February 2018

Cao J, Brouwer NJ, Jordanova ES, Marinkovic M, van Duinen SG, de Waard NE, Ksander BR, Mulder A, Claas FHJ, Heemskerk MHM, Jager MJ. HLA Class I Antigen Expression in Conjunctival Melanoma Is Not Associated With PD-L1/PD-1 Status. Invest Ophthalmol Vis Sci 2018;59(2):1005-1015.Abstract
Purpose: Antitumor T cells need expression of HLA class I molecules but can be inhibited by ligands such as programmed death ligand 1 (PD-L1). We determined expression and regulation of these molecules in human conjunctival melanoma (CM) samples, cell lines, and murine xenografts. Methods: Immunofluorescence staining was performed to examine the expression of HLA-A, HLA-B/C, and β-2-microglobulin (B2M) in 23 primary CM samples. HLA class I expression was compared with clinicopathologic characteristics, the presence of tumor-infiltrating leukocytes, and PD-L1/PD-1 status. The effect of interferon γ (IFN-γ) on HLA class I expression was tested on three CM cell lines using quantitative PCR and flow cytometry. Furthermore, HLA class I expression was determined in CM cell line-derived murine xenografts. Results: One third of tumors had positive HLA-A, HLA-B/C, and B2M expression. A positive expression was especially seen in thin and epibulbar tumors but was not associated with recurrences. HLA class I expression was correlated with M2 macrophage density and tended to associate with CD8+ T-cell density but was independent of PD-L1 or PD-1 expression. IFN-γ upregulated HLA class I expression and genes involved in HLA transcription and transportation on CM cell lines. Murine xenografts showed a comparable HLA class I expression as their respective cell lines. Conclusions: Our data indicate that subsets of CM have positive HLA class I expression, and HLA class I and PD-L1/PD-1 are expressed independently. When one considers immunotherapy, one should also analyze HLA class I expression, whose downregulation can limit the efficacy of T cell-mediated therapies.
Wan MJ, Chiu H, Shah AS, Hunter DG. Long-term Surgical Outcomes for Large-angle Infantile Esotropia. Am J Ophthalmol 2018;189:155-159.Abstract
PURPOSE: To report the long-term surgical outcomes for a cohort of children with large-angle infantile esotropia. DESIGN: Multicenter, nonrandomized clinical study. METHODS: Setting: Two tertiary-care pediatric hospitals. STUDY POPULATION: Children with large-angle (≥55 prism diopters) infantile esotropia. INTERVENTION: Surgical treatment of infantile esotropia. MAIN OUTCOME MEASURE: Success rate at final follow-up (postoperative deviation ≤ 10 prism diopters and no need for retreatment). RESULTS: A total of 88 patients with large-angle infantile esotropia were treated during the 13-year study period. Treatment was bilateral medial rectus muscle recessions in 70 patients, botulinum toxin-augmented surgery in 15 patients, and 3-muscle surgery in 3 patients. After a mean follow-up of 40 months, 20 patients (23%) had a successful outcome compared to 68 treatment failures (77%). Of the 68 treatment failures, 59 had residual or recurrent esotropia and 9 had sequential exotropia. On multivariate logistic regression, treatment modality was the only factor significantly associated with a successful outcome. Specifically, patients treated with botulinum toxin-augmented surgery were more likely to have a successful outcome compared to patients treated with bilateral medial rectus muscle recessions. For the 26 patients (30%) who underwent retreatment, the mean number of procedures was 2.1, and 7 (27%) had a deviation of ≤10 prism diopters at final follow-up. CONCLUSIONS: The overall success rate for treatment of large-angle infantile esotropia was poor in this cohort, with most failures owing to recurrent or residual esotropia. Botulinum toxin-augmented surgery was associated with a higher success rate at final follow-up.
Chen X, Sullivan DA, Sullivan AG, Kam WR, Liu Y. Toxicity of cosmetic preservatives on human ocular surface and adnexal cells. Exp Eye Res 2018;Abstract
Cosmetic products, such as mascara, eye shadow, eyeliner and eye makeup remover are used extensively to highlight the eyes or clean the eyelids, and typically contain preservatives to prevent microbial growth. These preservatives include benzalkonium chloride (BAK) and formaldehyde (FA)-releasing preservatives. We hypothesize that these preservatives, at concentrations (BAK = 1 mg/ml; FA = 0.74 mg/ml) approved for consumer use, are toxic to human ocular surface and adnexal cells. Accordingly, we tested the influence of BAK and FA on the morphology, survival, and proliferation and signaling ability of immortalized human meibomian gland (iHMGECs), corneal (iHCECs) and conjunctival (iHConjECs) epithelial cells. iHMGECs, iHCECs and iHConjECs were cultured with different concentrations of BAK (5 μg/ml to 0.005 μg/ml) or FA (1 mg/ml to 1 μg/ml) under basal, proliferating or differentiating conditions up to 7 days. We used low BAK levels, because we found that 0.5 mg/ml and 50 μg/ml BAK killed iHMGECs within 1 day after a 15 min exposure. Experimental procedures included analyses of cell appearance, cell number, and neutral lipid content (LipidTox), lysosome accumulation (LysoTracker) and AKT signaling in all 3 cell types. Our results demonstrate that BAK and FA cause dose-dependent changes in the morphology, survival, proliferation and AKT signaling of iHMGECs, iHCECs and iHConjECs. Many of the concentrations tested induced cell atrophy, poor adherence, decreased proliferation and death, after 5 days of exposure. Cellular signaling, as indicated by AKT phosphorylation after 15 (FA) or 30 (BAK) minutes of treatment, was also reduced in a dose-dependent fashion in all 3 cell types, irrespective of whether cells had been cultured under proliferating or differentiating conditions. Our results support our hypothesis and demonstrate that the cosmetic preservatives, BAK and FA, exert many toxic effects on cells of the ocular surface and adnexa.
Li W, Webster KA, LeBlanc ME, Tian H. Secretogranin III: a diabetic retinopathy-selective angiogenic factor. Cell Mol Life Sci 2018;75(4):635-647.Abstract
Secretogranin III (Scg3) is a member of the granin protein family that regulates the biogenesis of secretory granules. Scg3 was recently discovered as an angiogenic factor, expanding its functional role to extrinsic regulation. Unlike many other known angiogenic factors, the pro-angiogenic actions of Scg3 are restricted to pathological conditions. Among thousands of quantified endothelial ligands, Scg3 has the highest binding activity ratio to diabetic vs. healthy mouse retinas and lowest background binding to normal vessels. In contrast, vascular endothelial growth factor binds to and stimulates angiogenesis of both diabetic and control vasculature. Consistent with its role in pathological angiogenesis, Scg3-neutralizing antibodies alleviate retinal vascular leakage in mouse models of diabetic retinopathy and retinal neovascularization in oxygen-induced retinopathy mice. This review summarizes our current knowledge of Scg3 as a regulatory protein of secretory granules, highlights its new role as a highly disease-selective angiogenic factor, and envisions Scg3 inhibitors as "selective angiogenesis blockers" for targeted therapy.
Jakobiec FA, Ma L, Wolkow N, Cameron DJ, Maltry AC. Osseous and Adipocytic Differentiations in the Intraocular Lens and Vitreous. Am J Ophthalmol 2018;186:77-88.Abstract
PURPOSE: To analyze 3 unusual mesenchymal transformations within the eye: adipose or osseous metaplasia of the lens and adipose tissue in the vitreous cavity. DESIGN: Observational case series. METHODS: Reevaluation of clinicopathologic diagnoses and histopathologic findings in sections stained with hematoxylin-eosin, periodic acid-Schiff (PAS) reaction, and Masson trichrome method. RESULTS: The 3 cases of mesenchymal transformation occurred in microphthalmic eyes with persistent hyperplastic primary vitreous (more recently termed persistent fetal vasculature). In 1 case there was total lens replacement with lamellar bone; in another, total replacement of the crystalline lens by adipose tissue; and in a third, an anomalous pocket of adipose tissue in the central vitreous. Multifocal remnants of the lens capsule were seen in the osseous case but were absent from the adipocytic cases. The vitreous adipose tissue was surrounded by an elaborate capillary plexus with an empty, collapsed PAS-positive lens capsule in the pupillary region. Anterior pigmented neuroectodermal disorganization, dysgenesis of angle structures, and a hypoplastic or disorganized iris were also observed in the 3 cases. CONCLUSIONS: After review of the literature, it appears that lenticular osseous replacement occurs more often than adipocytic. In addition to vascularization of the lens through a capsular dehiscence, other causes are explored, including direct epithelial-mesenchymal transformations of the lens epithelium or, less likely, of the disorganized adjacent neuroectoderm. The focus of vitreous adipose tissue may represent a transformed luxated lens extruded from its capsule, which was left behind in the pupillary zone.
Cheng L, Wong LJ, Yan N, Han RC, Yu H, Guo C, Batsuuri K, Zinzuwadia A, Guan R, Cho K-S, Chen DF. Ezh2 does not mediate retinal ganglion cell homeostasis or their susceptibility to injury. PLoS One 2018;13(2):e0191853.Abstract
Epigenetic predisposition is thought to critically contribute to adult-onset disorders, such as retinal neurodegeneration. The histone methyltransferase, enhancer of zeste homolog 2 (Ezh2), is transiently expressed in the perinatal retina, particularly enriched in retinal ganglion cells (RGCs). We previously showed that embryonic deletion of Ezh2 from retinal progenitors led to progressive photoreceptor degeneration throughout life, demonstrating a role for embryonic predisposition of Ezh2-mediated repressive mark in maintaining the survival and function of photoreceptors in the adult. Enrichment of Ezh2 in RGCs leads to the question if Ezh2 also mediates gene expression and function in postnatal RGCs, and if its deficiency changes RGC susceptibility to cell death under injury or disease in the adult. To test this, we generated mice carrying targeted deletion of Ezh2 from RGC progenitors driven by Math5-Cre (mKO). mKO mice showed no detectable defect in RGC development, survival, or cell homeostasis as determined by physiological analysis, live imaging, histology, and immunohistochemistry. Moreover, RGCs of Ezh2 deficient mice revealed similar susceptibility against glaucomatous and acute optic nerve trauma-induced neurodegeneration compared to littermate floxed or wild-type control mice. In agreement with the above findings, analysis of RNA sequencing of RGCs purified from Ezh2 deficient mice revealed few gene changes that were related to RGC development, survival and function. These results, together with our previous report, support a cell lineage-specific mechanism of Ezh2-mediated gene repression, especially those critically involved in cellular function and homeostasis.
Nilsson AK, Löfqvist C, Najm S, Hellgren G, Sävman K, Andersson MX, Smith LEH, Hellström A. Long-chain polyunsaturated fatty acids decline rapidly in milk from mothers delivering extremely preterm indicating the need for supplementation. Acta Paediatr 2018;107(6):1020-1027.Abstract
AIM: Our aim was to perform an in-depth analysis of the composition of fatty acids in milk from mothers delivering extremely preterm babies. We investigated longitudinal changes in milk fatty acid profiles and the relationship between several types of fatty acids, including omega-3 and omega-6. METHODS: Milk samples were collected at three stages of lactation from 78 mothers who delivered at less than 28 weeks of pregnancy at the Sahlgrenska University Hospital, Gothenburg, Sweden, from April 2013 to September 2015. Fatty acid composition was analysed by gas chromatography-mass spectrometry. RESULTS: A reduction in long-chain polyunsaturated fatty acids (LCPUFAs) was observed during the lactation period. The concentrations of arachidonic acid and docosahexaenoic acid declined from medians of 0.34 to 0.22 mol% and 0.29 to 0.15 mol%, respectively, between postnatal day 7 and a postmenstrual age of 40 weeks. Strong correlations were found between the intermediates of several classes of fatty acids, including omega-3, omega-6 and omega-9. CONCLUSION: A rapid reduction in LCPUFA content in the mother's milk during the lactation period emphasises the importance of fatty acid supplementation to infants born extremely preterm, at least during the period corresponding to the third trimester, when rapid development of the brain and adipose tissue requires high levels of LCPUFAs.
Strainiene E, Binkis M, Urnikyte S, Stankevicius V, Sasnauskiene A, Kundrotas G, Kazlauskas A, Suziedelis K. Microenvironment dependent gene expression signatures in reprogrammed human colon normal and cancer cell lines. BMC Cancer 2018;18(1):222.Abstract
BACKGROUND: Since the first evidence suggesting existence of stem-like cancer cells, the process of cells reprogramming to the stem cell state remains as an attractive tool for cancer stemness research. Current knowledge in the field of cancer stemness, indicates that the microenvironment is a fundamental regulator of cell behavior. With regard to this, we investigated the changes of genome wide gene expression in reprogrammed human colon normal epithelial CRL-1831 and colon carcinoma DLD1 cell lines grown under more physiologically relevant three-dimensional (3D) cell culture microenvironment compared to 2D monolayer. METHODS: Whole genome gene expression changes were evaluated in both cell lines cultured under 3D conditions over a 2D monolayer by gene expression microarray analysis. To evaluate the biological significance of gene expression changes, we performed pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Gene network analysis was used to study relationships between differentially expressed genes (DEGs) in functional categories by the GeneMANIA Cytoscape toolkit. RESULTS: In total, we identified 3228 and 2654 differentially expressed genes (DEGs) for colon normal and cancer reprogrammed cell lines, respectively. Furthermore, the expression of 1097 genes was commonly regulated in both cell lines. KEGG enrichment analysis revealed that in total 129 and 101 pathways for iPSC-CRL-1831 and for CSC-DLD1, respectively, were enriched. Next, we grouped these pathways into three functional categories: cancer transformation/metastasis, cell interaction, and stemness. β-catenin (CTNNB1) was confirmed as a hub gene of all three functional categories. CONCLUSIONS: Our present findings suggest common pathways between reprogrammed human colon normal epithelium (iPSC-CRL-1831) and adenocarcinoma (CSC-DLD1) cells grown under 3D microenvironment. In addition, we demonstrated that pathways important for cancer transformation and tumor metastatic activity are altered both in normal and cancer stem-like cells during the transfer from 2D to 3D culture conditions. Thus, we indicate the potential of cell culture models enriched in normal and cancer stem-like cells for the identification of new therapeutic targets in cancer treatment.
Chen X, Liu Y, Zhang Y, Kam WR, Pasquale LR, Sullivan DA. Impact of aromatase absence on murine intraocular pressure and retinal ganglion cells. Sci Rep 2018;8(1):3280.Abstract
We hypothesize that aromatase, an enzyme that regulates estrogen production, plays a significant role in the control of intraocular pressure (IOP) and retinal ganglion cells (RGCs). To begin to test our hypothesis, we examined the impact of aromatase absence, which completely eliminates estrogen synthesis, in male and female mice. Studies were performed with adult, age-matched wild type (WT) and aromatase knockout (ArKO) mice. IOP was measured in a masked fashion in both eyes of conscious mice at 12 and 24 weeks of age. Retinas were obtained and processed for RGC counting with a confocal microscope. IOP levels in both 12- and 24-week old female ArKO mice were significantly higher than those of age- and sex-matched WT controls. The mean increase in IOP was 7.9% in the 12-week-, and 19.7% in the 24-week-old mice, respectively. These changes were accompanied by significant 9% and 7% decreases in RGC numbers in the ArKO female mice, relative to controls, at 12- and 24-weeks, respectively. In contrast, aromatase deficiency did not lead to an increased IOP in male mice. There was a significant reduction in RGC counts in the 12-, but not 24-, week-old male ArKO mice, as compared to their age- and sex-matched WT controls. Overall, our findings show that aromatase inhibition in females is associated with elevated IOP and reduced RGC counts.
Wan MJ, Gilbert A, Kazlas M, Wu C, Mantagos IS, Hunter DG, Shah AS. The Effect of Botulinum Toxin Augmentation on Strabismus Surgery for Large-Angle Infantile Esotropia. Am J Ophthalmol 2018;189:160-165.Abstract
PURPOSE: To determine whether botulinum toxin augments the effect of strabismus surgery in pediatric patients with large-angle infantile esotropia. DESIGN: Retrospective, comparative, case series. METHODS: Setting: Tertiary-care pediatric hospital. STUDY POPULATION: Patients with large-angle infantile esotropia. INTERVENTION: Treatment with botulinum toxin-augmented bilateral medial rectus muscle recessions ("augmented-surgery group") or traditional bilateral medial rectus muscle recessions ("surgery-only group"). MAIN OUTCOME MEASURE: The effect of surgery on ocular alignment at 4 months, measured in prism diopters of change per mm of surgery (PD/mm). RESULTS: There were 14 patients in the augmented-surgery group and 16 patients in the surgery-only group. The mean effect on alignment was significantly greater in the augmented-surgery group compared to the surgery-only group at 4 months (5.7 ± 1.3 vs 4.0 ± 1.4 PD/mm, P = .002) and at 1 year (5.4 ± 1.2 vs 3.7 ± 1.2 PD/mm, P = .002). There was a partial loss of treatment effect between 4 months and 1 year in both groups, which was similar in magnitude (P = .57). On linear regression, there was a trend toward a positive correlation between botulinum toxin dose and treatment effect, but this was not statistically significant (P = .09). CONCLUSIONS: Botulinum toxin augments the surgical effect of medial rectus muscle recession. Botulinum toxin-augmented surgery may be an alternative to traditional options for large-angle infantile esotropia. A surgical dosing table is proposed for this technique.
Trakhtenberg EF, Li Y, Feng Q, Tso J, Rosenberg PA, Goldberg JL, Benowitz LI. Zinc chelation and Klf9 knockdown cooperatively promote axon regeneration after optic nerve injury. Exp Neurol 2018;300:22-29.Abstract
The inability of axons to regenerate over long-distances in the central nervous system (CNS) limits the recovery of sensory, motor, and cognitive functions after various CNS injuries and diseases. Although pre-clinical studies have identified a number of manipulations that stimulate some degree of axon growth after CNS damage, the extent of recovery remains quite limited, emphasizing the need for improved therapies. Here, we used traumatic injury to the mouse optic nerve as a model system to test the effects of combining several treatments that have recently been found to promote axon regeneration without the risks associated with manipulating known tumor suppressors or oncogenes. The treatments tested here include TPEN, a chelator of mobile (free) zinc (Zn); shRNA against the axon growth-suppressing transcription factor Klf9; and the atypical growth factor oncomodulin combined with a cAMP analog. Whereas some combinatorial treatments produced only marginally stronger effects than the individual treatments alone, co-treatment with TPEN and Klf9 knockdown had a substantially stronger effect on axon regeneration than either one alone. This combination also promoted a high level of cell survival at longer time points. Thus, Znchelation in combination with Klf9 suppression holds therapeutic potential for promoting axon regeneration after optic nerve injury, and may also be effective for treating other CNS injuries and diseases.
Bailey JCN, Gharahkhani P, Kang JH, Butkiewicz M, Sullivan DA, Weinreb RN, Aschard H, Allingham RR, Ashley-Koch A, Lee RK, Moroi SE, Brilliant MH, Wollstein G, Schuman JS, Fingert JH, Budenz DL, Realini T, Gaasterland T, Scott WK, Singh K, Sit AJ, Igo RP, Song YE, Hark L, Ritch R, Rhee DJ, Vollrath D, Zack DJ, Medeiros F, Vajaranant TS, Chasman DI, Christen WG, Pericak-Vance MA, Liu Y, Kraft P, Richards JE, Rosner BA, Hauser MA, Craig JE, Burdon KP, Hewitt AW, Mackey DA, Haines JL, Macgregor S, Wiggs JL, Pasquale LR, and of Consortium ANZRAG (ANZRAG). Testosterone Pathway Genetic Polymorphisms in Relation to Primary Open-Angle Glaucoma: An Analysis in Two Large Datasets. Invest Ophthalmol Vis Sci 2018;59(2):629-636.Abstract
Purpose: Sex hormones may be associated with primary open-angle glaucoma (POAG), although the mechanisms are unclear. We previously observed that gene variants involved with estrogen metabolism were collectively associated with POAG in women but not men; here we assessed gene variants related to testosterone metabolism collectively and POAG risk. Methods: We used two datasets: one from the United States (3853 cases and 33,480 controls) and another from Australia (1155 cases and 1992 controls). Both datasets contained densely called genotypes imputed to the 1000 Genomes reference panel. We used pathway- and gene-based approaches with Pathway Analysis by Randomization Incorporating Structure (PARIS) software to assess the overall association between a panel of single nucleotide polymorphisms (SNPs) in testosterone metabolism genes and POAG. In sex-stratified analyses, we evaluated POAG overall and POAG subtypes defined by maximum IOP (high-tension [HTG] or normal tension glaucoma [NTG]). Results: In the US dataset, the SNP panel was not associated with POAG (permuted P = 0.77), although there was an association in the Australian sample (permuted P = 0.018). In both datasets, the SNP panel was associated with POAG in men (permuted P ≤ 0.033) and not women (permuted P ≥ 0.42), but in gene-based analyses, there was no consistency on the main genes responsible for these findings. In both datasets, the testosterone pathway association with HTG was significant (permuted P ≤ 0.011), but again, gene-based analyses showed no consistent driver gene associations. Conclusions: Collectively, testosterone metabolism pathway SNPs were consistently associated with the high-tension subtype of POAG in two datasets.
Rasool N, Boudreault K, Lessell S, Prasad S, Cestari DM. Tacrolimus Optic Neuropathy. J Neuroophthalmol 2018;38(2):160-166.Abstract
BACKGROUND: Tacrolimus (FK506, Prograf) is a potent immunosuppressant, which inhibits cytokine synthesis and blocks T-cell development. Optic neuropathy from tacrolimus toxicity is very uncommon but, when present, can result in severe vision loss. METHODS: Case series and review of the literature. RESULTS: We present 3 patients with tacrolimus optic neuropathy after bone marrow transplantation complicated by graft-vs-host disease and demonstrate the differing clinical and radiologic presentation of this presumed toxic optic neuropathy. CONCLUSIONS: Tacrolimus optic neuropathy can manifest in a multitude of clinical presentations and can have devastating visual consequences.
Han X, Liu Y, Kam WR, Sullivan DA. Effect of brimonidine, an α2 adrenergic agonist, on human meibomian gland epithelial cells. Exp Eye Res 2018;170:20-28.Abstract
We recently discovered that the anti-glaucoma pharmaceuticals timolol, a β adrenergic antagonist, and pilocarpine, a cholinergic compound, negatively influence the morphology, proliferative capacity and survival of human meibomian gland epithelial cells (HMGECs). We hypothesize that another class of anti-glaucoma drugs, the α2 adrenergic agonists, also acts directly on HMGECs to affect their structure and function. We tested this hypothesis. Immortalized (i) HMGECs were cultured with brimonidine, as well as clonidine (α2 agonist), phenylephrine (α1 agonist), RX821002 (inverse α2 agonist) and MK912 (neutral α2 agonist) for up to 7 days. Cells were counted with a hemocytometer, and evaluated for morphology, signaling pathway activity, protein biomarker expression, and the accumulation of neutral lipids, phospholipids and lysosomes. Our findings demonstrate that brimondine treatment induces a dose-dependent decrease in Akt signaling and proliferation of iHMGECs. In contrast, brimonidine also promotes a dose-dependent differentiation of iHMGECs, including an increase in neutral lipid, phospholipid and lysosome levels. These effects were paralleled by an inhibition of p38 signaling, and duplicated by cellular exposure to clonidine, but not phenylephrine. Brimonidine also enhanced the cellular content of sterol regulatory binding protein-1, a master regulator of lipid synthesis. Of particular interest, the putative α2 antagonists, RX821002 and MK912, did not interfere with brimonidine action, but rather stimulated IHMGEC differentiation. Our results support our hypothesis and demonstrate that α2 adrenergic agonists act directly on iHMGECs. However, these compounds do not elicit an overall negative effect. Rather, the α2 agonists promote the differentiation of iHMGECs.

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