Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 123
Filtrar
Más filtros

Banco de datos
Tipo del documento
Intervalo de año de publicación
1.
Mol Vis ; 29: 338-356, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38264613

RESUMEN

Purpose: Inflammation and oxidative stress contribute to age-related macular degeneration (AMD) and other retinal diseases. We tested a cell-penetrating peptide from the kinase inhibitory region of an intracellular checkpoint inhibitor suppressor of cytokine signaling 3 (R9-SOCS3-KIR) peptide for its ability to blunt the inflammatory or oxidative pathways leading to AMD. Methods: We used anaphylatoxin C5a to mimic the effect of activated complement, lipopolysaccharide (LPS), and tumor necrosis factor alpha (TNFα) to stimulate inflammation and paraquat to induce mitochondrial oxidative stress. We used a human retinal pigment epithelium (RPE) cell line (ARPE-19) as proliferating cells and a mouse macrophage cell line (J774A.1) to follow cell propagation using microscopy or cell titer assays. We evaluated inflammatory pathways by monitoring the nuclear translocation of NF-κB p65 and mitogen-activated protein kinase p38. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the induction of inflammatory markers. In differentiated ARPE-19 monolayers, we evaluated the integrity of tight junction proteins through microscopy and the measurement of transepithelial electrical resistance (TEER). We used intraperitoneal injection of sodium iodate in mice to test the ability of R9-SOC3-KIR to prevent RPE and retinal injury, as assessed by fundoscopy, optical coherence tomography, and histology. Results: R9-SOCS3-KIR treatment suppressed C5a-induced nuclear translocation of the NF-kB activation domain p65 in undifferentiated ARPE-19 cells. TNF-mediated damage to tight junction proteins in RPE, and the loss of TEER was prevented in the presence of R9-SOCS3-KIR. Treatment with the R9-SOCS3-KIR peptide blocked the C5a-induced expression of inflammatory genes. The R9-SOCS3-KIR treatment also blocked the LPS-induced expression of interleukin-6, MCP1, cyclooxygenase 2, and interleukin-1 beta. R9-SOCS3-KIR prevented paraquat-mediated cell death and enhanced the levels of antioxidant effectors. Daily eye drop treatment with R9-SOCS3-KIR protected against retinal injury caused by i.p. administration of sodium iodate. Conclusions: R9-SOCS3-KIR blocks the induction of inflammatory signaling in cell culture and reduces retinal damage in a widely used RPE/retinal oxidative injury model. As this peptide can be administered through corneal instillation, this treatment may offer a convenient way to slow down the progression of ocular diseases arising from inflammation and chronic oxidative stress.


Asunto(s)
Yodatos , Degeneración Macular , Enfermedades de la Retina , Humanos , Animales , Ratones , Lipopolisacáridos , Paraquat , Retina , Estrés Oxidativo , Péptidos , Inflamación , Proteínas de Uniones Estrechas , Citocinas
2.
Exp Eye Res ; 203: 108416, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33359513

RESUMEN

Microglia are immune cells of the central nervous system capable of distinct phenotypic changes and migration in response to injury. These changes most notably include the retraction of fine dendritic structures and adoption of a globular, phagocytic morphology. Due to their characteristic responses, microglia frequently act as histological indicators of injury progression. While algorithms seeking to automate microglia counts and morphological analysis are becoming increasingly popular, few exist that are adequate for use within the retina and manual analysis remains prevalent. To address this, we propose a novel segmentation routine, implemented within FIJI-ImageJ, to perform automated segmentation and cell counting of retinal microglia. We show that our routine could perform cell counts with accuracy similar to manual observers using the I307N Rho model. Tracking cell position relative to retinal vasculature, we observed population migration towards the photoreceptor layer beginning 12 h post light damage. Using feature selection with Chi2 and principal component analysis, we resolved cells along a morphological gradient, demonstrating that extracted features were sufficiently descriptive to capture subtle morphological changes within cell populations in I307N Rho and Balb/c TLR2-/- retinal degeneration models. Taken together, we introduce a novel automated routine capable of efficient image processing and segmentation. Using data retrieved following segmentation, we perform morphological analysis simultaneously on whole populations of cells, rather than individually. Our algorithm was built entirely with open-source software, for use on retinal microglia.


Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Luz/efectos adversos , Microglía/patología , Traumatismos Experimentales por Radiación/etiología , Retina/efectos de la radiación , Degeneración Retiniana/etiología , Algoritmos , Animales , Recuento de Células , Modelos Animales de Enfermedad , Imagenología Tridimensional , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Traumatismos Experimentales por Radiación/patología , Degeneración Retiniana/patología , Vasos Retinianos/patología
3.
Proc Natl Acad Sci U S A ; 115(36): E8547-E8556, 2018 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-30127005

RESUMEN

Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (RHO) gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) RHO in a mutation-independent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human RHO replacement cDNA made resistant to RNA interference and tested this construct in a naturally occurring canine model of RHO-adRP. Subretinal vector injections led to nearly complete suppression of endogenous canine RHO RNA, while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with RHO-adRP.


Asunto(s)
Dependovirus , Técnicas de Sustitución del Gen/métodos , Técnicas de Silenciamiento del Gen/métodos , Terapia Genética/métodos , Vectores Genéticos , ARN Catalítico , Células Fotorreceptoras Retinianas Bastones/metabolismo , Retinitis Pigmentosa , Rodopsina , Animales , Perros , Células HEK293 , Humanos , ARN Catalítico/biosíntesis , ARN Catalítico/genética , Células Fotorreceptoras Retinianas Bastones/patología , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Rodopsina/biosíntesis , Rodopsina/genética
4.
J Biol Chem ; 294(21): 8480-8489, 2019 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-30940649

RESUMEN

Among the repertoire of immunoregulatory proteins encoded by myxoma virus, M013 is a viral homologue of the viral pyrin domain-only protein (vPOP) family. In myeloid cells, M013 protein has been shown to inhibit both the inflammasome and NF-κB signaling pathways by direct binding to ASC1 and NF-κB1, respectively. In this study, a three-dimensional homology model of the M013 pyrin domain (PYD) was built based on similarities to known PYD structures. A distinctive feature of the deduced surface electrostatic map of the M013 PYD is the presence of a negatively region consisting of numerous aspartate and glutamate residues in close proximity. Single-site mutations of aspartate and glutamate residues reveal their role in interactions with ASC-1. The biological significance of charge complementarity in the M013-ASC-1 interaction was further confirmed by functional assays of caspase-1 activation and subsequent secretion of cytokines. M013 also has a unique 33-residue C-terminal tail that follows the N-terminal PYD, and it is enriched in positively charged residues. Deletion of the tail of M013 significantly inhibited the interactions between M013 and NF-κB1, thus compromising the ability of the viral protein to suppress the secretion of pro-inflammatory cytokines. These results demonstrate that vPOP M013 exploits distinct structural motifs to regulate both the inflammasome and NF-κB pathways.


Asunto(s)
Myxoma virus , FN-kappa B/inmunología , Transducción de Señal/inmunología , Proteínas Virales , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Caspasa 1/genética , Caspasa 1/inmunología , Células HeLa , Humanos , Inflamasomas/genética , Mutagénesis Sitio-Dirigida , Mutación Missense , Myxoma virus/química , Myxoma virus/genética , Myxoma virus/inmunología , FN-kappa B/genética , Dominios Proteicos , Transducción de Señal/genética , Células THP-1 , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/inmunología
5.
J Virol ; 92(16)2018 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-29875240

RESUMEN

During herpes simplex virus (HSV) latency, most viral genes are silenced, with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long noncoding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilized an adeno-associated virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition, these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness.IMPORTANCE Herpes simplex virus (HSV) establishes a lifelong infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency, with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This work shows that the LAT RNA is important for reactivation and suggests the potential of this treatment as a therapy for treating HSV infections.


Asunto(s)
Regulación Viral de la Expresión Génica , Herpesvirus Humano 1/fisiología , ARN Largo no Codificante/metabolismo , ARN Viral/metabolismo , Activación Viral , Latencia del Virus , Animales , Células Cultivadas , Dependovirus/genética , Vectores Genéticos , Herpesvirus Humano 1/genética , Neuronas/virología , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Largo no Codificante/genética , ARN Viral/genética , Conejos , Transcripción Genética
6.
Adv Exp Med Biol ; 1185: 477-481, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31884657

RESUMEN

Evidence suggests that metabolic dysregulation plays an important role in disease etiology of retinal degenerations. Several studies suggest that preserving the retinal metabolic ecosystem may be protective against retinal degenerations. We investigated whether activation of 5' adenosine monophosphate protein kinase (AMPK) is protective to the retina in several preclinical mouse models of retinal degeneration and found that metformin-induced activation of AMPK was able to delay or prevent retinal degeneration in the rd10 model of retinitis pigmentosa, the NaIO3 model of RPE and retinal injury, and the light damage model of retinal degeneration. This protection was associated with increased mitochondrial DNA copy number, increased levels of ATP, and a reduction in oxidative stress and oxidative DNA damage. We propose that AMPK plays an important role in regulation of the retinal metabolic ecosystem and that activation of AMPK may promote metabolic processes to prevent retinal degeneration.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Retina/enzimología , Degeneración Retiniana/prevención & control , Animales , Daño del ADN , ADN Mitocondrial/genética , Modelos Animales de Enfermedad , Dosificación de Gen , Metformina/farmacología , Ratones , Estrés Oxidativo , Retinitis Pigmentosa/enzimología , Retinitis Pigmentosa/prevención & control
7.
Exp Eye Res ; 177: 12-22, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30048621

RESUMEN

We describe an immunosuppressive peptide corresponding to the kinase inhibitory region (KIR) of the intracellular checkpoint protein suppressor of cytokine signaling 1 (SOCS-1) that binds to the phospho-tyrosine containing regions of the tyrosine kinases JAK2 and TYK2 and the adaptor protein MAL, and thereby inhibits signaling downstream from these signaling mediators. The peptide, SOCS1-KIR, is thus capable of downregulating overactive JAK/STAT or NF-kB signaling in somatic cells, including those in many compartments of the eye. Attachment of poly-arginine to this peptide (R9-SOCS1-KIR) allows it to penetrate the plasma membrane in aqueous media. R9-SOCS1-KIR was tested in ARPE-19 cells and was found to attenuate mediators of inflammation by blocking the inflammatory effects of IFNγ, TNFα, or IL-17A. R9-SOCS1-KIR and also protected against TNFα or IL-17A mediated damage to the barrier properties of ARPE-19 cells, as evidenced by immunostaining with the tight junction protein, zona occludin 1 (ZO-1), and measurement of transepithelial electrical resistance (TEER). Experimental autoimmune uveitis (EAU) was generated in B10. RIII mice using a peptide of interphotoreceptor retinal binding protein (IRBP161-180) as immunogen. Topical administration of R9-SOCS1-KIR, 2 days before (prophylactic), or 7 days after immunization (therapeutic) protected ocular structure and function as seen by fundoscopy, optical coherence tomography (OCT), and electroretinography (ERG). The ability R9-SOCS1-KIR to suppress ocular inflammation and preserve barrier properties of retinal pigment epithelium makes it a potential candidate for treatment of autoimmune uveitis.


Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Proteínas del Ojo/farmacología , Inmunosupresores/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteína 1 Supresora de la Señalización de Citocinas/farmacología , Uveítis/tratamiento farmacológico , Animales , Enfermedades Autoinmunes/inmunología , Péptidos de Penetración Celular , Modelos Animales de Enfermedad , Interleucina-17/metabolismo , Ratones , Factor de Necrosis Tumoral alfa/metabolismo , Uveítis/inmunología
8.
Mol Ther ; 25(4): 917-927, 2017 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-28202390

RESUMEN

Bone marrow stem and progenitor cells can differentiate into a range of non-hematopoietic cell types, including retinal pigment epithelium (RPE)-like cells. In this study, we programmed bone marrow-derived cells (BMDCs) ex vivo by inserting a stable RPE65 transgene using a lentiviral vector. We tested the efficacy of systemically administered RPE65-programmed BMDCs to prevent visual loss in the superoxide dismutase 2 knockdown (Sod2 KD) mouse model of age-related macular degeneration. Here, we present evidence that these RPE65-programmed BMDCs are recruited to the subretinal space, where they repopulate the RPE layer, preserve the photoreceptor layer, retain the thickness of the neural retina, reduce lipofuscin granule formation, and suppress microgliosis. Importantly, electroretinography and optokinetic response tests confirmed that visual function was significantly improved. Mice treated with non-modified BMDCs or BMDCs pre-programmed with LacZ did not exhibit significant improvement in visual deficit. RPE65-BMDC administration was most effective in early disease, when visual function and retinal morphology returned to near normal, and less effective in late-stage disease. This experimental paradigm offers a minimally invasive cellular therapy that can be given systemically overcoming the need for invasive ocular surgery and offering the potential to arrest progression in early AMD and other RPE-based diseases.


Asunto(s)
Células de la Médula Ósea/metabolismo , Ingeniería Celular , Degeneración Retiniana/genética , cis-trans-Isomerasas/genética , Animales , Tratamiento Basado en Trasplante de Células y Tejidos , Modelos Animales de Enfermedad , Electrorretinografía , Femenino , Terapia Genética , Vectores Genéticos/genética , Lentivirus/genética , Ratones , Ratones Noqueados , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Degeneración Retiniana/terapia , Superóxido Dismutasa/deficiencia
9.
Adv Exp Med Biol ; 1074: 11-17, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29721922

RESUMEN

Age-related macular degeneration (AMD) is the leading cause of blindness in older adults in developed countries. The molecular mechanisms of disease pathogenesis remain poorly understood; however, evidence suggests that mitochondrial dysfunction may contribute to the progression of the disease. Studies have shown that mitochondrial DNA lesions are increased in the retinal pigment epithelium (RPE) of human patients with the disease and that the number of these lesions increases with disease severity. Additionally, microscopy of human RPE from patients with dry AMD shows severe disruptions in mitochondrial inner and outer membrane structure, mitochondrial size, and mitochondrial cellular organization. Thus, improving our understanding of mitochondrial dysfunction in dry AMD pathogenesis may lead to the development of targeted therapies. We propose that mitochondrial dysfunction in the RPE can lead to the chronic oxidative stress associated with the disease. Therefore, one protective strategy may involve the use of small molecule therapies that target the regulation of mitochondrial biogenesis and mitochondrial fission and mitophagy.


Asunto(s)
ADN Mitocondrial/metabolismo , Degeneración Macular/metabolismo , Mitocondrias/patología , Terapia Molecular Dirigida , Epitelio Pigmentado de la Retina/patología , Adenilato Quinasa/fisiología , Animales , ADN Mitocondrial/genética , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Atrofia Geográfica/patología , Humanos , Yodatos/toxicidad , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/genética , Metformina/farmacología , Ratones , Mitocondrias/efectos de los fármacos , Dinámicas Mitocondriales/efectos de los fármacos , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo
10.
Adv Exp Med Biol ; 1074: 185-191, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29721943

RESUMEN

The eye is an immuno-privileged organ. However, certain diseases such as uveitis are intrinsically linked to inflammation. In several retinal degenerative diseases, there is a unique damage at the onset of the disease, but evidence suggests that chronic and low-grade inflammatory processes play an important role in their progression. Studies have identified similar signaling pathways and changes in resident immune cells within the retina among these diseases. Herein, we will discuss some of these studies and propose how understanding this inflammatory response could aid in the development of therapies.


Asunto(s)
Retinopatía Diabética/inmunología , Degeneración Macular/inmunología , Retinitis Pigmentosa/inmunología , Animales , Antígenos de Neoplasias/fisiología , Citocinas/fisiología , Retinopatía Diabética/patología , Células Ependimogliales/inmunología , Células Ependimogliales/patología , Gliosis/inmunología , Gliosis/patología , Humanos , Inflamasomas/fisiología , Inflamación , Degeneración Macular/patología , Ratones , Microglía/inmunología , Microglía/patología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Receptor para Productos Finales de Glicación Avanzada/deficiencia , Retina/inmunología , Retina/patología , Drusas Retinianas/inmunología , Drusas Retinianas/patología , Retinitis Pigmentosa/patología
11.
Proc Natl Acad Sci U S A ; 112(42): E5689-98, 2015 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-26438859

RESUMEN

Considerable evidence supports mutations in mitochondrial genes as the cause of maternally inherited diseases affecting tissues that rely primarily on oxidative energy metabolism, usually the nervous system, the heart, and skeletal muscles. Mitochondrial diseases are diverse, and animal models currently are limited. Here we introduced a mutant human mitochondrial gene responsible for Leber hereditary optic neuropathy (LHON) into the mouse germ line using fluorescence imaging for tissue-specific enrichment in the target retinal ganglion cells. A mitochondria-targeted adeno-associated virus (MTS-AAV) containing the mutant human NADH ubiquinone oxidoreductase subunit 4 (ND4) gene followed by mitochondrial-encoded mCherry was microinjected into zygotes. Female founders with mCherry fluorescence on ophthalmoscopy were backcrossed with normal males for eight generations. Mutant human ND4 DNA was 20% of mouse ND4 and did not integrate into the host genome. Translated human ND4 protein assembled into host respiratory complexes, decreasing respiratory chain function and increasing oxidative stress. Swelling of the optic nerve head was followed by progressive demise of ganglion cells and their axons, the hallmarks of human LHON. Early visual loss that began at 3 mo and progressed to blindness 8 mo after birth was reversed by intraocular injection of MTS-AAV expressing wild-type human ND4. The technology of introducing human mitochondrial genes into the mouse germ line has never been described, to our knowledge, and has implications not only for creating animal models recapitulating the counterpart human disorder but more importantly for reversing the adverse effects of the mutant gene using gene therapy to deliver the wild-type allele.


Asunto(s)
ADN Mitocondrial/genética , Técnicas de Transferencia de Gen , Células Germinativas , Mutación , Cigoto , Animales , Axones , Encéfalo/patología , Transporte de Electrón , Humanos , Ratones , Ratones Transgénicos , NADH Deshidrogenasa/genética , Estrés Oxidativo , Degeneración Retiniana/genética
12.
Proc Natl Acad Sci U S A ; 112(43): E5844-53, 2015 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-26460017

RESUMEN

Inherited retinal degenerations cause progressive loss of photoreceptor neurons with eventual blindness. Corrective or neuroprotective gene therapies under development could be delivered at a predegeneration stage to prevent the onset of disease, as well as at intermediate-degeneration stages to slow the rate of progression. Most preclinical gene therapy successes to date have been as predegeneration interventions. In many animal models, as well as in human studies, to date, retinal gene therapy administered well after the onset of degeneration was not able to modify the rate of progression even when successfully reversing dysfunction. We evaluated consequences of gene therapy delivered at intermediate stages of disease in a canine model of X-linked retinitis pigmentosa (XLRP) caused by a mutation in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene. Spatiotemporal natural history of disease was defined and therapeutic dose selected based on predegeneration results. Then interventions were timed at earlier and later phases of intermediate-stage disease, and photoreceptor degeneration monitored with noninvasive imaging, electrophysiological function, and visual behavior for more than 2 y. All parameters showed substantial and significant arrest of the progressive time course of disease with treatment, which resulted in long-term improved retinal function and visual behavior compared with control eyes. Histology confirmed that the human RPGR transgene was stably expressed in photoreceptors and associated with improved structural preservation of rods, cones, and ON bipolar cells together with correction of opsin mislocalization. These findings in a clinically relevant large animal model demonstrate the long-term efficacy of RPGR gene augmentation and substantially broaden the therapeutic window for intervention in patients with RPGR-XLRP.


Asunto(s)
Terapia Genética , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Degeneración Retiniana/terapia , Visión Ocular , Animales , Modelos Animales de Enfermedad , Perros , Degeneración Retiniana/fisiopatología
13.
J Virol ; 90(17): 7894-901, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27334582

RESUMEN

UNLABELLED: Following infection of epithelial tissues, herpes simplex virus 1 (HSV-1) virions travel via axonal transport to sensory ganglia and establish a lifelong latent infection within neurons. Recent studies have revealed that, following intraganglionic or intrathecal injection, recombinant adeno-associated virus (rAAV) vectors can also infect sensory neurons and are capable of stable, long-term transgene expression. We sought to determine if application of rAAV to peripheral nerve termini at the epithelial surface would allow rAAV to traffic to sensory ganglia in a manner similar to that seen with HSV. We hypothesized that footpad or ocular inoculation with rAAV8 would result in transduction of dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. To test this, we inoculated the footpads of mice with various amounts of rAAV as well as rAAV capsid mutants. We demonstrated that this method of inoculation can achieve a transduction rate of >90% of the sensory neurons in the DRG that innervate the footpad. Similarly, we showed that corneal inoculation with rAAV vectors in the rabbit efficiently transduced >70% of the TG neurons in the optic tract. Finally, we demonstrated that coinfection of mouse footpads or rabbit eyes with rAAV vectors and HSV-1 resulted in colocalization in nearly all of the HSV-1-positive neurons. These results suggest that rAAV is a useful tool for the study of HSV-1 infection and may provide a means to deliver therapeutic cargos for the treatment of HSV infections or of dysfunctions of sensory ganglia. IMPORTANCE: Adeno-associated virus (AAV) has been shown to transduce dorsal root ganglion sensory neurons following direct intraganglionic sciatic nerve injection and intraperitoneal and intravenous injection as well as intrathecal injection. We sought to determine if rAAV vectors would be delivered to the same sensory neurons that herpes simplex virus (HSV-1) infects when applied peripherally at an epithelial surface that had been treated to expose the underlying sensory nerve termini. For this study, we chose two well-established HSV-1 infection models: mouse footpad infection and rabbit ocular infection. The results presented here provide the first description of AAV vectors transducing neurons following delivery at the skin/epithelium/eye. The ability of AAV to cotransduce HSV-1-infected neurons in both the mouse and the rabbit provides an opportunity to experimentally explore and disrupt host and viral proteins that are integral to the establishment of HSV-1 latency, to the maintenance of latency, and to reactivation from latency in vivo.


Asunto(s)
Dependovirus/crecimiento & desarrollo , Dependovirus/genética , Vectores Genéticos , Herpesvirus Humano 1/crecimiento & desarrollo , Células Receptoras Sensoriales/virología , Transducción Genética , Animales , Coinfección/virología , Ojo/virología , Pie/virología , Ganglios Espinales/virología , Herpes Simple/virología , Ratones , Infecciones por Parvoviridae/virología , Conejos , Ganglio del Trigémino/virología
14.
Mol Vis ; 22: 294-310, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27110092

RESUMEN

PURPOSE: Chronic oxidative stress and subacute inflammation have been implicated as causes of age-related macular degeneration (AMD). In this study, we tested whether an orally available 5-OH-tryptamine (5HT) 1a receptor agonist, xaliproden, could protect against retinal pigment epithelium (RPE) cell damage in culture and in a mouse model of geographic atrophy. METHODS: Paraquat was used to create mitochondrial oxidative stress in ARPE-19 cells, and tumor necrosis factor-α (TNF-α) was used to stimulate the production of inflammatory cytokines in these cells. The production of antioxidant proteins, metallothionein, and inflammatory cytokines was assayed with quantitative real-time PCR. Cell survival was analyzed with microscopy and a cell titer assay. Integrity of the RPE monolayer was determined by measuring the transepithelial electrical resistance (TEER) and with immunocytochemistry with zona occludens protein 1 (ZO-1) antibody. RPE atrophy was studied in mice deleted for Sod2 (the gene for mitochondrial superoxide dismutase) specifically in the RPE. The mice were treated orally with daily doses of xaliproden at 0.5 and 3 mg/kg for 4 months. The retinal structure was analyzed with spectral domain optical coherence tomography (SD-OCT) and with light and electron microscopy. Retinal function was assessed with full-field electroretinography (ERG) and with optokinetic measurements. RESULTS: Xaliproden led to a dose-dependent increase in cell survival following treatment with paraquat. Synthesis of the antioxidant response genes NqO1, GSTM1, CAT, HO-1, and Nrf2 was increased in response to the drug, as was the zinc chaperone metallothionein. Treatment of cells with TNF-α led to increased production of IL-1ß, IL-6, chemokine (C-C motif) ligand 20 (CCL20), and vascular endothelial growth factor (VEGF) by ARPE-19 cells, and this response was attenuated by treatment with xaliproden. TNF-α also led to a decrease in the TEER that was prevented by treatment with the 5HT1a agonist. Daily gavage with xaliproden at either dose induced the production of protective enzymes in the mouse retina, and treatment of the Sod2-deleted mice with the drug showed improved thickness of the outer nuclear layer and improved visual acuity relative to the control-treated mice. There was no significant difference in full-field scotopic ERG among the treatment groups, however. Vacuolization of the RPE and disorganization of the photoreceptor outer segments were reduced at both dose levels of xaliproden. CONCLUSIONS: Xaliproden protected RPE cells from oxidative and inflammatory insults and protected the mouse RPE and retina from RPE atrophy in the face of excess mitochondrial oxidative stress. These results suggest that this drug, which had a reasonable safety profile in clinical trials, may be used to prevent the progression of geographic atrophy in humans.


Asunto(s)
Atrofia Geográfica/prevención & control , Naftalenos/uso terapéutico , Piridinas/uso terapéutico , Epitelio Pigmentado de la Retina/efectos de los fármacos , Agonistas del Receptor de Serotonina 5-HT1/uso terapéutico , Administración Oral , Animales , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Reposicionamiento de Medicamentos , Impedancia Eléctrica , Electrorretinografía , Ensayo de Inmunoadsorción Enzimática , Atrofia Geográfica/metabolismo , Atrofia Geográfica/fisiopatología , Humanos , Metalotioneína/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Naftalenos/administración & dosificación , Piridinas/administración & dosificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/fisiología , Epitelio Pigmentado de la Retina/metabolismo , Agonistas del Receptor de Serotonina 5-HT1/administración & dosificación , Tomografía de Coherencia Óptica , Proteína de la Zonula Occludens-1/metabolismo
15.
Mol Ther ; 23(5): 875-884, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25698151

RESUMEN

Inflammation is a key component of chronic and acute diseases of the eye. Our goal is to test anti-inflammatory genes delivered by an adeno-associated virus (AAV) vector as potential treatments for retinal inflammation. We developed a secretable and cell penetrating form of the caspase activation and recruitment domain (CARD) from the apoptosis-associated speck-like protein containing a CARD (ASC) gene that binds caspase-1 and inhibits its activation by the inflammasome. The secretion and cell penetration characteristics of this construct were validated in vitro by measuring its effects on inflammasome signaling in a monocyte cell line and in an retinal pigmented epithelium (RPE) cell line. This vector was then packaged as AAV particles and tested in the endotoxin-induced uveitis mouse model. Gene expression was monitored one month after vector injection by fluorescence fundoscopy. Ocular inflammation was then induced by injecting lipopolysaccharide into the vitreous and was followed by enucleation 24 hours later. Eyes injected with the secretable and cell penetrating CARD AAV vector had both a significantly lower concentration of IL-1ß as well as a 64% reduction in infiltrating cells detected in histological sections. These results suggest that anti-inflammatory genes such as the CARD could be used to treat recurring inflammatory diseases like uveitis or chronic subacute inflammations of the eye.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Terapia Genética , Inflamación/genética , Dominios y Motivos de Interacción de Proteínas/genética , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD , Caspasa 1/metabolismo , Línea Celular , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Endotoxinas/efectos adversos , Expresión Génica , Orden Génico , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Inflamación/metabolismo , Inflamación/patología , Inflamación/terapia , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/metabolismo , Lentivirus/genética , Ratones , Reproducibilidad de los Resultados , Retina/metabolismo , Transducción Genética , Transgenes , Uveítis/inducido químicamente , Uveítis/genética , Uveítis/metabolismo , Uveítis/patología , Uveítis/terapia
16.
Adv Exp Med Biol ; 854: 59-65, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26427394

RESUMEN

Age related macular degeneration (AMD) is the most common cause of blindness among people of 65 years and older in developed countries (Klein and Klein, Invest Ophthalmol Vis Sci 54:7395-7401, 2013). Recent advances in dry AMD research points towards an important role of the inflammatory response in the development of the disease. The presence of inflammatory cells, antibodies, complement factors and pro-inflammatory cytokines in AMD retinas and drusen indicates that the immune system could be an important driving force in dry AMD. The NLRP3 inflammasome has been proposed as an integrator of process associated with AMD and the induction of inflammation. Herein we summarize the most recent studies that attempt to understand the role of the NLRP3 inflammasome in AMD.


Asunto(s)
Proteínas Portadoras/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Degeneración Macular/metabolismo , Animales , Citocinas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Retina/metabolismo , Transducción de Señal
17.
Adv Exp Med Biol ; 854: 509-15, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26427453

RESUMEN

The G90D mutation in the rhodopsin gene leads to autosomal dominant congenital stationary night blindness (CSNB) in patients. This occurs because the G90D mutant protein cannot efficiently bind chromophore and is constitutively active. To combat this mutation, we designed and characterized two different hammerhead ribozymes to cleave G90D transcript. In vitro testing showed that the G90D1 ribozyme efficiently and specifically cleaved the mutant transcript while G90D2 cleaved both WT and mutant transcript. AAV-mediated delivery of G90D1 under the control of the mouse opsin promoter (MOP500) to G90D transgenic eyes showed that the ribozyme partially retarded the functional degeneration (as measured by electroretinography [ERG]) associated with this mutation. These results suggest that with additional optimization, ribozymes may be a useful part of the gene therapy knockdown strategy for dominant retinal disease.


Asunto(s)
Enfermedades Hereditarias del Ojo/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Mutación , Miopía/genética , Ceguera Nocturna/genética , ARN Catalítico/metabolismo , Rodopsina/genética , Animales , Biocatálisis , Dependovirus/genética , Electrorretinografía , Enfermedades Hereditarias del Ojo/fisiopatología , Enfermedades Hereditarias del Ojo/terapia , Enfermedades Genéticas Ligadas al Cromosoma X/fisiopatología , Enfermedades Genéticas Ligadas al Cromosoma X/terapia , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Ratones Transgénicos , Proteínas Mutantes/genética , Miopía/fisiopatología , Miopía/terapia , Ceguera Nocturna/fisiopatología , Ceguera Nocturna/terapia , ARN/genética , ARN/metabolismo , ARN Catalítico/genética , Especificidad por Sustrato , Transcripción Genética/genética
18.
Adv Exp Med Biol ; 854: 31-7, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26427390

RESUMEN

An appropriate animal model is essential to screening drugs or designing a treatment strategy for geographic atrophy. Since oxidative stress contributes to the pathological changes of the retinal pigment epithelium (RPE), we are reporting a new mouse AMD model of retinal degeneration by inducing mitochondrial oxidative stress in RPE. Sod2 the gene for manganese superoxide dismutase (MnSOD) was deleted in RPE layer using conditional knockout strategy. Fundus microscopy, SD-OCT and electroretinography were used to monitor retinal structure and function in living animals and microscopy was used to assess pathology post mortem. Tissue specific deletion of Sod2 caused elevated signs of oxidative stress, RPE dysfunction and showed some key features of AMD. Due to induction of oxidative stress, the conditional knockout mice show progressive reduction in ERG responses and thinning of outer nuclear layer (ONL) compared to non-induced littermates.


Asunto(s)
Modelos Animales de Enfermedad , Degeneración Macular/genética , Estrés Oxidativo , Degeneración Retiniana/genética , Epitelio Pigmentado de la Retina/metabolismo , Superóxido Dismutasa/genética , Animales , Bestrofinas , Electrorretinografía , Proteínas del Ojo/genética , Femenino , Humanos , Inmunohistoquímica , Canales Iónicos/genética , Degeneración Macular/metabolismo , Degeneración Macular/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Oftalmoscopios , Oftalmoscopía/métodos , Degeneración Retiniana/metabolismo , Degeneración Retiniana/fisiopatología , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/fisiopatología , Superóxido Dismutasa/deficiencia , Superóxido Dismutasa/metabolismo , Tomografía de Coherencia Óptica
19.
Adv Exp Med Biol ; 854: 185-91, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26427410

RESUMEN

RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease -mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop (-/-) background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin.


Asunto(s)
Degeneración Retiniana/genética , Células Fotorreceptoras Retinianas Bastones/metabolismo , Rodopsina/genética , Factor de Transcripción CHOP/genética , Animales , Supervivencia Celular/genética , Electrorretinografía , Expresión Génica , Humanos , Ratones Noqueados , Ratones Transgénicos , Degeneración Retiniana/metabolismo , Degeneración Retiniana/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rodopsina/metabolismo , Factor de Transcripción CHOP/deficiencia , Transgenes/genética
20.
Exp Eye Res ; 140: 94-105, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26315784

RESUMEN

Chronic oxidative stress contributes to age related diseases including age related macular degeneration (AMD). Earlier work showed that the 5-hydroxy-tryptamine 1a (5HT1a) receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) protects retinal pigment epithelium (RPE) cells from hydrogen peroxide treatment and mouse retinas from oxidative insults including light injury. In our current experiments, RPE derived cells subjected to mitochondrial oxidative stress were protected from cell death by the up-regulation of anti-oxidant enzymes and of the metal ion chaperone metallothionein. Differentiated RPE cells were resistant to oxidative stress, and the expression of genes for protective proteins was highly increased by oxidative stress plus drug treatment. In mice treated with 8-OH-DPAT, the same genes (MT1, HO1, NqO1, Cat, Sod1) were induced in the neural retina, but the drug did not affect the expression of Sod2, the gene for manganese superoxide dismutase. We used a mouse strain deleted for Sod2 in the RPE to accelerate age-related oxidative stress in the retina and to test the impact of 8-OH-DPAT on the photoreceptor and RPE degeneration developed in these mice. Treatment of mice with daily injections of the drug led to increased electroretinogram (ERG) amplitudes in dark-adapted mice and to a slight improvement in visual acuity. Most strikingly, in mice treated with a high dose of the drug (5 mg/kg) the structure of the RPE and Bruch's membrane and the normal architecture of photoreceptor outer segments were preserved. These results suggest that systemic treatment with this class of drugs may be useful in preventing geographic atrophy, the advanced form of dry AMD, which is characterized by RPE degeneration.


Asunto(s)
8-Hidroxi-2-(di-n-propilamino)tetralin/uso terapéutico , Mitocondrias/metabolismo , Estrés Oxidativo , Retina/efectos de los fármacos , Agonistas de Receptores de Serotonina/uso terapéutico , Animales , Línea Celular , Electrorretinografía , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Eliminación de Gen , Metalotioneína/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidorreductasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Serotonina 5-HT1A/metabolismo , Retina/metabolismo , Retina/fisiopatología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Superóxido Dismutasa/genética , Tomografía de Coherencia Óptica , Agudeza Visual/efectos de los fármacos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA