Depistage mutationnel des genes de la peripherine/RDS, rhodopsine et ROM-1 dans 69 cas index de retinite pigmentaire et autres dystrophies retiniennes [Mutational screening of peripherin/RDS genes, rhodopsin and ROM-1 in 69 index cases with retinitis pigmentosa and other retinal dystrophies]

PURPOSE: Phenotypic, genetic and molecular characterization of 69 index patients with retinitis pigmentosa (RP) and various inherited retinal diseases. PATIENTS AND METHOD: patients went through complete ocular examination and blood samples were drawn for mutational screening of three candidate genes: rhodopsin (RHO), peripherin/RDS, and ROM-1. RESULTS: the most frequent type of RP among our population was the autosomal dominant (43.6%). Three RHO mutations were found among the RP patients. A RDS mutation was detected in three unrelated families segregating dominant macular dystrophy. DISCUSSION AND CONCLUSIONS: 18% of the autosomal dominant RP patients presented a RHO mutation; RDS R172W mutation was present in 25% of the dominant macular dystrophies.

Patient with Fanconi Syndrome (FS) and retinitis pigmentosa (RP) caused by a deletion and duplication of mitochondrial DNA (mtDNA).

BACKGROUND: We report a patient with a highly unusual presentation of a mitochondrial disorder. HISTORY AND SIGNS: An 8-year old girl presented with muscular cramps as well as height and weight deceleration. Investigations revealed lactic acidosis, electrolytic imbalance and urinary loss of glucose and electrolytes secondary to proximal renal tubulopathy consistent with Fanconi syndrome (FS). Ophthalmic examination revealed asymptomatic retinitis pigmentosa (RP) with no other ocular manifestations. A mitochondriopathy was suspected and genetic analysis performed. THERAPY AND OUTCOME: Southern blotting documented a heteroplasmic mutation of mtDNA with deletion/duplication. Three discrete mitochondrial genomes were detected: normal; deletion of 6.7 kb and a deletion/duplication consisting of 1 normal and 1 deleted genome. The relative proportions varied considerably between tissues. CONCLUSIONS: The association of FS and RP combines features of Kearns-Sayre syndrome and Pearson marrow-pancreas syndrome, without being typical of either. This highly unusual clinical presentation emphasises the need for systemic investigation of patients with FS and further underlines the importance of mtDNA analysis in patients with unexpected associations of affected tissues.

Identification of the minimal promoter region of the mouse NKX5-3, a transcription factor implicated in eye development.

Early ocular development is controlled by a complex network of transcription factors, cell cycle regulators, and diffusible signalling molecules. Together, these molecules regulate cell proliferation and apoptosis, and specify retinal fate. NKX5-3 is a homeobox transcription factor implicated in eye development. The analysis of the 5'-flanking region of the mouse Nkx5-3 gene revealed a predicted TATA-less promoter sequence between -416 and -166 of the translation start site. To functionally characterise Nkx5-3 promoter activity, serial deletions of the promoter sequence were introduced in pGL-3 basic vector and promoter activity of these 5'- and 3'-deleted constructions was tested in HeLa and CHO cells. Transactivation assays identified a region between -350 and -296 exhibiting promoter-like activity. Combined analysis by deletions and point mutations showed that this sequence, containing multiple Sp1 binding sites was necessary to promote transcriptional activity. Binding of Sp1 to this region was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation, using an antibody specific for Sp1. Altogether, these results demonstrated that the immediate upstream region of Nkx5-3 gene possessed a strong intrinsic promoter activity in vitro, suggesting a potential role in Nkx5-3 transcription in vivo.

Iontophoresis: from the lab to the bed side.

Pioneer work on iontophoresis undertaken by David Maurice during the 1970s and 1980s laid the initial groundwork for its potential implementation as a promising ocular therapeutic modality. A better understanding of tissue interactions within the eye during electric current application, along with better designs of drug delivery devices have enabled us to pursue David Maurice's original ideas and take them from the bench to the bed side. In the present study we demonstrate the potential application of an iontophoresis device (Eyegate, Optis, France) for the treatment of certain human eye diseases. Seventeen patients received a penetrating keratoplasty (PKP) at various intervals before presentation with active graft rejection in our clinic and were treated using this iontophoresis device. Methylprednisolone sodium succinate (MP) 62.5 mg/ml was infused within the Eyegate ocular probe container and an electrical current of 1.5 mA was delivered for 4 min with the negative pole connected to the ocular probe. Patients were treated on an ambulatory basis and received a standard course of three iontophoresis applications given once a day over 3 consecutive days. After treatment, 15 of the 17 treated eyes (88%) demonstrated a complete reversal of the rejection processes. In two eyes, only a partial and temporary improvement was observed. The mean best corrected visual acuity of all 17 patients during the last follow up visit was 0.37 +/- 0.2 compared to 0.06 +/- 0.05 before initiation of the iontophoresis treatment. The mean follow-up time was 13.7 months with a range of 5-29 months for the 17 patients. No significant side-effects associated with the iontophoresis treatment were observed. Thus, for the management of active corneal graft rejection, iontophoresis of MP can be an alternative to very frequent instillations of eye drops, or to pulsed intravenous therapy of corticosteroids.

Protective effect of intravitreal injection of vasoactive intestinal peptide-loaded liposomes on experimental autoimmune uveoretinitis.

PURPOSE: The aim of this study was to investigate the effect of a single intravitreal (i.v.t.) injection of vasoactive intestinal peptide (VIP) loaded in rhodamine-conjugated liposomes (VIP-Rh-Lip) on experimental autoimmune uveoretinitis (EAU). METHODS: An i.v.t. injection of VIP-Rh-Lip, saline, VIP, or empty-(E)-Rh-Lip was performed simultaneously, either 6 or 12 days after footpad immunization with retinal S-antigen in Lewis rats. Clinical and histologic scores were determined. Immunohistochemistry and cytokine quantification by multiplex enzyme-linked immunosorbent assay were performed in ocular tissues. Systemic immune response was determined at day 20 postimmunization by measuring proliferation and cytokine secretion of cells from inguinal lymph nodes (ILNs) draining the immunization site, specific delayed-type hypersensitivity (DTH), and the serum concentration of cytokines. Ocular and systemic biodistribution of VIP-Rh-Lip was studied in normal and EAU rats by immunofluorescence. RESULTS: The i.v.t. injection of VIP-Rh-Lip performed during the afferent, but not the efferent, phase of the disease reduced clinical EAU and protected against retinal damage. No effect was observed after saline, E-Rh-Lip, or VIP injection. VIP-Rh-Lip and VIP were detected in intraocular macrophages and in lymphoid organs. In VIP-Rh-Lip-treated eyes, macrophages expressed transforming growth factor-beta2, low levels of major histocompatibility complex class II, and nitric oxide synthase-2. T-cells showed activated caspase-3 with the preservation of photoreceptors. Intraocular levels of interleukin (IL)-2, interferon-gamma (IFN-gamma), IL-17, IL-4, GRO/KC, and CCL5 were reduced with increased IL-13. At the systemic level, treatment reduced retinal soluble autoantigen lymphocyte proliferation, decreased IL-2, and increased IL-10 in ILN cells, and diminished specific DTH and serum concentration of IL-12 and IFN-gamma. CONCLUSIONS: An i.v.t. injection of VIP-Rh-Lip, performed during the afferent stage of immune response, reduced EAU pathology through the immunomodulation of intraocular macrophages and deviant stimulation of T-cells in ILN. Thus, the encapsulation of VIP within liposomes appears as an effective strategy to deliver VIP into the eye and is an efficient means of the prevention of EAU severity.

Suprachoroidal electrotransfer: a nonviral gene delivery method to transfect the choroid and the retina without detaching the retina.

Photoreceptors and retinal pigment epithelial cells (RPE) targeting remains challenging in ocular gene therapy. Viral gene transfer, the only method having reached clinical evaluation, still raises safety concerns when administered via subretinal injections. We have developed a novel transfection method in the adult rat, called suprachoroidal electrotransfer (ET), combining the administration of nonviral plasmid DNA into the suprachoroidal space with the application of an electrical field. Optimization of injection, electrical parameters and external electrodes geometry using a reporter plasmid, resulted in a large area of transfected tissues. Not only choroidal cells but also RPE, and potentially photoreceptors, were efficiently transduced for at least a month when using a cytomegalovirus (CMV) promoter. No ocular complications were recorded by angiographic, electroretinographic, and histological analyses, demonstrating that under selected conditions the procedure is devoid of side effects on the retina or the vasculature integrity. Moreover, a significant inhibition of laser induced-choroidal neovascularization (CNV) was achieved 15 days after transfection of a soluble vascular endothelial growth factor receptor-1 (sFlt-1)-encoding plasmid. This is the first nonviral gene transfer technique that is efficient for RPE targeting without inducing retinal detachment. This novel minimally invasive nonviral gene therapy method may open new prospects for human retinal therapies.

Cyclosporine A delivery to the eye: a pharmaceutical challenge.

Systemic administration of cyclosporine A (CsA) is commonly used in the treatment of local ophthalmic conditions involving cytokines, such as corneal graft rejection, autoimmune uveitis and dry eye syndrome. Local administration is expected to avoid the various side effects associated with systemic delivery. However, the currently available systems using oils to deliver CsA topically are poorly tolerated and provide a low bioavailability. These difficulties may be overcome through formulations aimed at improving CsA water solubility (e.g. cyclodextrins), or those designed to facilitate tissue drug penetration using penetration enhancers. The use of colloidal carriers (micelles, emulsions, liposomes and nanoparticles) as well as the approach using hydrosoluble prodrugs of CsA have shown promising results. Solid devices such as shields and particles of collagen have been investigated to enhance retention time on the eye surface. Some of these topical formulations have shown efficacy in the treatment of extraocular diseases but were inefficient at reaching intraocular targets. Microspheres, implants and liposomes have been developed to be directly administered subconjunctivally or intravitreally in order to enhance CsA concentration in the vitreous. Although progress has been made, there is still room for improvement in CsA ocular application, as none of these formulations is ideal.

Differential diagnosis of leukocoria and strabismus, first presenting signs of retinoblastoma.

Leukocoria in infants is always a danger signal as retinoblastoma, a malignant retinal tumor, is responsible for half of the cases in this age group. More common signs should also be considered suspicious until proved otherwise, such as strabismus, the second most frequent sign of retinoblastoma. Less frequent manifestations are inflammatory conditions resistant to treatment, hypopyon, orbital cellulitis, hyphema or heterochromia. Other causal pathologies, including persistent hyperplastic primary vitreous (PHPV), Coats' disease, ocular toxocariasis or retinopathy of prematurity, may also manifest the same warning signs and require specialized differential diagnosis. Members of the immediate family circle are most likely to notice the first signs, the general practitioner, pediatrician or general ophthalmologist the first to be consulted. On their attitude will depend the final outcome of this vision and life-threatening disease. Early diagnosis is vital.

Comment explorer une exophtalmie? [How to investigate a patient with exophthalmos?]

Exophthalmos is the main symptom revealing orbital masses. This sign needs to be imaged mainly by MRI and/or CT. As Graves disease is the main etiology of exophthalmos, CT scan should be performed as the initial imaging modality. Indications for US and Doppler are mostly limited to the study of ocular masses, and eventually may help the characterization of extra-ocular lesions. In all cases, imaging is useful to characterize: the precise location of the lesion which can be the intra-conal space (including muscles), the extra-conal space (associated or not to an extra-orbital lesion), or the eyeball; the features of the lesion (density, signal, enhancement.). These findings are used to generate a differential diagnosis. Imaging is also useful to precise the extension of the mass, and in some cases to select the appropriate surgical approach, and for follow-up.

Glucocorticoids exert direct toxicity on microvasculature: analysis of cell death mechanisms.

Glucocorticoids (GCs) are routinely administered systemically or injected into the eye when treating numerous ocular diseases; however, their toxicity on the retinal microvasculature has not been previously investigated. In this article, the effects of hydrocortisone (Hydro), dexamethasone, dexamethasone-phosphate and triamcinolone acetonide (TA) were evaluated in vitro on human skin microcirculation cells and, bovine endothelial retinal cells, ex-vivo, on flat mounted rat retinas. The degree of GCs induced endothelial cell death varied according to the endothelial cell type and GCs chemical properties. GCs toxicity was higher in skin microvascular endothelial cells and for hydrophobic GC formulations. The mechanism of cell death differed between GCs, Hydro and TA activated the leukocyte elastase inhibitor/L-DNase II pathways but did not activate caspases. The mechanisms of cell death observed in cell cultures were similar to those observed in rat retinal explants. Taken together these results indicate that particular attention should be paid to the potential vascular side effects when administrating GCs clinically and in particular when developing sustained-release intraocular devices.

Nanoparticles for gene delivery to retinal pigment epithelial cells.

PURPOSE: To evaluate the safety and potential use of poly(lactic) acid (PLA) and poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) as vectors for gene transfer to RPE cells. METHODS: Experiments were conducted with primary bovine RPE cells and with the ARPE-19 human RPE cell line. Rhodamine loaded NPs were used to study factors influencing the internalization process by the various RPE cells: concentrations of NPs, duration of contact time, stage of cell culture and ambient temperature. The extent of NPs internalization was evaluated by fluorescence and phase microscopy. Potential NP toxicity was measured by the trypan blue exclusion dye test and the MTT method. Green fluorescent protein (GFP) plasmid or red nuclear fluorescent protein (RNFP) plasmid were sequestered in NPs. The ability ot these "loaded" NPs to generate gene transfection and protein expression in RPE cells was assessed both in vivo and in vitro by fluorescence and confocal microscopy. RESULTS: The extent of NP internalization in cultured cells increases with their concentration reaching a plateau at 1 mg/ml and a contact time of up to 6 h. Temperature and culture stage did not influence the in vitro internalization process. No toxic effects on RPE cells could be detected when these were incubated with up to 4 mg/ml of NPs. In human and bovine RPE cells incubated with GFP loaded NPs, cytoplasmic green fluorescence was observed in 14+/-1.65% of the cultured cells. Incubation with RNFP loaded NPs yielded a nuclear red fluorescence in 18.9+/-1.6% of the cells. These percentage levels of expression initially detected after 48 h of incubation remained unchanged during the following 8 additional days in culture. No significant differences in the extent of cytoplasm or nuclear fluorescence expression were observed between bovine or human RPE cultured cells. In vivo, a preferential RNFP expression within the RPE cell layer was detected after intra vitreous injection of RNFP plasmid loaded NPs. CONCLUSIONS: The ability of PLGA NPs to sequester plasmids, their nontoxic characteristics, and rapid internalization enables gene transfer and expression in RPE cells. These findings may be of potential use when designing future gene therapy strategies for ocular diseases of the posterior segment.

A new poly(ortho ester)-based drug delivery system as an adjunct treatment in filtering surgery.

PURPOSE: Pharmacologic modulation of wound healing after glaucoma filtering surgery remains a major clinical challenge in ophthalmology. Poly(ortho ester) (POE) is a bioerodible and biocompatible viscous polymer potentially useful as a sustained drug delivery system that allows the frequency of intraocular injections to be reduced. The purpose of this study was to determine the efficacy of POE containing a precise amount of 5-fluorouracil (5-FU) in an experimental model of filtering surgery in the rabbit. METHODS: Trabeculectomy was performed in pigmented rabbit eyes. An ointmentlike formulation of POE containing 1% wt/wt 5-FU was injected subconjunctivally at the site of surgery, during the procedure. Intraocular pressure (IOP), bleb persistence, and ocular inflammatory reaction were monitored until postoperative day 30. Quantitative analysis of 5-FU was performed in the anterior chamber. Histologic analysis was used to assess the appearance of the filtering fistula and the polymer's biocompatibility. RESULTS: The decrease in IOP from baseline and the persistence of the filtering bleb were significantly more marked in the 5-FU-treated eyes during postoperative days 9 through 28. Corneal toxicity triggered by 5-FU was significantly lower in the group that received 5-FU in POE compared with a 5-FU tamponade. Histopathologic evaluation showed that POE was well tolerated, and no fibrosis occurred in eyes treated with POE containing 5-FU. CONCLUSIONS: In this rabbit model of trabeculectomy, the formulation based on POE and containing a precise amount of 5-FU reduced IOP and prolonged bleb persistence in a way similar to the conventional method of a 5-FU tamponade, while significantly reducing 5-FU toxicity.

Intraocular implants for extended drug delivery: therapeutic applications.

An overview of ocular implants with therapeutic application potentials is provided. Various types of implants can be used as slow release devices delivering locally the needed drug for an extended period of time. Thus, multiple periocular or intraocular injections of the drug can be circumvented and secondary complications minimized. The various compositions of polymers fulfilling specific delivery goals are described. Several of these implants are undergoing clinical trials while a few are already commercialized. Despite the paramount progress in design, safety and efficacy, the place of these implants in our clinical therapeutic arsenal remains limited. Miniaturization of the implants allowing for their direct injection without the need for a complicated surgery is a necessary development avenue. Particulate systems which can be engineered to target specifically certain cells or tissues are another promising alternative. For ocular diseases affecting the choroid and outer retina, transscleral or intrasscleral implants are gaining momentum.

Etude clinique et angiographique lors de la syphilis oculaire

UNIVERSITE DE LAUSANNE - FACULTE DE BIOLOGIE ET DE MEDECINE Médecine Ophtalmologie Etude clinique et angiographique lors de la Syphilis Oculaire THESE préparée sous la direction du Docteur Yan Guex-Crosier et présentée à la Faculté de biologie et de médecine de l'Université de Lausanne pour l'obtention du grade de DOCTEUR EN MEDECINE par Konstantinos BALASKAS Médecin diplômé de la Grèce Originaire d'Athènes (Grèce) ι Lausanne 2012  Une recrudescence des cas de syphilis a été observée ces dernières années. Son diagnostic clinique reste particulièrement difficile en l'absence de signe pathognomonique. Sur le plan oculaire elle présente un vaste spectre de manifestations diverses lui ayant valu le surnom de « la grande imitatrice ». Sa rareté et son hétérogénéité ont empêché l'identification et la vérification statistique des facteurs liés à un pronostic défavorable. Le but de cette étude a été d'explorer les paramètres cliniques et para-cliniques déterminant la sévérité et influençant l'évolution de la maladie. Ce travail comprend deux volets, la première partie intitulée : «Analysis of significant factors influencing visual acuity in ocular syphilis» publiée dans le British Journal of Ophthalmology a démontré qu'une mauvaise acuité visuelle initiale est associée de façon statistiquement significative à la sévérité de l'amputation du champ visuel, à la présence d'un oedème maculaire ou d'une neuropathie optique. Une amélioration de l'acuité visuelle grâce au traitement antibiotique est associée à la présence d'une vasculite (visible à l'angiographie fluorescéinique), d'une neurosyphilis ou d'une uvéite antérieure. Une récidive inflammatoire est fortement associée à une durée prolongée des symptômes avant l'introduction du traitement et à la présence de douleur comme signe d'appel. L'utilité clinique principale de ces résultats statistiques permet de conclure que les formes inflammatoires sévères associées à la triade (vasculite, neurosyphilis et uvéite antérieure) constituent un phénomène réversible pour autant qu'un traitement précoce et adéquat ait été introduit. Le deuxième volet de l'étude analyse les manifestations angiographiques (à la fluorescéine (AF) et au vert d'indocyanine (AICG)) de la syphilis oculaire et a été publié dans le journal du Graefes Archives of Clinical and Experimental Ophthalmology sous le titre suivant : "Fluorescein and indocyanine-green angiography in ocular syphilis: an exploratory study". Des associations faibles ont été démontrées entre la présence d'une vasculite lors de AF et d'une uvéite antérieure, d'une hyalite et d'un âge plus jeune. Une association forte est identifiée entre la présence des «dark dots» sur l'AICG et d'une uvéite antérieure ainsi qu'entre la présence des «hot spots» sur l'AICG et d'une durée prolongée des symptômes. Les conclusions d'importance clinique significative lors de la syphilis oculaire sont que les « dark dots » ou hypofluorescences en AICG évoquent la présence d'une atteinte inflammatoire oculaire sévère et que les « hot spots » ou hyperfluorescences en AICG sont significatifs d'une atteinte chronique. Cette étude contribue à modifier les attitudes cliniques lors de la syphilis oculaire surtout en ce qui concerne l'urgence à traiter afin d'assurer une récupération optimale. Elle aide aussi à redéfinir le rôle de l'angiographie dans la syphilis oculaire pour déterminer la sévérité, la durée de la maladie ainsi que le pronostic visuel.

Metastatic choroidal paraganglioma.

PURPOSE: To describe a patient with metastatic choroidal paraganglioma that was locally controlled with radiotherapy. DESIGN: Interventional clinicopathologic case report. PARTICIPANT: One patient with metastatic choroidal paraganglioma. METHODS: Interventional clinicopathologic case report and systematic search of the literature. MAIN OUTCOME MEASURES: Description of clinicopathologic features, treatment methods, and outcome. RESULTS: A 50-year-old man had a nonpigmented atypical choroidal mass with secondary retinal detachment in the left eye. After incisional biopsy, the diagnosis of paraganglioma was established. Metastatic work-up revealed vertebral, mediastinal, and pulmonary metastases of a nonsecretory, malignant paraganglioma without tracer uptake. The primary tumor was not identified. The ocular tumor regressed after stereotaxic radiotherapy. Two years later, recurrent lesions developed in the contralateral eye, which also was irradiated. CONCLUSIONS: Malignant paraganglioma can metastasize in the choroid and should be included in the differential diagnosis of a nonpigmented choroidal mass. Stereotaxic radiation therapy is an effective treatment method. To the authors' knowledge, this is the first report of a patient with choroidal paraganglioma. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.