Homozygosity has long been associated with rare, often devastating, Mendelian disorders, and Darwin was one of the first to recognize that inbreeding reduces evolutionary fitness. However, the effect of the more distant parental relatedness that is common in modern human populations is less well understood. Genomic data now allow us to investigate the effects of homozygosity on traits of public health importance by observing contiguous homozygous segments (runs of homozygosity), which are inferred to be homozygous along their complete length. Given the low levels of genome-wide homozygosity prevalent in most human populations, information is required on very large numbers of people to provide sufficient power. Here we use runs of homozygosity to study 16 health-related quantitative traits in 354,224 individuals from 102 cohorts, and find statistically significant associations between summed runs of homozygosity and four complex traits: height, forced expiratory lung volume in one second, general cognitive ability and educational attainment (P < 1 × 10(-300), 2.1 × 10(-6), 2.5 × 10(-10) and 1.8 × 10(-10), respectively). In each case, increased homozygosity was associated with decreased trait value, equivalent to the offspring of first cousins being 1.2 cm shorter and having 10 months' less education. Similar effect sizes were found across four continental groups and populations with different degrees of genome-wide homozygosity, providing evidence that homozygosity, rather than confounding, directly contributes to phenotypic variance. Contrary to earlier reports in substantially smaller samples, no evidence was seen of an influence of genome-wide homozygosity on blood pressure and low density lipoprotein cholesterol, or ten other cardio-metabolic traits. Since directional dominance is predicted for traits under directional evolutionary selection, this study provides evidence that increased stature and cognitive function have been positively selected in human evolution, whereas many important risk factors for late-onset complex diseases may not have been.

OBJECTIVE: Patients suffering from corneal neuropathy may present with photoallodynia; i.e., increased light sensitivity, frequently with a normal slit-lamp examination. This study aimed to evaluate the efficacy of autologous serum tears (AST) for treatment of severe photoallodynia in corneal neuropathy and to correlate clinical findings with corneal subbasal nerve alterations by in vivo confocal microscopy (IVCM). METHODS: Retrospective case control study with 16 patients with neuropathy-induced severe photoallodynia compared to 16 normal controls. Symptom severity, clinical examination and bilateral corneal IVCM scans were recorded. RESULTS: All patients suffered from extreme photoallodynia (8.8±1.1) with no concurrent ocular surface disease. Subbasal nerves were significantly decreased at baseline in patients compared to controls; total nerve length (9208±1264 vs 24714±1056 μm/mm(2); P<.0001) and total nerve number (9.6±1.4 vs 28.6±2.0; P<.0001), respectively. Morphologically, significantly increased reflectivity (2.9±0.2 vs 1.8±0.1; P<.0001), beading (in 93.7%), and neuromas (in 62.5%) were seen. AST (3.6±2.1 months) resulted in significantly decreased symptom severity (1.6±1.7; P=.02). IVCM demonstrated significantly improved nerve parameters (P<.005), total nerve length (15451±1595 μm/mm(2)), number (13.9±2.1), and reflectivity (1.9±0.1). Beading and neuromas were seen in only 56.2% and 7.6% of patients. CONCLUSION: Patients with corneal neuropathy-induced photoallodynia show profound alterations in corneal nerves. AST restores nerve topography through nerve regeneration, and this correlated with improvement in patient-reported photoallodynia. The data support the notion that corneal nerve damage results in alterations in afferent trigeminal pathways to produce photoallodynia.

Impaired corneal wound healing that occurs with ocular surface disease, trauma, systemic disease, or surgical intervention can lead to persistent corneal epithelial defects (PCED), which result in corneal scarring, ulceration, opacification, corneal neovascularization, and, ultimately, visual compromise and vision loss. The current standard of care can include lubricants, ointments, bandage lenses, amniotic membranes, autologous serum eye drops, and corneal transplants. Various inherent problems exist with application and administration of these treatments, which often may not result in a completely healed surface. A topically applicable compound capable of promoting corneal epithelial cell proliferation and/or migration would be ideal to accelerate healing. We hypothesize that human growth hormone (HGH) is such a compound. In a recent study, HGH was shown to activate signal transducer and activators of transcription-5 (STAT5) signaling and promote corneal wound healing by enhancing corneal epithelial migration in a co-culture system of corneal epithelial cells and fibroblasts. These effects require an intact communication between corneal epithelia and fibroblasts. Further, HGH promotes corneal wound healing in a rabbit debridement model, thus demonstrating the effectiveness of HGH in vivo as well. In conclusion, HGH may represent an exciting and effective topical therapeutic to promote corneal wound healing.

PURPOSE: To describe the clinical and histopathologic features distinguishing an extensive complex choristoma of the epibulbar surface and to address the management of such lesions. METHODS: Clinical history, diagnostic imaging studies, and histopathologic sections stained with hematoxylin and eosin were reviewed from a 2-year-old girl with a congenital conjunctival lesion of the right eye that was surgically excised. RESULTS: The patient clinically displayed an extensive, vascularized amelanotic conjunctival lesion located superotemporally with extension onto the cornea. Her visual acuity was reduced to 20/670. The clinical diagnosis was a large lacrimal gland choristoma with corneal involvement and resulting deprivation amblyopia. The patient underwent an excision of the lesion including the corneal portion, and the ocular surface was reconstructed with amniotic membrane. Histopathologic evaluation disclosed lobules of lacrimal tissue and cartilage plaques, smooth muscle, and nerves consistent with a complex choristoma. Six weeks postoperatively, the visual acuity had improved to 20/180. The patient returned to her local ophthalmologist for amblyopia management. CONCLUSIONS: We emphasize the importance of recognizing lesion-induced amblyopia and the timely performance of appropriate surgery for complex epibulbar choristomas. A differential diagnosis of other congenital epibulbar lesions is provided.

BACKGROUND: Susac syndrome (SS) is a rare, presumed autoimmune condition characterized by the clinical triad of branch retinal artery occlusions (BRAOs), encephalopathy, and sensorineural hearing loss. The aim of this study was to evaluate clinical features, diagnostic results, treatment, and outcomes in SS. METHODS: Five patients with SS were referred to three tertiary care centers in Boston. The observation period across these patients was 7-57months. RESULTS: At initial presentation, none of the patients demonstrated the complete triad of BRAO, sensorineural hearing loss, and encephalopathy. The interval between symptom onset and diagnosis of SS was 4-30weeks. Brain MRI findings thought to be characteristic of SS (including callosal fluid-attenuated inversion recovery (FLAIR) hyperintense and T1 hypointense lesions) were frequently absent. Microinfarcts noted on diffusion-weighted imaging (DWI), BRAOs and vessel wall hyperfluorescence on fluorescein angiography (FA) were present in all cases in the acute encephalopathic phase. All patients treated with glucocorticoids and intravenous immunoglobulins (IVIg) alone experienced further clinical progression until additional immunosuppressive therapy was instituted. CONCLUSIONS: The rarity of SS, its incomplete and variable presentation, and the nonspecific imaging findings invariably led to delayed diagnosis. DWI and FA should be used to identify the acute microvascular injury and monitor treatment response. Immunomodulatory agents more potent than glucocorticoids and IVIg might be required to control the disease.

PURPOSE: To illustrate that corneal neuralgia may be the basis for refractory dry eye syndrome after laser-assisted in situ keratomileusis (LASIK). METHODS: The methodology used is that of a retrospective medical record review of a small case series. RESULTS: Three male patients, aged 30 to 48 years, referred in 2012 for dry eye syndrome refractory to treatment within 1 year of LASIK or LASIK enhancement are reported. Each patient gave history of eye pain, light sensitivity, and difficulty with visual activities beginning within 2 months of LASIK or LASIK enhancement. Best-corrected visual acuity was 20/15 or 20/20 in each of the six eyes. Tear-centered models and metrics did not explain persistent symptoms, which was consistent with inadequate response to standard dry eye treatments used before referral and reported here. In vivo confocal microscopy was abnormal at presentation in each case and was followed over time. Treatments undertaken subsequent to referral included autologous serum tears (three cases), PROSE (Prosthetic Replacement of the Ocular Surface Ecosystem) treatment (two cases), and systemic agents for pain, anxiety, or depression (three cases). By the end of 2013, at a mean of 23 months after LASIK or LASIK enhancement, symptoms improved in all three patients. CONCLUSIONS: Patients with persistent dry eye symptoms out of proportion to clinical signs after LASIK have a syndrome that may best be classified as corneal neuralgia. In vivo confocal microscopy can be informative as to the neuropathic basis of this condition. In keeping with current understanding of complex regional pain syndrome, early multimodal treatment directed toward reducing peripheral nociceptive signaling is warranted to avoid subsequent centralization and persistence of pain. Distinguishing this syndrome from typical post-LASIK dry eye remains a challenge.

The etiology of Posner-Schlossman syndrome (PSS) remains unknown. The association of human leukocyte antigens (HLA) allelic diversity with PSS has been poorly investigated. To evaluate the association of allelic polymorphisms of class I HLA-A, -B and -C and class II HLA-DRB1 and -DQB1 with PSS, 100 unrelated patients with PSS and 128 age- and ethnically matched control subjects were recruited from a southern Chinese Han population. Polymorphisms in exons 2-4 for HLA-A, -B, -C loci, exon 2 for HLA-DRB1 and exons 2,3 for HLA-DQB1 were analyzed for association with PSS at allele and haplotype levels. The allele frequency of HLA-C*1402 in PSS patients was significantly higher than that in controls (P = 0.002, OR = 4.12). This association survived the Bonferroni correction (Pc = 0.04). The allele frequency of HLA-B*1301 in PSS patients was lower than that in the control group (P = 0.003, OR = 0.21), although this association did not survive the Bonferroni correction (Pc = 0.16). In PSS patients, the haplotype frequencies of HLA-A*1101~C*1402 and B*5101~C*1402 were higher than that in controls (P = 0.03, OR = 4.44; P = 0.02, OR = 3.20; respectively), while the HLA-B*1301~C*0304 was lower than that in controls (P = 0.007, OR = 0.23), although these associations did not survive the Bonferroni correction (Pc > 0.16). This study for the first time demonstrated that polymorphisms at the HLA-B and HLA-C loci were nominally associated with PSS in the southern Chinese Han population. Our results suggest that HLA-C*1402, A*1101~C*1402 and B*5101~C*1402 might be risk factors for PSS, whereas HLA-B*1301 plus B*1301~C*0304 might be protective factors against PSS, but even larger datasets are required to confirm these findings. Findings from this study provide valuable new clues for investigating the mechanisms and development of new diagnosis and treatment for PSS.

PURPOSE: Each year, over 8,000 corneal transplantation surgeries are performed in China. Unlike developed countries, which have established standard requirements for operative experience for corneal specialists, little information exists on surgical training for keratoplasty in China. The aim of this study was to assess the keratoplasty experience of Chinese corneal specialists and to characterize their surgical patterns. METHODS: One hundred and twenty-one corneal specialists in 16 provinces (65 cities) in China were invited to complete an anonymous survey at the 2014 Chinese Corneal Society annual meeting, which consisted of questions with single or multiple-choice answers. Demographics, the number and type of keratoplasties performed, and the perceived limiting factors for performing keratoplasties were analyzed. RESULTS: An overwhelming 89% response rate was achieved. Of the 108 respondents, 76% worked in tertiary centers, and only 23% held a medical doctorate degree. Furthermore, 69% of the participants had received corneal fellowship training of less than one year. Only 71% were capable of keratoplasties. Among those doing keratoplasty, 68% performed less than 50 keratoplasties each year. Of the same group of keratoplasty surgeons, 88% of corneal specialists capable of keratoplasties had performed penetrating keratoplasties, 87% had performed lamellar keratoplasties, 12% had performed deep anterior lamellar keratoplasties, and 5% had performed Descemet's stripping endothelial keratoplasties. When questioned on the reasons for the low number of keratoplasties performed in China, the respondents deemed the following factors most important: lack of surgical training (71%), a shortage of donor supply (52%), and a lack of curricula (42%). A multivariate logistic regression analysis showed that corneal transplantation capabilities are significantly associated with responders' education levels and training time. CONCLUSION: Keratoplasty surgery experience is suboptimal for Chinese corneal specialists. Penetrating and lamellar keratoplasties are the preferred surgical patterns. Our findings raise concerns about the adequacy of keratoplasty training in China.

The mouse model of laser-induced choroidal neovascularization (CNV) has been used in studies of the exudative form of age-related macular degeneration using both the conventional slit lamp and a new image-guided laser system. A standardized protocol is needed for consistent results using this model, which has been lacking. We optimized details of laser-induced CNV using the image-guided laser photocoagulation system. Four lesions with similar size were consistently applied per eye at approximately double the disc diameter away from the optic nerve, using different laser power levels, and mice of various ages and genders. After 7 days, the mice were sacrificed and retinal pigment epithelium/choroid/sclera was flat-mounted, stained with Isolectin B4, and imaged. Quantification of the area of the laser-induced lesions was performed using an established and constant threshold. Exclusion criteria are described that were necessary for reliable data analysis of the laser-induced CNV lesions. The CNV lesion area was proportional to the laser power levels. Mice at 12-16 weeks of age developed more severe CNV than those at 6-8 weeks of age, and the gender difference was only significant in mice at 12-16 weeks of age, but not in those at 6-8 weeks of age. Dietary intake of omega-3 long-chain polyunsaturated fatty acid reduced laser-induced CNV in mice. Taken together, laser-induced CNV lesions can be easily and consistently applied using the image-guided laser platform. Mice at 6-8 weeks of age are ideal for the laser-induced CNV model.

PURPOSE: Retinitis pigmentosa (RP) is a group of inherited retinal degenerations leading to blindness due to photoreceptor loss. Retinitis pigmentosa is a rare disease, affecting only approximately 100 000 people in the United States. There is no cure and no approved medical therapy to slow or reverse RP. The purpose of this clinical trial was to evaluate the safety, reliability, and benefit of the Argus II Retinal Prosthesis System (Second Sight Medical Products, Inc, Sylmar, CA) in restoring some visual function to subjects completely blind from RP. We report clinical trial results at 1 and 3 years after implantation. DESIGN: The study is a multicenter, single-arm, prospective clinical trial. PARTICIPANTS: There were 30 subjects in 10 centers in the United States and Europe. Subjects served as their own controls, that is, implanted eye versus fellow eye, and system on versus system off (native residual vision). METHODS: The Argus II System was implanted on and in a single eye (typically the worse-seeing eye) of blind subjects. Subjects wore glasses mounted with a small camera and a video processor that converted images into stimulation patterns sent to the electrode array on the retina. MAIN OUTCOME MEASURES: The primary outcome measures were safety (the number, seriousness, and relatedness of adverse events) and visual function, as measured by 3 computer-based, objective tests. RESULTS: A total of 29 of 30 subjects had functioning Argus II Systems implants 3 years after implantation. Eleven subjects experienced a total of 23 serious device- or surgery-related adverse events. All were treated with standard ophthalmic care. As a group, subjects performed significantly better with the system on than off on all visual function tests and functional vision assessments. CONCLUSIONS: The 3-year results of the Argus II trial support the long-term safety profile and benefit of the Argus II System for patients blind from RP. Earlier results from this trial were used to gain approval of the Argus II by the Food and Drug Administration and a CE mark in Europe. The Argus II System is the first and only retinal implant to have both approvals.

PURPOSE: To describe the anatomical and functional outcomes in a cohort of subjects undergoing vitrectomy for myopic foveoschisis, and to analyse the factors predicting foveal reattachment and visual improvement. METHODS: This retrospective case series evaluated case records and optical coherence tomography images 6 months after surgery. Multivariate linear and logistic regressions were performed to assess the factors predicting anatomical and visual improvement. RESULTS: In total, 55 eyes of 54 patients were analysed. The mean spherical equivalent refraction was -11.83±4.94D. Foveal detachment was present in 63.5% of eyes preoperatively and subjects with foveal detachment had 0.70 logMAR units (95% CI 0.02 to 1.39) poorer visual acuity than subjects without (p=0.046). The mean preoperative visual acuity was 0.84±0.59 logMAR units and the mean postoperative visual acuity was 0.64±0.64 logMAR units (mean difference 0.20±0.68 logMAR units (p=0.04)). The proportion of eyes with foveal detachment was significantly lower after surgery (12.5%; p<0.001). However, the proportion of eyes with ellipsoid zone disruption was significantly higher after surgery (59.6% vs 34.0%; p<0.001). In multivariate analyses, the preoperative central foveal thickness significantly predicted postoperative visual improvement by two or more lines (OR 1.004 (95% CI 1.000 to 1.007), per μm increase; p=0.049). The presence of ellipsoid zone disruption preoperatively was associated with 0.96 logMAR (95% CI 0.2 to 1.72) poorer final acuity (p=0.02). CONCLUSIONS: Eyes with myopic foveoschisis with preoperative ellipsoid disruption and thinner central foveal thickness tend to have poorer visual outcomes. While current surgical manoeuvres are effective in reattaching the fovea, they may also cause iatrogenic injury to the photoreceptors.

PURPOSE/AIMS: To assess the effect of storage temperature on the viability, phenotype, metabolism, and morphology of cultured human oral keratinocytes (HOK). MATERIALS AND METHODS: Cultured HOK cells were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium (MEM) at nine temperatures in approximately 4°C increments from 4°C to 37°C for seven days. Cells were characterized for viability by calcein fluorescence, phenotype retention by immunocytochemistry, metabolic parameters (pH, glucose, lactate, and O2) within the storage medium by blood gas analysis, and morphology by scanning electron microscopy and light microscopy. RESULTS: Relative to the cultured, but non-stored control cells, a high percentage of viable cells were retained only in the 12°C and 16°C storage groups (85%±13% and 68%±10%, respectively). Expression of ABCG2, Bmi1, C/EBPδ, PCNA, cytokeratin 18, and caspase-3 were preserved after storage in the 5 groups between 4°C and 20°C, compared to the non-stored control. Glucose, pH and pO2 in the storage medium declined, whereas lactate increased with increasing storage temperature. Morphology was best preserved following storage of the three groups between 12°C, 16°C, and 20°C. CONCLUSION: We conclude that storage temperatures of 12°C and 16°C were optimal for maintenance of cell viability, phenotype, and morphology of cultured HOK. The storage method described in the present study may be applicable for other cell types and tissues; thus its significance may extend beyond HOK and the field of ophthalmology.

PURPOSE: Amino-amide or amino-ester local anesthetics, which are currently used for topical ocular anesthesia, are short acting and may delay corneal healing with long-term use. In contrast, site 1 sodium channel blockers (S1SCBs) are potent local anesthetics with minimal adverse tissue reaction. In this study, we examined topical local anesthesia with two S1SCBs, tetrodotoxin (TTX) or saxitoxin (STX) individually or in combination with α2-adrenergic receptor agonists (dexmedetomidine or clonidine), and compared them with the amino-ester ocular anesthetic proparacaine. The effect of test solutions on corneal healing was also studied. METHODS: Solutions of TTX ± dexmedetomidine, TTX ± clonidine, STX ± dexmedetomidine, dexmedetomidine, or proparacaine were applied to the rat cornea. Tactile sensitivity was measured by recording the blink response to probing of the cornea with a Cochet-Bonnet esthesiometer. The duration of corneal anesthesia was calculated. Cytotoxicity from anesthetic solutions was measured in vitro. The effect on corneal healing was measured in vivo after corneal debridement followed by repeated drug administration. RESULTS: Addition of dexmedetomidine to TTX or STX significantly prolonged corneal anesthesia beyond that of either drug alone, whereas clonidine did not. Tetrodotoxin or STX coadministered with dexmedetomidine resulted in two to three times longer corneal anesthesia than did proparacaine. S1SCB-dexmedetomidine formulations were not cytotoxic. Corneal healing was not delayed significantly by any of the test solutions. CONCLUSIONS: Coadministration of S1SCBs with dexmedetomidine provided prolonged corneal anesthesia without delaying corneal wound healing. Such formulations may be useful for the management of acute surgical and nonsurgical corneal pain.

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.

IMPORTANCE: Subspecialty surgical training is an important part of resident education. We changed the glaucoma rotation in which postgraduate year 4 residents worked with multiple attending physicians with varying teaching styles to a structured surgical curriculum led by 2 dedicated preceptors, and we evaluated the effect on residents' surgical performance prospectively. OBSERVATIONS: A curriculum consisting of preoperative training, intraoperative teaching, postoperative feedback, and repetition was implemented for postgraduate year 4 residents between July 2, 2012, and June 30, 2014. In a class of 8 residents per year, the mean (SD) glaucoma surgical volume increased from 8.9 (0.8) cases in the prior year to 13.6 (2.5) in 2013 (mean difference, 4.8 cases; 95% CI, 2.4-7.1; P = .001) and 14.8 (4.2) in 2014 (mean difference, 5.9 cases; 95% CI, 2.1-9.6; P = .007). A self-assessment survey showed improvement in suturing (scores for each section range from 1 [worst] to 5 [best]; mean rating, 3.9 in the prior year vs 4.4 in 2013 [P = .04] and 4.5 in 2014 [P = .02]). A validated survey assessing overall surgical competency revealed improvement in handling adverse events (mean rating, 4.1 in the prior year vs 5.0 for both 2013 and 2014; both P < .001). CONCLUSIONS AND RELEVANCE: Despite the small sample size and nonrandomized study design, these data suggest that a structured surgical curriculum has advantages in teaching subspecialty surgery and might be considered by other ophthalmology training programs.

PurposeThe utility of in vivo confocal microscopy (IVCM) in the investigation of palpebral conjunctival and corneal inflammation in patients with meibomian gland dysfunction (MGD)-associated refractory dry eye symptoms following gland expression, despite objective clinical improvement.MethodsA retrospective, observational pilot study was conducted evaluating five patients with MGD-associated refractory dry eye symptoms and three control groups: symptomatic untreated MGD patients (n=3), treatment-responsive MGD patients with improved symptoms (n=3) and asymptomatic healthy normals (n=11). Ocular surface disease index (OSDI) scores, tear break-up time (TBUT), the number of meibomian glands yielding liquid secretion (MGYLS), palpebral conjunctival epithelial and substantia propria immune cell (EIC, SIC), and corneal dendritic cell (DC) densities were measured.ResultsDespite clinical improvement (TBUT: 6.4±1.2 s to 10.1±2.1 s, P=0.03; MGYLS: 3.5±0.8 glands to 7.0±1.1 glands, P=0.13) and a normal clinical examination post treatment, MGD patients remained symptomatic. IVCM revealed increased immune cells in the palpebral conjunctiva (refractory MGD EIC=592.6±110.1 cells/mm(2); untreated MGD EIC=522.6±104.7 cells/mm(2), P=0.69; responsive MGD EIC=194.9±119.4 cells/mm(2), P<0.01; normals EIC=123.7±19.2 cells/mm(2), P< 0.001), but not the cornea (refractory MGD DC=60.9±28.3 cells/mm(2); normals DC=25.9±6.3 cells/mm(2); P=0.43). EIC did not correlate with TBUT (Rs=-0.26, P=0.33). OSDI scores correlated with both EIC (Rs=0.76, P<0.001) and TBUT (Rs=-0.69, P<0.01) but not SIC. Intraglandular immune cells were also seen.ConclusionMGD-associated refractory symptoms and the symptom-sign disparity may be explained by clinically non-apparent, active inflammation of the palpebral conjunctiva as detected by IVCM. These patients may benefit from anti-inflammatory therapy.

The astrocytes of the optic nerve head are a specialized subtype of white matter astrocytes that form the direct cellular environment of the unmyelinated ganglion cell axons. Due to their potential involvement in glaucoma, these astrocytes have become a target of research. Due to the heterogeneity of the optic nerve tissue, which also contains other cell types, in some cases it may be desirable to conduct gene expression studies on small numbers of well-characterized astrocytes or even individual cells. Here, we describe a simple method to isolate individual astrocytes. This method permits obtaining astrocytes with intact morphology from the adult mouse optic nerve and reduces contamination of the isolated astrocytes by other cell types. Individual astrocytes can be recognized by their morphology and collected under microscopic control. The whole procedure can be completed in 2-3 h. We also discuss downstream applications like multiplex single-cell PCR and quantitative PCR (qPCR).

PURPOSE: Epiretinal fibrovascular membranes (FVMs) are a hallmark of proliferative diabetic retinopathy (PDR). Surgical removal of FVMs is often indicated to treat tractional retinal detachment. This potentially informative pathological tissue is usually disposed of after surgery without further examination. We developed a method for isolating and characterizing cells derived from FVMs and correlated their expression of specific markers in culture with that in tissue. METHODS: FVMs were obtained from 11 patients with PDR during diabetic vitrectomy surgery and were analyzed with electron microscopy (EM), comparative genomic hybridization (CGH), immunohistochemistry, and/or digested with collagenase II for cell isolation and culture. Antibody arrays and enzyme-linked immunosorbent assay (ELISA) were used to profile secreted angiogenesis-related proteins in cell culture supernatants. RESULTS: EM analysis of the FVMs showed abnormal vessels composed of endothelial cells with large nuclei and plasma membrane infoldings, loosely attached perivascular cells, and stromal cells. The cellular constituents of the FVMs lacked major chromosomal aberrations as shown with CGH. Cells derived from FVMs (C-FVMs) could be isolated and maintained in culture. The C-FVMs retained the expression of markers of cell identity in primary culture, which define specific cell populations including CD31-positive, alpha-smooth muscle actin-positive (SMA), and glial fibrillary acidic protein-positive (GFAP) cells. In primary culture, secretion of angiopoietin-1 and thrombospondin-1 was significantly decreased in culture conditions that resemble a diabetic environment in SMA-positive C-FVMs compared to human retinal pericytes derived from a non-diabetic donor. CONCLUSIONS: C-FVMs obtained from individuals with PDR can be isolated, cultured, and profiled in vitro and may constitute a unique resource for the discovery of cell signaling mechanisms underlying PDR that extends beyond current animal and cell culture models.

Galectins have emerged as potent immunoregulatory agents that control chronic inflammation through distinct mechanisms. Here, we report that treatment with Galectin-8 (Gal-8), a tandem-repeat member of the galectin family, reduces retinal pathology and prevents photoreceptor cell damage in a murine model of experimental autoimmune uveitis. Gal-8 treatment increased the number of regulatory T cells (Treg) in both the draining lymph node (dLN) and the inflamed retina. Moreover, a greater percentage of Treg cells in the dLN and retina of Gal-8 treated animals expressed the inhibitory coreceptor cytotoxic T lymphocyte antigen (CTLA)-4, the immunosuppressive cytokine IL-10, and the tissue-homing integrin CD103. Treg cells in the retina of Gal-8-treated mice were primarily inducible Treg cells that lack the expression of neuropilin-1. In addition, Gal-8 treatment blunted production of inflammatory cytokines by retinal T helper type (TH) 1 and TH17 cells. The effect of Gal-8 on T cell differentiation and/or function was specific for tissues undergoing an active immune response, as Gal-8 treatment had no effect on T cell populations in the spleen. Given the need for rational therapies for managing human uveitis, Gal-8 emerges as an attractive therapeutic candidate not only for treating retinal autoimmune diseases, but also for other TH1- and TH17-mediated inflammatory disorders.

PURPOSE: Although the impact of homonymous visual field defects (HFDs) on mobility has been investigated previously, the emphasis has been on obstacle detection. Relatively little is known about HFD patients' ability to judge collisions once an obstacle is detected. We investigated this using a walking simulator. METHODS: Patients with HFDs (n = 29) and subjects with normal vision (NV; n = 21) were seated in front of a large screen on which a visual simulation of walking was displayed. They made collision judgments for a human figure that appeared for 1 second at lateral offsets from the virtual walking path. A perceived-collision threshold was calculated for right and left sides. RESULTS: Symmetrical collision thresholds (same on left and right sides) were measured for participants with NV (n = 21), and right (n = 9) and left (n = 7) HFD without hemispatial neglect. Participants with left neglect (n = 10) showed significant asymmetry with thresholds smaller (compared to the NV group and other HFD groups) on the blind (P < 0.001) and larger on the seeing (P = 0.05) sides. Despite the asymmetry, the overall width of the zone of perceived collision risk was not different, suggesting a relatively uniform rightward deviation in judgments of the left neglect group. CONCLUSIONS: Left neglect was associated with rightward asymmetry in collision judgments, which may cause collisions on the left side even when an obstacle is detected. These behaviors may represent the spatial misperceptions in body midline described previously in patients with left neglect.

PURPOSE: To examine the relationship between proportion of African ancestry (PAA) and proliferative diabetic retinopathy (PDR) and to identify genetic loci associated with PDR using admixture mapping in African Americans with type 2 diabetes (T2D). METHODS: Between 1993 and 2013, 1440 participants enrolled in four different studies had fundus photographs graded using the Early Treatment Diabetic Retinopathy Study scale. Cases (n = 305) had PDR while controls (n = 1135) had nonproliferative diabetic retinopathy (DR) or no DR. Covariates included diabetes duration, hemoglobin A1C, systolic blood pressure, income, and education. Genotyping was performed on the Affymetrix platform. The association between PAA and PDR was evaluated using logistic regression. Genome-wide admixture scanning was performed using ANCESTRYMAP software. RESULTS: In the univariate analysis, PDR was associated with increased PAA (odds ratio [OR] = 1.36, 95% confidence interval [CI] = 1.16-1.59, P = 0.0002). In multivariate regression adjusting for traditional DR risk factors, income and education, the association between PAA and PDR was attenuated and no longer significant (OR = 1.21, 95% CI = 0.59-2.47, P = 0.61). For the admixture analyses, the maximum genome-wide score was 1.44 on chromosome 1. CONCLUSIONS: In this largest study of PDR in African Americans with T2D to date, an association between PAA and PDR is not present after adjustment for clinical, demographic, and socioeconomic factors. No genome-wide significant locus (defined as having a locus-genome statistic > 5) was identified with admixture analysis. Further analyses with even larger sample sizes are needed to definitively assess if any admixture signal for DR is present.

PURPOSE: To identify the factors responsible for the poor validity of the most common aniseikonia tests, which involve size comparisons of red-green stimuli presented haploscopically. METHODS: Aniseikonia was induced by afocal size lenses placed before one eye. Observers compared the sizes of semicircles presented haploscopically via color filters. The main factor under study was viewing mode (free viewing versus short presentations under central fixation). To eliminate response bias, a three-response format allowed observers to respond if the left, the right, or neither semicircle appeared larger than the other. To control decisional (criterion) bias, measurements were taken with the lens-magnified stimulus placed on the left and on the right. To control for size-color illusions, measurements were made with color filters in both arrangements before the eyes and under binocular vision (without color filters). RESULTS: Free viewing resulted in a systematic underestimation of lens-induced aniseikonia that was absent with short presentations. Significant size-color illusions and decisional biases were found that would be mistaken for aniseikonia unless appropriate action is taken. CONCLUSIONS: To improve their validity, aniseikonia tests should use short presentations and include control conditions to prevent contamination from decisional/response biases. If anaglyphs are used, presence of size-color illusions must be checked for. TRANSLATIONAL RELEVANCE: We identified optimal conditions for administration of aniseikonia tests and appropriate action for differential diagnosis of aniseikonia in the presence of response biases or size-color illusions. Our study has clinical implications for aniseikonia management.