PURPOSE: To establish the efficacy of topical N-acetylcysteine (NAC) as a treatment to reduce protein deposition on the contact lens surface. METHODS: In this prospective, nonrandomized clinical trial, a total of 10 eyes (9 patients) were enrolled from a single center. All patients had a prior ocular history of either a Boston Keratoprosthesis type I or trichiasis from Stevens-Johnson syndrome, which necessitated full-time contact lens wear. Four visits were required to complete the study. During visit 1, a new contact lens was inserted and a baseline examination was performed. Visit 2 served as the control month, whereas visits 3 and 4 were month 1 and 2 on treatment with 20% NAC. At the end of each visit the contact lens was replaced. The lenses from visit 2 (control month-without NAC) and from visit 3 (treatment month-with NAC) were collected for proteomic analysis. The main outcome measures were to quantify protein deposition, as well as to assess the visual acuity and ocular surface symptoms before and after treatment. RESULTS: Topical NAC resulted in a 20% decrease in protein deposition. This correlated with a trend for improvement in visual acuity and increased subjective improvement in vision at month 1 (P=0.0153) and 2 (P=0.0016). CONCLUSIONS: NAC reduced protein deposition, decreased ocular surface symptoms, and improved contact lens transparency, thereby providing increased optical clarity.

Adult neural stem/progenitor (B1) cells within the walls of the lateral ventricles generate different types of neurons for the olfactory bulb (OB). The location of B1 cells determines the types of OB neurons they generate. Here we show that the majority of mouse B1 cell precursors are produced between embryonic days (E) 13.5 and 15.5 and remain largely quiescent until they become reactivated postnatally. Using a retroviral library carrying over 100,000 genetic tags, we found that B1 cells share a common progenitor with embryonic cells of the cortex, striatum, and septum, but this lineage relationship is lost before E15.5. The regional specification of B1 cells is evident as early as E11.5 and is spatially linked to the production of neurons that populate different areas of the forebrain. This study reveals an early embryonic regional specification of postnatal neural stem cells and the lineage relationship between them and embryonic progenitor cells.

PURPOSE: To describe 7 patients with paraproteinemic keratopathy and to highlight the clinical and pathologic diversity of this rare entity and the importance of timely, systemic evaluation. DESIGN: Retrospective, multicenter collaborative case series. PARTICIPANTS: Seven patients with paraproteinemic keratopathy. METHODS: Clinical and pathologic records were reviewed to identify patients with well-documented corneal immunoglobulin deposits. Detailed ophthalmologic and medical histories were assembled. In 6 patients, corneal tissue was evaluated histochemically and immunohistochemically; in selected cases, corneal tissue was evaluated by in situ hybridization and ultrastructurally. MAIN OUTCOME MEASURES: Visual acuity and anterior segment examination at presentation and follow-up; local therapy; systemic diagnosis and management; and histopathologic, immunohistochemical, in situ hybridization, and ultrastructural findings. RESULTS: Seven patients were identified with corneal immunoglobulin deposition. In addition to previously reported crystalline, nummular, patch-like, and lattice-like corneal opacities, prominent corneal vascularization was present in 2 patients mimicking interstitial keratitis and limbal stem cell deficiency. All patients had evidence of paraproteinemia in a setting of monoclonal gammopathy of undetermined significance, smoldering plasma cell myeloma, or Waldenström macroglobulinemia. Corneal findings were the first manifestation of systemic disease in 4 patients, and the diagnosis was not suspected in 3 of these patients. Pathologic evaluation of biopsied corneal and conjunctival tissues demonstrated immunoglobulin deposits. Previously unreported ultrastructural patterns in the cornea were noted: large scroll-like immunotactoid deposits, immune complex-like deposits, and randomly arranged fibrils morphologically intermediate between amyloid and immunotactoid deposits. Surgical intervention to improve vision was performed in 4 patients, with recurrence of deposits in 3 patients. Three patients underwent systemic therapy with diminution of the deposits and improvement in vision in 1 patient. CONCLUSIONS: The clinical and pathologic expressions of corneal immunoglobulin deposits are protean and present a diagnostic challenge. Early recognition of this rare entity is important to address the potentially serious associated systemic disease.

This study characterizes the kallikrein-kinin system in vitreous from individuals with diabetic macular edema (DME) and examines mechanisms contributing to retinal thickening and retinal vascular permeability (RVP). Plasma prekallikrein (PPK) and plasma kallikrein (PKal) were increased twofold and 11.0-fold (both P < 0.0001), respectively, in vitreous from subjects with DME compared with those with a macular hole (MH). While the vascular endothelial growth factor (VEGF) level was also increased in DME vitreous, PKal and VEGF concentrations do not correlate (r = 0.266, P = 0.112). Using mass spectrometry-based proteomics, we identified 167 vitreous proteins, including 30 that were increased in DME (fourfold or more, P < 0.001 vs. MH). The majority of proteins associated with DME displayed a higher correlation with PPK than with VEGF concentrations. DME vitreous containing relatively high levels of PKal and low VEGF induced RVP when injected into the vitreous of diabetic rats, a response blocked by bradykinin receptor antagonism but not by bevacizumab. Bradykinin-induced retinal thickening in mice was not affected by blockade of VEGF receptor 2. Diabetes-induced RVP was decreased by up to 78% (P < 0.001) in Klkb1 (PPK)-deficient mice compared with wild-type controls. B2- and B1 receptor-induced RVP in diabetic mice was blocked by endothelial nitric oxide synthase (NOS) and inducible NOS deficiency, respectively. These findings implicate the PKal pathway as a VEGF-independent mediator of DME.

The cornea functions as the major refractive interface for vision and protects the internal eye from insult. Current understanding of innate immune responses to corneal infection derives from a synthesis of in vitro and in vivo analyses. However, monolayer cell cultures and mouse models do not accurately duplicate all aspects of innate immunity in human patients. Here, we describe a three-dimensional culture system that incorporates human cells and extracellular matrix to more completely simulate the human cornea for studies of infection. Human corneal stromal fibroblasts were mixed with type I collagen in 3-μm pore size transwell inserts, and overlayed with Matrigel to simulate a human corneal stroma and epithelial basement membrane. These were then infected with a cornea-tropic adenovirus, and exposed on their inferior side to leukocytes derived from human peripheral blood. Subsequent analyses were performed with histology, confocal microscopy, ELISA, and fluorescence-activated cell sorting (FACS). CXCL8, a neutrophil chemokine shown previously as the first cytokine induced in infection of human corneal cells, increased upon adenovirus infection of facsimiles in a dose-responsive fashion. Myeloperoxidase-positive cells infiltrated infected corneal facsimiles in a sub-Matrigel location, possibly due to CXCL8 colocalization with heparan sulfate, a Matrigel constituent. Cellular infiltration was significantly inhibited by treatment with chemical inhibitors of p38 MAPK and Src kinase, both constituents of a signaling cascade previously suggested to regulate inflammation after adenovirus infection. FACS analysis determined that both virus and corneal fibroblasts were necessary for the induction of leukocyte migration into the facsimiles. The corneal facsimile, literally a cornea in a test tube, permits mechanistic studies on human tissue in a highly tractable system.

IMPORTANCE: Somatic mutations in BAP1 (BRCA1-associated protein 1 gene) are frequently identified in uveal melanoma. To date, the role of germline BAP1 mutations in uveal melanoma has not been characterized. OBJECTIVE: To characterize the clinical phenotype of uveal melanoma in patients with germline BAP1 mutations. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cohort study at an academic ophthalmology referral center among 507 patients with uveal melanoma who consented for collection of blood samples. The study dates were June 22, 1992, to December 14, 2010. MAIN OUTCOMES AND MEASURES: Clinical characteristics of uveal melanoma and the development of metastases. BAP1 gene sequencing from blood samples of patients with uveal melanoma was correlated with clinical characteristics. RESULTS: Of 507 blood samples analyzed, 25 patients (4.9%) exhibited 18 BAP1 polymorphisms, of which 9 were novel. Computational analyses predicted that 8 BAP1 mutations in 8 patients (1.6%) were likely to result in damaged BAP1 protein. Five of these 8 mutations were novel. These 8 patients were compared with 482 patients in whom no BAP1 polymorphisms were identified. In univariate analyses, patients with germline BAP1 mutations exhibited larger tumor diameters (mean, 15.9 vs 12.3 mm; P = .004) and higher rates of ciliary body involvement (75.0% vs 21.6%, P = .002) and metastases (71.4% vs 18.0%, P = .003) compared with control subjects. Patients with germline BAP1 mutations exhibited increased frequency of family history of cancer (100% vs 65.9%, P = .06), particularly cutaneous melanoma (62.5% vs 9.9%, P < .001) and ocular melanoma (25.0% vs 1.9%, P = .01). No differences were identified in age at diagnosis, sex, history of other malignant neoplasm, presenting visual acuity, distance of the tumor from the optic nerve or fovea, iris involvement, extrascleral extension, or tumor pigmentation. Germline BAP1 mutations increased risk of metastasis independent of ciliary body involvement (P = .02). Germline BAP1 mutation approached significance as an independent risk factor for metastasis (P = .09). CONCLUSIONS AND RELEVANCE: These data suggest that germline BAP1 mutations occur infrequently in uveal melanoma and are associated with larger tumors and higher rates of ciliary body involvement, 2 known risk factors for metastasis.

PURPOSE: There is a need for automated retinal optical coherence tomography (OCT) image analysis tools for quantitative measurements in small animals. Some image processing techniques for retinal layer analysis have been developed, but reports about how useful those techniques are in actual animal studies are rare. This paper presents the use of a retinal layer detection method we developed in an actual mouse study that involves wild type and mutated mice carrying photoreceptor degeneration. METHODS: Spectral domain OCT scanning was performed by four experimenters over 12 months on 45 mouse eyes that were wild-type, deficient for ephrin-A2 and ephrin-A3, deficient for rhodopsin, or deficient for rhodopsin, ephrin-A2 and ephrin-A3. The thickness of photoreceptor complex between the outer plexiform layer and retinal pigment epithelium was measured on two sides of the optic disc as the biomarker of retinal degeneration. All the layer detection results were visually confirmed. RESULTS: Overall, 96% (8519 out of 9000) of the half-side images were successfully processed using our technique in a semi-automatic manner. There was no significant difference in success rate between mouse lines (p = 0.91). Based on a human observer's rating of image quality for images successfully and unsuccessfully processed, the odds ratios for 'easily visible' images and 'not clear' images to be successfully processed is 62 and 4, respectively, against 'indistinguishable' images. Thickness of photoreceptor complex was significantly different across the quadrants compared (p < 0.001). It was also found that the average thickness based on 4-point sparse sampling was not significantly different from the full analysis, while the range of differences between the two methods could be up to about 6 μm or 16% for individual eyes. Differences between mouse lines and progressive thickness reduction were revealed by both sampling measures. CONCLUSIONS: Although the thickness of the photoreceptor complex layer is not even, manual sparse sampling may be as sufficiently accurate as full analysis in some studies such as ours, where the error of sparse sampling was much smaller than the effect size of rhodopsin deficiency. It is also suggested that the image processing method can be useful in actual animal studies. Even for images poorly visible to human eyes the image processing method still has a good chance to extract the complex layer.

We conducted a nested candidate gene study and pathway-based enrichment analysis on data from a multi-national 77,000-person project on the molecular genetics of age-related macular degeneration (AMD) to identify AMD-associated DNA-sequence variants in genes encoding constituents of a netrin-1 (NTN1)-based signaling pathway that converges on DNA-binding transcription complexes through a 3'-5'-cyclic adenosine monophosphate-calcineurin (cAMP-CN)-dependent axis. AMD-associated single nucleotide polymorphisms (SNPs) existed in 9 linkage disequilibrium-independent genomic regions; these included loci overlapping NTN1 (rs9899630, P ≤ 9.48 x 10-5), DCC (Deleted in Colorectal Cancer)-the gene encoding a primary NTN1 receptor (rs8097127, P ≤ 3.03 x 10-5), and 6 other netrin-related genes. Analysis of the NTN1-DCC pathway with exact methods demonstrated robust enrichment with AMD-associated SNPs (corrected P-value = 0.038), supporting the idea that processes driven by NTN1-DCC signaling systems operate in advanced AMD. The NTN1-DCC pathway contains targets of FDA-approved drugs and may offer promise for guiding applied clinical research on preventive and therapeutic interventions for AMD.