RESUMEN
PURPOSE: To evaluate the technique, safety, and efficacy of the retropupillary implantation of iris-claw intraocular lenses in a long-term follow-up study. PATIENTS AND METHODS: This retrospective study included 31 eyes of 31 patients who underwent an Artisan aphakic intraocular lens implantation between January 2006 and February 2011 at the University Hospital Essen, Essen, Germany and at the Zentrum für Augenheilkunde PD Dr Laube, Düsseldorf, Germany. Preoperative data collected included demographics, etiology of aphakia, previous surgeries, preoperative eye pathology, intraocular pressure, clinical signs of endothelial cell loss, and best corrected visual acuity. Operative data and postoperative outcomes included the best corrected visual acuity, lens position, intraocular pressure, pigment dispersion, clinical signs of endothelial cell loss, development of macular edema, and other complications. RESULTS: Thirty-one patients were included. The mean follow-up was 25.2 months (range: 4-48 months). The mean best corrected visual acuity postoperatively was 0.64 logarithm of the minimum angle of resolution (logMAR) and varied from 0 logMAR to 3 logMAR. Some patients had a low visual acuity preoperatively because of preoperative eye pathologies. In 22 patients the visual acuity improved, in two patients the visual acuity remained unchanged, and seven patients showed a decreased visual acuity. Complications were peaked pupils (n=10) and retinal detachment in one case. Four patients showed an iris atrophy and high intraocular pressure was observed only in one patient. Subluxation of the intraocular lens, endothelial cell loss, and macular edema were not observed. CONCLUSION: The presented long-term results demonstrate that retropupillary iris-claw lens implantation is a safe and effective method for the correction of aphakia in patients without capsule support. This surgical procedure has the advantages of a posterior chamber implantation with a low intraoperative and postoperative risk profile.
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A requisite component of nervous system development is the achievement of cellular recognition and spatial segregation through competition-based refinement mechanisms. Competition for available axon space by myelinating oligodendrocytes ensures that all relevant CNS axons are myelinated properly. To ascertain the nature of this competition, we generated a transgenic mouse with sparsely labeled oligodendrocytes and establish that individual oligodendrocytes occupying similar axon tracts can greatly vary the number and lengths of their myelin internodes. Here we show that intercellular interactions between competing oligodendroglia influence the number and length of myelin internodes, referred to as myelinogenic potential, and identify the amino-terminal region of Nogo-A, expressed by oligodendroglia, as necessary and sufficient to inhibit this process. Exuberant and expansive myelination/remyelination is detected in the absence of Nogo during development and after demyelination, suggesting that spatial segregation and myelin extent is limited by microenvironmental inhibition. We demonstrate a unique physiological role for Nogo-A in the precise myelination of the developing CNS. Maximizing the myelinogenic potential of oligodendrocytes may offer an effective strategy for repair in future therapies for demyelination.
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Sistema Nervioso Central/patología , Enfermedades Desmielinizantes/fisiopatología , Proteínas de la Mielina/metabolismo , Vaina de Mielina/fisiología , Oligodendroglía/fisiología , Animales , Western Blotting , Sistema Nervioso Central/citología , Técnicas de Silenciamiento del Gen , Técnicas Histológicas , Ratones , Ratones Transgénicos , Microscopía Electrónica , Microesferas , Proteínas de la Mielina/genética , Proteínas Nogo , Oligodendroglía/metabolismo , Oligodendroglía/ultraestructura , Poliestirenos , ARN Interferente Pequeño/genética , UltracentrifugaciónRESUMEN
Activation of interferon regulatory factors (IRFs) 3 and 7 is essential for the induction of Type I interferons (IFN) and innate antiviral responses, and herpesviruses have evolved mechanisms to evade such responses. We previously reported that Epstein-Barr virus BZLF1, an immediate-early (IE) protein, inhibits the function of IRF7, but the role of BRLF1, the other IE transactivator, in IRF regulation has not been examined. We now show that BRLF1 expression decreased induction of IFN-beta, and reduced expression of IRF3 and IRF7; effects were dependent on N- and C-terminal regions of BRLF1 and its nuclear localization signal. Endogenous IRF3 and IRF7 RNA and protein levels were also decreased during cytolytic EBV infection. Finally, production of IFN-beta was decreased during lytic EBV infection and was associated with increased susceptibility to superinfection with Sendai virus. These data suggest a new role for BRLF1 with the ability to evade host innate immune responses.
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Proteínas Aviares/antagonistas & inhibidores , Herpesvirus Humano 4/patogenicidad , Interacciones Huésped-Patógeno , Proteínas Inmediatas-Precoces/fisiología , Factor 7 Regulador del Interferón/antagonistas & inhibidores , Factores Reguladores del Interferón/antagonistas & inhibidores , Interferón beta/antagonistas & inhibidores , Transactivadores/fisiología , Proteínas Aviares/inmunología , Línea Celular , Herpesvirus Humano 4/inmunología , Humanos , Factor 7 Regulador del Interferón/inmunología , Factores Reguladores del Interferón/inmunología , Interferón beta/inmunología , Señales de Localización Nuclear , Mapeo de Interacción de Proteínas , Virus Sendai/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.
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Inestabilidad Genómica , ARN Interferente Pequeño/fisiología , Transducción de Señal , Enfermedad de Charcot-Marie-Tooth/genética , Biología Computacional , Daño del ADN , Reparación del ADN/genética , Replicación del ADN/genética , Regulación hacia Abajo , Genes cdc , Biblioteca Genómica , Genómica , Células HeLa , Histonas/metabolismo , Humanos , Fosforilación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
PURPOSE: Pathologic corneal neovascularization not only reduces corneal transparency and visual acuity, but also is one of the most significant preoperative and postoperative risk factors for graft rejection after corneal transplantation. The aim of this study was to test tolerability and efficacy of gene signal (GS)-101 eye drops, an antisense oligonucleotide against insulin receptor substrate-1, versus placebo on inhibition of progressive corneal neovascularization. DESIGN: Randomized, double-blind, multicenter, phase II clinical study. PARTICIPANTS AND CONTROLS: Interim analysis on 40 patients with progressive corneal neovascularization resulting from various underlying diseases being nonresponsive to conventional therapy. INTERVENTIONS: Four groups of 10 patients were treated for 3 months in this dose-finding study comparing 3 doses of GS-101 (eye drops twice daily; 43, 86, and 172 microg/day total) with placebo (10 patients per group). MAIN OUTCOME MEASURES: The primary end point was the area covered by pathologic corneal blood vessels, which was measured morphometrically on digitized slit-lamp pictures using image analysis techniques. RESULTS: GS-101 eye drops were well tolerated. All serious and 95% of all other adverse events were categorized by the investigators as unrelated. In 3 patients, there was a potentially related side effect of ocular surface discomfort. At a dose of 86 microg/day (43 microg/drop), GS-101 eye drops produced a significant inhibition and regression of corneal neovascularization (-2.04+/-1.57% of total corneal area; P = 0.0047), whereas the low dose tended to stabilize it (0.07+/-2.94%; P = 0.2088) compared with placebo (0.89+/-2.15%), where corneal neovascularization progressed in all patients. There was no apparent benefit to the higher dose (1.60+/-7.63%). CONCLUSIONS: The interim results of this phase II study suggest that GS-101 eye drops at an optimal dose of 86 microg/day are an effective and noninvasive approach specifically to inhibit and regress active corneal angiogenesis, a major risk factor for corneal graft transplantation and graft rejection. Safety concerns were not detected. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.
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Inhibidores de la Angiogénesis/administración & dosificación , Neovascularización de la Córnea/tratamiento farmacológico , Oligonucleótidos/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Inhibidores de la Angiogénesis/efectos adversos , Neovascularización de la Córnea/patología , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oligonucleótidos/efectos adversos , Soluciones Oftálmicas/administración & dosificación , Soluciones Oftálmicas/efectos adversos , Resultado del Tratamiento , Agudeza Visual/efectos de los fármacosRESUMEN
Cell cycle analysis typically relies on fixed time-point measurements of cells in particular phases of the cell cycle. The cell cycle, however, is a dynamic process whose subtle shifts are lost by fixed time-point methods. Live-cell fluorescent biosensors and time-lapse microscopy allows the collection of temporal information about real time cell cycle progression and arrest. Using two genetically-encoded biosensors, we measured the precision of the G(1), S, G(2) and M cell cycle phase durations in different cell types and identified a bimodal G(1) phase duration in a fibroblast cell line that is not present in the other cell types. Using a cell line model for neuronal differentiation, we demonstrated that NGF-induced neurite extension occurs independently of NGF-induced cell cycle G(1) phase arrest. Thus, we have begun to use cell cycle fluorescent biosensors to examine the proliferation of cell populations at the resolution of individual cells and neuronal differentiation as a dynamic process of parallel cell cycle arrest and neurite outgrowth.
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Técnicas Biosensibles/métodos , Ciclo Celular/fisiología , División Celular/fisiología , Neuritas/fisiología , Neurogénesis/fisiología , Animales , Fibroblastos/citología , Fibroblastos/fisiología , Fluorescencia , Células HeLa , Humanos , Ratones , Factor de Crecimiento Nervioso/metabolismo , Neuronas/citología , Neuronas/fisiología , Células PC12 , RatasRESUMEN
High content cell-based genetic and small molecule library screens are powerful strategies in drug discovery and investigations of disease mechanisms. We report that primary cells derived from a transgenic mouse model expressing a fluorescence mitosis biosensor provide unambiguous phenotype readouts without the need for transfection or immunocytochemistry. Phenotype profiles of cell cycle disruption and of apoptosis are easily detectable at a single time point selected from time-lapse live fluorescence microscopy. Most importantly, this transgenic mouse model may be crossed with cancer mouse models to derive biosensor-expressing primary cancer cells for use in high content screening strategies targeting discovery of tumor-specific chemotherapeutic compounds.
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Ciclo Celular/genética , Fibroblastos/fisiología , Pruebas Genéticas/métodos , Modelos Animales , Fenotipo , Animales , Femenino , Fibroblastos/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Embarazo , Especificidad de la EspecieRESUMEN
Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.
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Centrosoma/metabolismo , Ciclina A/fisiología , Ciclina B/fisiología , Mitosis/fisiología , Membrana Nuclear/metabolismo , Núcleo Celular/metabolismo , Ciclina A2 , Ciclina B/metabolismo , Ciclina B1 , Ciclina B2 , Quinasas Ciclina-Dependientes/metabolismo , Células HeLa , HumanosRESUMEN
Virus infection stimulates potent antiviral responses; specifically, Epstein-Barr virus (EBV) infection induces and activates interferon regulatory factor 7 (IRF-7), which is essential for production of alpha/beta interferons (IFN-alpha/beta) and upregulates expression of Tap-2. Here we present evidence that during cytolytic viral replication the immediate-early EBV protein BZLF-1 counteracts effects of IRF-7 that are central to host antiviral responses. We initiated these studies by examining IRF-7 protein expression in vivo in lesions of hairy leukoplakia (HLP) in which there is abundant EBV replication but the expected inflammatory infiltrate is absent. This absence might predict that factors involved in the antiviral response are absent or inactive. First, we detected significant levels of IRF-7 in the nucleus, as well as in the cytoplasm, of cells in HLP lesions. IRF-7 activity in cell lines during cytolytic viral replication was examined by assay of the IRF-7-responsive promoters, IFN-alpha4, IFN-beta, and Tap-2, as well as of an IFN-stimulated response element (ISRE)-containing reporter construct. These reporter constructs showed consistent reduction of activity during lytic replication. Both endogenous and transiently expressed IRF-7 and EBV BZLF-1 proteins physically associate in cell culture, although BZLF-1 had no effect on the nuclear localization of IRF-7. However, IRF-7-dependent activity of the IFN-alpha4, IFN-beta, and Tap-2 promoters, as well as an ISRE promoter construct, was inhibited by BZLF-1. This inhibition occurred in the absence of other EBV proteins and was independent of IFN signaling. Expression of BZLF-1 also inhibited activation of IRF-7 by double-stranded RNA, as well as the activity of a constitutively active mutant form of IRF-7. Negative regulation of IRF-7 by BZLF-1 required the activation domain but not the DNA-binding domain of BZLF-1. Thus, EBV may subvert cellular antiviral responses and immune detection by blocking the activation of IFN-alpha4, IFN-beta, and Tap-2 by IRF-7 through the medium of BZLF-1 as a negative regulator.
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Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 4/fisiología , Transactivadores/genética , Transcripción Genética , Proteínas Virales/genética , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Biopsia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulación hacia Abajo , Factor 7 Regulador del Interferón , Interferón-alfa/genética , Interferón-alfa/metabolismo , Interferón beta/genética , Interferón beta/metabolismo , Leucoplasia Vellosa/metabolismo , Leucoplasia Vellosa/patología , Leucoplasia Vellosa/virología , Regiones Promotoras Genéticas , Piel/metabolismo , Piel/patología , Piel/virología , Transactivadores/metabolismo , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
We have shown previously that interferon regulatory factor 7 (IRF7), a multifunctional protein intimately involved in latent Epstein-Barr virus (EBV) infection, is induced as well as activated by EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein. Since the LMP1 promoter (LMP1p) contains an interferon-stimulated response element (ISRE), we hypothesized that IRF7 might be able to regulate LMP1 expression and thus participate in a regulatory circuit between these two genes. In this study, IRF7 was shown first to activate LMP1p in transient transfection assays. Compared with EBV nuclear antigen 2 (EBNA2), the most potent viral transactivator of LMP1p, IRF7 has a lesser effect (approximately 10% that of EBNA2) on induction of LMP1p. Study with IRF7 deletion mutants showed that IRF7 functional domains have similar effects on both the beta interferon (IFN-beta) and LMP1 promoters in BJAB and 293 cells, and study with IRF7 phosphomimetic mutants showed that IRF7 phosphorylation may be involved in the activation of these two promoters. Further, the ISRE in LMP1p responds to IRF7 induction and IRF7 binds to this element. In the EBV-positive cell line P3HR1, which lacks the complete EBNA2 and EBV-encoded leader protein genes and hence expresses low-level LMP1, IRF7 alone can notably increase the endogenous LMP1 mRNA and protein levels. These results indicate that LMP1 is regulated by this host cell gene in addition to the viral factor, EBNA2, and may help to explain how LMP1 is expressed in type II latency in the absence of EBNA2. Moreover, IRF7 can regulate a viral gene in addition to a host cellular gene such as the IFN-beta gene. Together with the previous data that LMP1 can induce IRF7 expression and facilitate IRF7 phosphorylation and nuclear translocation, these results suggest a positive regulatory circuit between IRF7 and LMP1.