Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system

PURPOSE: To evaluate the expression and presence of surfactant protein (SP) A and SP-D in the lacrimal apparatus, at the ocular surface, and in tears in healthy and pathologic states. METHODS: Expression of mRNA for SP-A and SP-D was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts as well as in a spontaneously immortalized conjunctival epithelial cell line (HCjE; IOBA-NHC) and a SV40-transfected cornea epithelial cell line (HCE). Deposition of SP-A and SP-D was determined by Western blot, dot blot, and immunohistochemistry in healthy tissues, in tears, aqueous humor, and in sections of different corneal abnormalities (keratoconus, herpetic keratitis, and Staphylococcus aureus-based ulceration). Cell lines were stimulated with different cytokines and bacterial components and were analyzed for the production of SP-A and SP-D by immunohistochemistry. RESULTS: The presence of SP-A and SP-D on mRNA and protein levels was evidenced in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal duct samples. Moreover, both proteins were present in tears but were absent in aqueous humor. Immunohistochemistry revealed the production of both peptides by acinar epithelial cells of the lacrimal gland and epithelial cells of the conjunctiva and nasolacrimal ducts, whereas goblet cells revealed no reactivity. Healthy cornea revealed weak reactivity on epithelial surface cells only. In contrast, SP-A and SP-D revealed strong reactivity in patients with herpetic keratitis and corneal ulceration surrounding lesions and in several immigrated defense cells. Reactivity in corneal epithelium and endothelium was also seen in patients with keratoconus. Cell culture experiments revealed that SP-A and SP-D are produced by both epithelial cell lines without and after stimulation with cytokines and bacterial components. CONCLUSIONS: These results show that SP-A, in addition to SP-D, is a peptide of the tear film. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D seem to be involved in several ocular surface diseases.

Is pseudophakia a risk factor for neovascular age-related macular degeneration?

PURPOSE: To examine the possible association between pseudophakia and neovascular age-related macular degeneration (AMD). METHODS: Reports of all patients undergoing fluorescein angiography in the authors' department over a 6-year period were retrospectively reviewed. Four hundred ninety-nine patients with recent onset of neovascular AMD in one eye and early age-related maculopathy (ARM) in the fellow eye were included in the study. Lens status (phakic or pseudophakic) in both eyes at the time of onset of neovascular AMD and the time between cataract surgeries (if performed) and onset of neovascular AMD were determined. RESULTS: There was no significant difference in lens status between eyes with neovascular AMD and fellow eyes with early ARM (115/499 [23.0%] vs. 112/499 [22.4%] pseudophakic; P = 0.88, odds ratio 1.035, 95% CI 0.770-1.391). Subgroup analysis revealed no difference between the groups with large drusen, small drusen, or pigmentary changes only (respectively, 20.3% vs. 19.6% pseudophakic, P = 0.92; 20.5% vs. 23.3% pseudophakic, P = 0.84; 33.3% vs. 31.7% pseudophakic, P = 1.0). Pseudophakic eyes with neovascular AMD had not been pseudophakic for a significantly longer period at the time of onset of neovascular AMD than their pseudophakic fellow eyes at the same time point (225.9 +/- 170.4 vs. 209.9 +/- 158.2 weeks, P = 0.27). CONCLUSIONS: The results do not support the hypothesis that pseudophakia is a major risk factor for the development of neovascular AMD.

Endogenous bone marrow derived cells express retinal pigment epithelium cell markers and migrate to focal areas of RPE damage

PURPOSE: The aim of the present study was to investigate whether bone marrow-derived cells (BMCs) can be induced to express retinal pigment epithelial (RPE) cell markers in vitro and can home to the site of RPE damage after mobilization and express markers of RPE lineage in vivo. METHODS: Adult RPE cells were cocultured with green fluorescence protein (GFP)-labeled stem cell antigen-1 positive (Sca-1(+)) BMCs for 1, 2, and 3 weeks. Cell morphology and expression of RPE-specific markers and markers for other retinal cell types were studied. Using an animal model of sodium iodate (NaIO(3))-induced RPE degeneration, BMCs were mobilized into the peripheral circulation by granulocyte-colony stimulating factor, flt3 ligand, or both. Immunocytochemistry was used to identify and characterize BMCs in the subretinal space in C57BL/6 wild-type (wt) mice and GFP chimeric mice. RESULTS: In vitro, BMCs changed from round to flattened, polygonal cells and expressed cytokeratin, RPE65, and microphthalmia transcription factor (MITF) when cocultured in direct cell-cell contact with RPE. In vivo, BMCs were identified in the subretinal space as Sca-1(+) or c-kit(+) cells. They were also double labeled for GFP and RPE65 or MITF. These cells formed a monolayer on the Bruch membrane in focal areas of RPE damage. CONCLUSIONS: Thus, it appears that BMCs, when mobilized into the peripheral circulation, can home to focal areas of RPE damage and express cell markers of RPE lineage. The use of endogenous BMCs to replace damaged retinal tissue opens new possibilities for cell replacement therapy in ophthalmology.

Ethnic differences in macular pigment density and distribution

PURPOSE: Many epidemiologic studies suggest a number of risk factors that may be associated with progression of age-related maculopathy (ARM). In this study, the authors investigate ethnic differences in macular pigment density (MPD) and macular pigment (MP) distribution. METHODS: Inclusion criteria were healthy subjects, aged 35 to 49 years, visual acuity >or=20/20, race ethnicity white non-Hispanic (WNH) or African. All subjects underwent the following examinations: best-corrected ETDRS visual acuity (VA), measurements of MPD, and spatial distribution of MP with a modified confocal scanning laser ophthalmoscope according to a standard protocol. MPD maps were calculated from autofluorescence images recorded at 488 nm and 514 nm. Central macular pigment density (MPDc) was quantified from MPD maps within 0.5 degrees around the center of the fovea. RESULTS: In total, 118 healthy subjects (61 women, 57 men) aged 35 to 49 years (mean, 42.5 +/- 3.6 years) were recruited for the study. Sixty-seven healthy subjects were WNH and 51 were African. Visual acuity ranged from 20/20 to 20/16 in the study eye. Significant differences were found among MPDc between the group of WNH (MPDc, 0.36 +/- 0.13 density units [DU]; P < 0.0001) and African subjects (MPDc, 0.59 +/- 0.14 DU). A parafoveal ring was significantly more frequent in African subjects than in WNH subjects (86% [African] vs. 68% [WNH]; P < 0.0001). CONCLUSIONS: This study demonstrates that ethnicity plays a role in MPD values and in MP distribution. The association of different distribution patterns and their relevance as possible prognostic factors for diseases leading to oxidative retinal damage requires further studies.

Distribution of amyloid precursor protein and amyloid-beta immunoreactivity in DBA/2J glaucomatous mouse retinas

PURPOSE: Evidence suggests that altered metabolism of amyloid precursor protein (APP) may play a role in the pathophysiology of retinal ganglion cell (RGC) death in the etiology of glaucoma. The authors sought to determine the distribution of APP and amyloid-beta (Abeta) in DBA/2J glaucomatous mouse retinas. METHODS: The retinas of 3- and 15-month-old DBA/2J mice and C57/BL-6 mice (control group) were fixed with 4% paraformaldehyde and processed for immunohistochemistry. Antibodies used included a polyclonal antibody to the C terminus of Abeta 40 and a polyclonal antibody to the APP ectodomain. Immunohistochemically stained tissue was graded using light microscopy. Distribution and semiquantitative expression of APP and Abeta in young and old glaucomatous and normal retinas were determined and compared. RESULTS: Strong APP and Abeta immunoreactivity was found in the RGC layer, optic nerve, and pia/dura of old DBA/2J retinas, with considerably higher intensity found in the old compared with the young DBA/2J mice. In contrast to glaucomatous mice, the control group did not show any notable age-related difference. CONCLUSIONS: Disruption of the homeostatic properties of secreted APP with consecutive Abeta cytotoxicity might be a contributing factor of ganglion cell loss in glaucomatous mouse retinas.

Novel TULP1 mutation causing leber congenital amaurosis or early onset retinal degeneration

PURPOSE: To report a large, consanguineous Algerian family affected with Leber congenital amaurosis (LCA) or early-onset retinal degeneration (EORD). METHODS: All accessible family members underwent a complete ophthalmic examination, and blood was obtained for DNA extraction. Homozygosity mapping was performed with markers flanking 12 loci associated with LCA. The 15 exons of TULP1 were sequenced. RESULTS: Seven of 30 examined family members were affected, including five with EORD and two with LCA. All patients had nystagmus, hemeralopia, mild myopia, and low visual acuity without photophobia. Fundus features were variable among EORD patients: typical spicular retinitis pigmentosa or clumped pigmented retinopathy with age-dependent macular involvement. A salt-and-pepper retinopathy with midperipheral retinal pigment epithelium (RPE) atrophy was present in the older patients with LCA, whereas the retina appeared virtually normal in the younger ones. Both scotopic and photopic electroretinograms were nondetectable. Fundus imaging revealed a perifoveal ring of increased fundus autofluorescence (FAF) in the proband, and optical coherence tomography disclosed a thinned retina, mainly due to photoreceptor loss. Linkage analysis identified a region of homozygosity on chromosome 6, region p21.3, and mutation screening revealed a novel 6-base in-frame duplication, in the TULP1 gene. CONCLUSIONS: Mutation in the TULP1 gene is a rare cause of LCA/EORD, with only 14 mutations reported so far. The observed intrafamilial phenotypic variability could be attributed to disease progression or possibly modifier alleles. This study provides the first description of FAF and quantitative reflectivity profiles in TULP1-related retinopathy.

Dynamic registration using ultrasound for anatomical referencing

This paper proposes methods to circumvent the need to attach physical markers to bones for anatomical referencing in computer-assisted orthopedic surgery. Using ultrasound, a bone could be non-invasively referenced, and so the problem is formulated as the need for dynamic registration. A method for correspondence establishment is presented, and the matching step is based on three least-squares algorithms: two that are typically used in registration methods such as ICP, and the third is a form of the Unscented Kalman filter that was adapted to work in this context. A simulation was developed in order to reliably evaluate and compare the dynamic registration methods

Nonpeptide somatostatin receptor agonists specifically target ocular neovascularization via the somatostatin type 2 receptor

PURPOSE: To define the molecular pharmacology underlying the antiangiogenic effects of nonpeptide imidazolidine-2,4-dione somatostatin receptor agonists (NISAs) and evaluate the efficacy of NISA in ocular versus systemic delivery routes in ocular disease models. METHODS: Functional inhibitory effects of the NISAs and the somatostatin peptide analogue octreotide were evaluated in vitro by chemotaxis, proliferation, and tube-formation assays. The oxygen-induced retinopathy (OIR) model and the laser model of choroidal neovascularization (CNV) were used to test the in vivo efficacy of NISAs. Transscleral permeability of a candidate NISA was also measured. RESULTS: NISAs inhibited growth factor-induced HREC proliferation, migration and tube formation with submicromolar potencies (IC(50), 0.1-1.0 microM) comparable to octreotide. In the OIR model, systemic administration of the NISAs RFE-007 and RFE-011 inhibited retinal neovascularization in a dose-dependent manner, comparable to octreotide. In the CNV model, intravitreal RFE-011 resulted in a 56% reduction (P < 0.01) in CNV lesion area, whereas systemic administration resulted in a 35% reduction (P < 0.05) in lesion area. RFE-011 demonstrated transscleral penetration. CONCLUSIONS: Micromolar concentrations of octreotide and NISAs are necessary for antiangiogenic effects, whereas nanomolar concentrations are effective for endocrine inhibition. This suggests that the antiangiogenic activity of NISAs and octreotide is mediated by an overall much less efficient downstream coupling mechanism than is growth hormone release. As a result, the intravitreal or transscleral route of administration should be seriously considered for future clinical studies of SSTR2 agonists used for treatment of ocular neovascularization to ensure efficacious concentrations in the target retinal and choroidal tissue.

Morphologic changes in patients with geographic atrophy assessed with a novel spectral OCT-SLO combination

To investigate the appearance of geographic atrophy in high-resolution optical coherence tomography (OCT) images, the fundus autofluorescence (FAF) pattern, and infrared images simultaneously recorded with a novel combined OCT-scanning laser ophthalmology (SLO) system.

Differential optical densities of intraretinal spaces

PURPOSE: To test the hypothesis that hyporeflective spaces in the neuroretina found on optical coherence tomography (OCT) examination have different optical reflectivities according to whether they are associated with exudation or degeneration. METHODS: Retrospective analysis of eyes with idiopathic perifoveal telangiectasia (IPT), diabetic macular edema (DME), idiopathic central serous chorioretinopathy (CSC), retinitis pigmentosa (RP), or cone dystrophy (CD) and eyes of healthy control subjects. OCT scans were performed. Raw scan data were exported and used to calculate light reflectivity profiles. Reflectivity data were acquired by projecting three rectangular boxes, each 50 pixels long and 5 pixels wide, into the intraretinal cystoid spaces, centrally onto unaffected peripheral RPE, and onto the prefoveolar vitreous. Light reflectivity in the retinal pigment epithelium (RPE), vitreous, and intraretinal spaces for the different retinal conditions and control subjects were compared. RESULTS: Reflectivities of the vitreous and the RPE were similar among the groups. Hyporeflective spaces in eyes with exudation (DME, RP, and CSC) had higher reflectivity compared with the mean reflectivity of the vitreous, whereas the cystoid spaces in the maculae of the eyes without exudation (CD and IPT) had a lower reflectivity than did the normal vitreous. CONCLUSIONS: Analysis of the light reflectivity profiles may be a tool to determine whether the density of hyporeflective spaces in the macula is greater or less than that of the vitreous, and may be a way to differentiate degenerative from exudative macular disease.

Impact of optic media opacities and image compression on quantitative analysis of optical coherence tomography

To analyze the impact of opacities in the optical pathway and image compression of 32-bit raw data to 8-bit jpg images on quantified optical coherence tomography (OCT) image analysis.

Macular thickness measurements in healthy eyes using six different optical coherence tomography instruments

To compare central retinal thickness (CRT) measurements in healthy eyes by different commercially available OCT instruments and to compare the intersession reproducibility of such measurements.