Low reduction potential of Ero1alpha regulatory disulphides ensures tight control of substrate oxidation.

Formation of disulphide bonds within the mammalian endoplasmic reticulum (ER) requires the combined activities of Ero1alpha and protein disulphide isomerase (PDI). As Ero1alpha produces hydrogen peroxide during oxidation, regulation of its activity is critical in preventing ER-generated oxidative stress. Here, we have expressed and purified recombinant human Ero1alpha and shown that it has activity towards thioredoxin and PDI. The activity towards PDI required the inclusion of glutathione to ensure sustained oxidation. By carrying out site-directed mutagenesis of cysteine residues, we show that Ero1alpha is regulated by non-catalytic disulphides. The midpoint reduction potential (E degrees') of the regulatory disulphides was calculated to be approximately -275 mV making them stable in the redox conditions prevalent in the ER. The stable regulatory disulphides were only partially reduced by PDI (E degrees' approximately -180 mV), suggesting either that this is a mechanism for preventing excessive Ero1alpha activity and oxidation of PDI or that additional factors are required for Ero1alpha activation within the mammalian ER.

Retinal nerve fiber layer thickness in normal children measured with optical coherence tomography.

PURPOSE: To measure the peripapillary retinal nerve fiber layer (RNFL) thickness in normal children. DESIGN: Observational cross-sectional study. PARTICIPANTS: Ninety-two eyes of 92 normal children ages 4 to 17 years presenting to the Ophthalmology Clinic at the Harkness Eye Institute, Department of Ophthalmology, Columbia University. METHODS: Retinal nerve fiber layer thickness was measured with optical coherence tomography (OCT). Patient cooperation and signal strength of the OCT scans were assessed. Optic disc photographs were evaluated by a glaucoma specialist in a masked fashion. Eyes with abnormal optic discs were excluded. One eye of each subject was randomly selected for statistical analysis. The effect of several factors on RNFL thickness was investigated statistically. MAIN OUTCOME MEASURES: Retinal nerve fiber layer thickness. RESULTS: Ninety-one percent of the study subjects were Hispanic; 8%, African American; and 1%, Caucasian. Optical coherence tomography measurements were obtained in 117 of 121 (96.7%) subjects, and disc photographs were available for 92 of them. Mean age (+/-standard deviation [SD]) was 9.7+/-2.7 years. Mean global RNFL thickness (+/-SD) was 107.0+/-11.1 microm (range, 78.1-134.6). The RNFL was thickest inferiorly (136.9+/-16.9 microm) and superiorly (135.4+/-19.3 microm), thinner nasally (83.0+/-18.0 microm), and thinnest temporally (72.5+/-13.4 microm). In univariate regression analysis, age (P = 0.013) and refraction (P<0.001) had a significant effect on RNFL thickness; age had a significant effect on refraction (P<0.001). When controlling for refraction, age no longer had a significant effect. CONCLUSIONS: Optical coherence tomography can be used to measure RNFL thickness in children. Refraction had an effect on RNFL thickness. In normal children, variation in RNFL thickness is large. The normative data provided by this study may assist in identifying changes in RNFL thickness in children.

Sorsby's fundus dystrophy mutations impair turnover of TIMP-3 by retinal pigment epithelial cells.

Sorsby's fundus dystrophy (SFD) is an autosomal dominant degenerative disease of the retina, caused by mutations in exon 5 of the gene for tissue inhibitor of metalloproteinases-3 (TIMP-3). The mechanism by which these mutations give rise to the disease phenotype is unknown. In an attempt to identify common properties of these molecules that might underlie the disease phenotype, a range of SFD mutants were expressed from human retinal pigment epithelial (RPE) cells. This showed that resistance to turnover, resulting from intermolecular disulfide bond formation, was a common property of all the SFD mutants examined, providing a possible explanation for the increased deposition of the protein observed in eyes from SFD patients. In contrast, SFD mutants varied in their ability to inhibit cell-surface activation of matrix metalloproteinase-2 (MMP-2), a potent mediator of angiogenesis, ranging from being fully active to totally inactive. These data show that increased deposition of active TIMP-3, rather than dysregulation of metalloproteinase inhibition, is likely to be the primary, initiating event in SFD.

Autofluorescence characteristics of normal foveas and reconstruction of foveal autofluorescence from limited data subsets.

PURPOSE: To develop mathematical and geometric models of the nonuniform autofluorescence (AF) patterns of foveas of normal subjects and to reconstruct these models from limited subsets of data. METHODS: Confocal scanning laser ophthalmoscope (cSLO) AF fundus images of normal maculae were obtained from both eyes of 10 middle-aged subjects. They were filtered and contrast enhanced, to obtain elliptical isobars of equal gray levels (GLs) and determine the isobars' resolutions, eccentricities, and angles of orientation. The original image data were fit with a mathematical model of elliptic quadratic polynomials in two equal zones: the center and the remaining annulus. RESULTS: The AF images segmented into nested concentric GL isobars with GLs that increased radially from the least-fluorescent center. The mean isobar resolution was 31 +/- 7 mum. The geometric eccentricity of the ellipses increased from 0.42 +/- 0.12 centrally to 0.52 +/- 0.14 peripherally (P = 0.0005), with mean axes of orientation peripherally 97.12 +/- 15.46 degrees . The model fits to the complete image data had mean absolute normalized errors ranging from 3.6% +/- 3.7% to 7.3% +/- 7.1%. The model fits to small subsets (1% to 2% of total image data) had mean absolute errors ranging from 3.7% +/- 3.8% to 7.3% +/- 7.2%. CONCLUSIONS: Normal AF fundus images show finely resolved, concentric, elliptical foveal patterns consistent with the anatomic distribution of fluorescent lipofuscin, light-attenuating macular pigment (MP), cone photopigment, and retinal pigment epithelial (RPE) pigment in the fovea. A two-zone, elliptic, quadratic polynomial model can accurately model foveal data. This model may be useful for image analysis and for automated segmentation of pathology.

Measurement of retinal thickness by ocular coherence tomography in a case of scleral transparency in high myopia.

PURPOSE: Staphylomatous thinning of the sclera and retina are among the numerous changes associated with the elongated eye of high myopia. We report a case of scleral and retinal transparency in high myopia in which ocular coherence tomography was used to measure retinal and scleral thickness in vivo. DESIGN: Observational case report. METHODS: The clinical presentation and evaluation of a case of scleral transparency in high myopia are presented. RESULTS: This case demonstrates numerous anatomical changes to the sclera and retina known to occur in association with high myopia. Initial evaluation by optical coherence tomography demonstrated a retinal thickness of 74 microm and a scleral thickness of 80 microm. CONCLUSIONS: Optical coherence tomography can be used to measure scleral and retinal thickness in vivo in a patient with high myopia. It is not known at this time what precautions are necessary in such a patient should intraocular surgery be required.

Autofluorescence as a novel approach to diagnosing macular holes.

PURPOSE: To report the capability of autofluorescence as a novel approach to diagnosing macular holes. DESIGN: Observational case report. METHODS: In an institutional study, autofluorescence photos and optical coherence tomography scans of two patients who had surgery for macular holes were reviewed. RESULTS: Macular hole cases had a strong subfoveal autofluorescence signal, which disappeared after surgical closure of the macular hole. CONCLUSIONS: Autofluorescence is a novel approach to demonstrating a macular hole. This technique can also be used to confirm postoperative outcome after macular hole repair. A lack of autofluorescence corresponds to macular hole closure.

Intraocular dispersion of perfluorocarbon liquids in silicone oil.

PURPOSE: To report one case of intraocular emulsification of perfluorocarbon liquids (PFCLs) and silicone oil after vitrectomy surgery. DESIGN: Interventional case report. METHODS: Retrospective review of the clinic, and surgical record of a 39-year old female referred for persistent retinal detachment after vitrectomy surgery; PFCLs and silicone oil were left in the eye at the end of the surgery. RESULTS: There was intraocular emulsification of PFCLs into silicone oil. Electron microscope examination of the silicone oil surgically extracted from the eye demonstrated emulsification of small PFCL bubbles into the silicone oil. CONCLUSION: Perfluorocarbon liquids are surgical tools. They should be completely removed from the eye at the end of the surgery; PFCLs and silicone oil tend to emulsify together, originating an opaque fluid, which contains both silicone oil and dispersed PFCL bubbles.

Cytomegalovirus retinitis and FK 506.

PURPOSE: To report a case of cytomegalovirus (CMV) retinitis in a patient immunosuppressed with FK 506. DESIGN: Interventional case report. METHODS: Retrospective institutional study. One patient, diagnosed with bilateral CMV retinitis while on immunosuppressive treatment with FK 506, received systemic ganciclovir and intravitreal, slow-release ganciclovir implant in one eye, combined with reduction in FK 506 dose. Main outcome measures were visual acuity and regression of CMV retinitis. RESULTS: Visual acuity at presentation was 20/20 in the right eye and 20/50 in the left eye. During the 6 months of follow-up, there was no change in visual acuity in either eye. The areas of CMV retinitis demonstrated progressive regression, leaving diffuse retinal atrophy. CONCLUSIONS: Cytomegalovirus retinitis can be a sight-threatening complication of immunosuppressive treatment with FK 506. Ganciclovir treatment together with a reduction in FK 506 dose was effective in preserving vision.

Persistent indocyanine green fluorescence after vitrectomy for macular hole.

PURPOSE: To evaluate the persistence of indocyanine green (ICG) autofluorescence after ICG-assisted internal limiting membrane peeling for macular hole surgery. DESIGN: Interventional case series. METHODS: Retrospective institutional study. Four eyes of four patients who underwent pars plana vitrectomy with ICG-assisted internal limiting membrane peeling for macular hole repair were imaged for ICG autofluorescence at 795 nm with a scanning laser ophthalmoscope. The main outcome measure was persistence of ICG autofluorescence. RESULTS: All four patients demonstrated persistent ICG fluorescence in the central macula up to 8 months postsurgery. CONCLUSIONS: Persistent ICG signal was noted in the macula months after vitrectomy for macular hole surgery. The persistence of ICG autofluorescence could be responsible for delayed photochemical damage to the retinal pigment epithelium. Further studies must quantify the risk of retinal pigment epithelium injury when ICG-assisted internal limiting membrane peeling is used in macular hole surgery.

Ruptured retinal arterial macroaneurysm associated with a stage IV macular hole.

PURPOSE: To report a case of stage IV macular hole associated with ruptured retinal arterial macroaneurysm. DESIGN: Observational case report. METHOD: The patient's clinical, angiographic, and ophthalmic coherent tomography data were reviewed. RESULTS: A stage IV macular hole was observed in association with a ruptured retinal arterial macroaneurysm in a 79-year-old patient. After vitrectomy surgery, the macular hole closed and vision improved from counting fingers to 20/100. CONCLUSIONS: Macular hole associated with ruptured retinal macroaneurysm can be closed successfully to result in improved vision.

Expression of mutant and wild-type TIMP3 in primary gingival fibroblasts from Sorsby's fundus dystrophy patients.

Gingival fibroblast cell lines were derived from Sorsby's fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1alpha. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context.

Expression of ADAMTS metalloproteinases in the retinal pigment epithelium derived cell line ARPE-19: transcriptional regulation by TNFalpha.

ADAMTS (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type I motifs) are multidomain proteins with demonstrated metalloproteinase functionality and have potential roles in embryonic development, angiogenesis and cartilage degradation. We present here investigations of ADAMTS expression in an ocular cell type, ARPE-19, with a view to implicating them in retinal matrix turnover. Expression analysis was undertaken using a combination of reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting experiments, which together detected the expression of mRNAs for several ADAMTS proteins, all of which have active site motifs characteristic of matrix metalloproteases (MMPs). These included ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS5, ADAMTS6, ADAMTS7 and ADAMTS9. The expression of mRNA isoforms for ADAMTS7 and ADAMTS9 were also detected. Following stimulation with TNFalpha, ADAMTS1, ADAMTS6 and both ADAMTS9 transcripts expressed in ARPE-19 cells showed a potent upregulation. The expression of ADAMTS genes in ARPE-19 cells and the transcriptional stimulation of some family members by TNFalpha may implicate them in inflammatory eye disease and the compromise of retinal matrix structure, which is evident in age-related macular degeneration (ARMD) and other retinal pathologies.

Simultaneous occurrence of neuroretinitis and optic perineuritis in a single eye.

A 52-year-old woman presented with pain, decreased visual acuity, decreased color vision, and visual field loss in the OD. Fundus examination revealed optic disc edema and later a macular star figure consistent with neuroretinitis. Magnetic resonance imaging showed enhancement of the orbital optic nerve sheath and perineural fat suggestive of optic perineuritis. The patient was not treated. At 11 months, visual function and fundus examination had returned to normal. This unusual simultaneous occurrence of neuroretinitis and optic perineuritis suggests that these two entities may have a similar pathogenesis.

Spontaneous resolution of bilateral stage 1 macular holes documented by optical coherence tomography.

PURPOSE: To describe a case of spontaneous resolution of bilateral stage 1 macular holes documented using optical coherence tomography. DESIGN: Observational case report. METHODS: A patient presented with blurring and distortion in the right eye. Optical coherence tomography showed a stage 1 macular hole manifested as a foveal pseudocyst. The left eye appeared normal. RESULTS: On 6-month follow-up, symptoms had resolved in the right eye but had developed in the left eye. Optical coherence tomography revealed normal anatomy in the right eye with a foveal pseudocyst in the left eye. At 10 months, the patient was asymptomatic and had normal foveal anatomy in both eyes on optical coherence tomography. CONCLUSION: Stage 1 macular holes may appear as foveal pseudocysts on optical coherence tomography. In such cases, optical coherence tomography can provide definitive diagnostic information when clinical examination is equivocal. The observation and spontaneous resolution of bilateral stage 1 holes documented by optical coherence tomography are unique and interesting events of which a MEDLINE search found no previous reports.