We describe the design and construction of a headlight glare simulator to be used with a driving simulator. The system combines a modified programmable off-the-shelf LED display board and a beamsplitter so that the LED lights, representing the headlights of oncoming cars, are superimposed over the driving simulator headlights image. Ideal spatial arrangement of optical components to avoid misalignments of the superimposed images is hard to achieve in practice and variations inevitably introduce some parallax. Furthermore, the driver's viewing position varies with driver's height and seating position preferences exacerbate such misalignment. We reduce the parallax errors using an intuitive calibration procedure (simple drag-and-drop alignment of nine LED positions with calibration dots on the screen). To simulate the dynamics of headlight brightness changes when two vehicles are approaching, LED intensity control algorithms based on both headlight and LED beam shapes were developed. The simulation errors were estimated and compared to real-world headlight brightness variability.

Synonymous single nucleotide polymorphisms (SNPs) within a transcript's coding region produce no change in the amino acid sequence of the protein product and are therefore intuitively assumed to have a neutral effect on protein function. We report that two common variants of high-temperature requirement A1 (HTRA1) that increase the inherited risk of neovascular age-related macular degeneration (NvAMD) harbor synonymous SNPs within exon 1 of HTRA1 that convert common codons for Ala34 and Gly36 to less frequently used codons. The frequent-to-rare codon conversion reduced the mRNA translation rate and appeared to compromise HtrA1's conformation and function. The protein product generated from the SNP-containing cDNA displayed enhanced susceptibility to proteolysis and a reduced affinity for an anti-HtrA1 antibody. The NvAMD-associated synonymous polymorphisms lie within HtrA1's putative insulin-like growth factor 1 (IGF-1) binding domain. They reduced HtrA1's abilities to associate with IGF-1 and to ameliorate IGF-1-stimulated signaling events and cellular responses. These observations highlight the relevance of synonymous codon usage to protein function and implicate homeostatic protein quality control mechanisms that may go awry in NvAMD.

PURPOSE: To evaluate clinicopathologically and immunohistochemically a spectrum of conjunctival squamous proliferations. DESIGN: Retrospective clinicopathologic study. METHODS: One large cell acanthoma, 7 epidermoid dysplasias, and 4 squamous papillomas were evaluated with microscopy and biomarkers Ki-67, p53, epithelial membrane antigen (EMA), Ber-EP4, AE1, AE3, and 8 individual cytokeratins. Normal associated conjunctiva served as a baseline for interpretation. RESULTS: The large cell acanthoma recurred 4 times but retained its benign histopathologic features. The cells were 2-3 times larger than the keratinocytes of the normal conjunctiva and did not display atypia. Immunohistochemistry revealed a low Ki-67 proliferation index (PI) in the large cell acanthoma compared with high indices in dysplasias and papillomas. p53 was negative in the nuclei of normal epithelium while positive in all neoplasms, most intensely in the dysplasias. Immunostaining showed similar staining patterns for cytokeratins in large cell acanthoma and normal conjunctiva, except for full-thickness CK14 positivity and CK7 negativity in the lesion. Dysplasias generally lost normal CK7 expression and frequently abnormally expressed CK17. The papillomas displayed a normal cytokeratin pattern but exhibited a higher than normal PI and weak p53 positivity. CONCLUSIONS: Conjunctival large cell acanthoma is a morphologically distinctive clonal entity with clinical and immunohistochemical phenotypic characteristics denoting a dysplasia of minimal severity. Because of recurrences without invasion, it requires treatment. Dysplasias exhibited more deviant biomarker abnormalities including frequent aberrant full-thickness CK17 positivity and CK7 negativity. The absence of major cytokeratin derangements in the squamous papillomas may be of ancillary diagnostic value for lesions displaying borderline cytologic features.

A 31-year-old woman developed left frontal headache and retrobulbar pain with exotropia and mild limitation of upgaze without proptosis. Imaging studies disclosed a circumscribed mass measuring 1.5 cm in the greatest diameter. At surgery, the lesion was adherent to the surrounding normal orbital tissues, making for a difficult and dangerous resection. Histopathologically and immunohistochemically, the lesion was a mixed cavernous angiomatous (CD31-positive) and lymphangiomatous (D2-40-positive) tumor with abundant interstitial smooth muscle. Such lesions can present significant surgical challenges due to incomplete pseudoencapsulation.

PURPOSE: To define the cytologic composition of the double-layered epithelial lining of dacryops (lacrimal duct cyst), improve histopathologic diagnosis, and better understand pathogenesis. DESIGN: Clinicopathologic retrospective study with immunohistochemical studies of 15 lesions compared with normal lacrimal gland. METHODS: Clinical data from 14 patients were reviewed and microscopy was performed with routine stains and immunohistochemical probes for epithelial membrane antigen (EMA), gross cystic disease fluid protein-15 (GCDFP-15), cytokeratin 7 (CK7), and smooth muscle actin (SMA). RESULTS: The major lacrimal gland was involved in 13 lesions; 2 lesions arose in an accessory gland of Krause. One case was bilateral; the average age of the patients was 50.7 years. Neither visual acuity nor motility was disturbed. No lesion was discovered to have recurred after excision. Microscopically, in all dacryops specimens goblet cells and luminal pseudoapocrine apical cytoplasmic projections were identified. Lacrimal acinar cells immunoreacted with GCDFP-15 and CK7, whereas the normal ducts and the epithelium of the dacryops lesions reacted diffusely only with CK7. SMA-positive myoepithelial cells were found in the acini but not in the normal ducts or dacryops epithelium. CONCLUSIONS: Negative GCDFP-15 staining ruled out apocrine metaplasia in dacryops. Normal ducts and dacryops showed no immunohistochemical evidence for the presence of myoepithelial cells. Pathogenetic theories of dacryops that implicate a failure of ductular "neuromuscular" contractility must therefore be revised. A dysfunction of the rich neural plexus around the ductules may play a role in the development of dacryops in conjunction with periductular inflammation and induced scarring.

PURPOSE: To demonstrate the value of a diagnostic biopsy of a fixed episcleral nodule overlying a uveal mass. METHOD: Clinicopathologic report with immunohistochemical investigations. RESULTS: B-scan ultrasonographic biomicroscopy disclosed in a 67-year-old man an asymptomatic placoid ciliary body tumor measuring 1.28 mm in thickness underlying a poorly pigmented, fixed episcleral nodule 0.56 mm in thickness. Biopsy of the episcleral nodule displayed benign nevus-type spindle cells with small nuclei, punctate nucleoli, no mitoses, and scattered melanophages. Immunohistochemistry demonstrated that the tumor cells were Ki67 negative (proliferation index, 0) and MART-1, HMB-45, and microphthalmia-associated transcription factor positive, all melanocytic markers. The melanophages but not the tumor cells were CD68 positive, a histiocytic marker. CONCLUSIONS: The results from biopsying an episcleral nodule can help to select among therapeutic options in managing an associated ciliary body tumor. A 1-year follow-up study and 3 sequential ultrasonographic studies in the current patient have failed to document the growth of the intraocular tumor, further confirming that it is a nevus. The excised epibulbar tumor has not recurred.

PURPOSE: An increased aqueous level of TGF-β2 has been found in many primary open-angle glaucoma patients. Secreted Protein, Acidic, and Rich in Cysteine (SPARC)-null mice have a lower intraocular pressure. The mechanistic relationship between SPARC and TGF-β2 in trabecular meshwork (TM) is unknown. We hypothesized that TGF-β2 upregulates SPARC expression in TM. METHODS: Cultured TM cells were incubated with selective inhibitors for p38 MAP kinase (p38), Smad3, p42, JNK, RhoA, PI3K, or TGF-β2 receptor for 2 hours, and then TGF-β2 was added for 24 hours in serum-free media. Quantitative polymerase chain reaction (qPCR) and immunoblot analysis were performed. Immunofluorescent microscopy was used to determine nuclear translocation of signaling proteins. Ad5.hSPARC and Lentiviral shRNA for p38 and Smad3 were constructed, and infected human TM cells. RESULTS: SPARC was upregulated by TGF-β2 in the human TM cells (3.8 ± 1.7-fold, n = 6, P = 0.01 for protein and 7.1 ± 3.7-fold, n = 6, P = 0.01 for mRNA), while upregulation of SPARC had no effect on TGF-β2. TGF-β2-induced SPARC expression was suppressed by inhibitors against p38 (-40.3 ± 20.9%, n = 10, P = 0.0001), Smad3 (-56.2 ± 18.9%, n = 10, P = 0.0001), JNK (-49.1 ± 24.6%, n = 10, P = 0.0001), and TGF-β2 receptor (-83.6 ± 14.4%, n = 6, P = 0.003). Phosphorylation and translocation of Smad3, p38, and MAPKAPK2 were detected at 30 minutes and 1 hour, respectively, following TGF-β2 treatment. Phosphorylation of JNK and c-jun was detected before TGF-β2 treatment. SPARC was suppressed 31 ± 13% (n = 5, P < 0.0001) by shRNA-p38 and 41 ± 3% (n = 5, P < 0.0001) by shRNA-Smad3. CONCLUSIONS: TGF-β2 upregulates SPARC expression in human TM through Smad-dependent (Smad2/3) or -independent (p38) signaling pathways. SPARC may be a downstream regulatory node of TGF-β2-mediated IOP elevation.

INTRODUCTION: Age-related macular degeneration is a major cause of blindness among people aged 50 and older in industrialized countries. Anti-VEGF therapy has been tremendously successful in the treatment of neovascular macular degeneration. Examining the pharmacogenetics of patients' response to the anti-VEGF molecules could allow for a tailored treatment strategy based on patients' underlying genetics rather than the "one-size fits all" approach currently used. METHODS: Review of the English literature for papers examining the pharmacogenetics of treatment response of neovascular macular degeneration to either ranibizumab or bevacizumab. Polymorphisms in CFH, ARMS2, HTRA1 and VEGF A were examined and reviewed. RESULTS: Patients with the high-risk CC genotype in complement factor H (CFH) had a worse response to therapy with ranibizumab and bevacizumab. No clear trends were found with ARMS2, HTRA1 and VEGF A. CONCLUSIONS: The goal of personalized medicine is to craft a treatment program that is ideally suited to an individual patient's disease and genetic make-up rather than simply what works for a large population who share similar disease characteristics. Continued research is needed to achieve this goal for the treatment of age-related macular degeneration.

PURPOSE: We have previously shown that TGF-β3 (T3) stimulates extracellular matrix (ECM) assembly while maintaining antifibrotic characteristics in a model using human corneal fibroblasts (HCFs). This model, however, requires non-physiological levels of serum. In the current study, we tested whether T3 could stimulate human corneal keratocytes (HCKs) in vitro to assemble a functional ECM, while maintaining their characteristics. METHODS: Human corneal keratocytes and HCFs were isolated and cultured using 1% or 10% serum, respectively ±T3. The constructs were processed for indirect immunofluorescence (IF), transmission electron microscopy (TEM), and qRT-PCR, analyzing for keratocyte marker, keratocan, and ECM components, collagen (col) types I, III, and V. RESULTS: Quantitative reverse transcriptase PCR data showed that keratocan, col I, and V were all upregulated in HCKs compared with HCFs, whereas col III was expressed at low levels in HCKs. Transforming growth factor beta 3 stimulation further enhanced the level of change. Without T3, HCK constructs were very thin, approximately 5 μm; however, as with HCFs, upon stimulation with T3, HCK constructs increased in thickness by approximately 5-fold. Cell counts and ECM production revealed that HCKs assembled more ECM per unit area compared with HCFs, and IF revealed downregulation of fibrotic markers, col III, and thrombospondin-1, with T3 stimulation. Transmission electron microscopy data revealed aligned ECM with long fibrils for all conditions except HCK Controls. Human corneal keratocytes+T3 also showed denser collagen fibrils with more consistent fibril diameter. CONCLUSIONS: Overall, the data suggests that it is possible to stimulate matrix secretion and assembly by HCKs in vitro by using a single growth factor, T3.

Steady state dendritic cells (DC) found in non-lymphoid tissue sites under normal physiologic conditions play a pivotal role in triggering T cell responses upon immune provocation. CD11b+ and CD103+ DC have received considerable attention in this regard. However, still unknown is whether such CD11b+ and CD103+ DC even exist in the ocular mucosa, and if so, what functions they have in shaping immune responses. We herein identified in the ocular mucosa of normal wild-type (WT) and Flt3-/- mice the presence of a CD11b+ DC (i.e., CD11c+ MHCII+ CD11b+ CD103- F4/80+ Sirp-a+). CD103+ DC (i.e. CD11c+ MHCII+ CD11b low CD103+ CD8a+ DEC205+ Langerin+) were also present in WT, but not in Flt3-/- mice. These CD103+ DC expressed high levels of Id2 and Flt3 mRNA; whereas CD11b+ DC expressed high Irf4, Csfr, and Cx3cr1 mRNA. Additionally, the functions of these DC differed in response to allergic immune provocation. This was assessed utilizing a previously validated model, which includes transferring specific populations of exogenous DC into the ocular mucosa of ovalbumin (OVA)/alum-primed mice. Interestingly, in such mice, topical OVA instillation following engraftment of exogenous CD11b+ DC led to dominant allergic T cell responses and clinical signs of ocular allergy relative to those engrafted with CD103+ DC. Thus, although CD11b+ and CD103+ DC are both present in the normal ocular mucosa, the CD11b+ DC subset plays a dominant role in a mouse model of ocular allergy.