OMM lipids. The membrane association and the alpha-helical structure of the C terminus in the presence of OMM lipids are also pH-dependent. These results further support StAR undergoing a pH-dependent change in its conformation when interacting with the acidic phospholipid head groups of a membrane.
Assuntos
Concentração de Íons de Hidrogênio , Membranas Artificiais , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Hidrólise , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Fosfoproteínas/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Although cell membranes normally resist the hydrolytic action of secretory phospholipase A(2) (sPLA(2)), they become susceptible during apoptosis or after cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of the calcium ionophore to cause susceptibility depends on temperature, occurring best above approximately 35 degrees C. Considerable evidence from experiments with artificial bilayers suggests that hydrolysis of membrane lipids requires two steps. First, the enzyme adsorbs to the membrane surface, and second, a phospholipid diffuses from the membrane into the active site of the adsorbed enzyme. Analysis of kinetic experiments suggested that this mechanism can explain the action of sPLA(2) on erythrocyte membranes and that temperature and calcium loading promote the second step. This conclusion was further supported by binding experiments and assessment of membrane lipid packing. The adsorption of fluorescent-labeled sPLA(2) was insensitive to either temperature or ionophore treatment. In contrast, the fluorescence of merocyanine 540, a probe sensitive to lipid packing, was affected by both. Lipid packing decreased modestly as temperature was raised from 20 to 60 degrees C. Calcium loading enhanced packing at temperatures in the low end of this range, but greatly reduced packing at higher temperatures. This result was corroborated by measurements of the rate of extraction of a fluorescent phosphatidylcholine analog from erythrocyte membranes. Furthermore, drugs known to inhibit susceptibility in erythrocytes also prevented the increase in phospholipid extraction rate. These results argue that the two-step model applies to biological as well as artificial membranes and that a limiting step in the hydrolysis of erythrocyte membranes is the ability of phospholipids to migrate into the active site of adsorbed enzyme.