Culture of primary human meibomian gland cells from surgically excised eyelid tissue.

Meibomian gland dysfunction is one of the most common ocular diseases, with therapeutic treatment being primarily palliative due to our incomplete understanding of meibomian gland (MG) pathophysiology. To progress in vitro studies of human MG, this study describes a comprehensive protocol, with detailed troubleshooting, for the successful isolation, cultivation and cryopreservation of primary MG cells using biopsy-size segments of human eyelid tissue that would otherwise be discarded during surgery. MG acini were isolated and used to establish and propagate lipid-producing primary human MG cells. The primary cell viability during culture procedure was maintained through the application of Rho-associated coiled-coil containing protein kinase inhibitor (Y-27632, 10 µM) and collagen I from rat tails. Transcriptomic analysis of differentiated primary human MG cells confirmed cell origin and revealed high-level expression of many lipogenesis-related genes such as stearoyl-CoA desaturase (SCD), ELOVL Fatty Acid Elongase 1 (ELOVL1) and fatty acid synthase (FASN). Primary tarsal plate fibroblasts were also successfully isolated, cultured and cryopreserved. Established primary human MG cells and tarsal plate fibroblasts presented in this study have potential for applications in 3D models and bioengineered tissue that facilitate research in understanding of MG biology and pathophysiology.

Involvement of mutant and wild-type CYSLTR2 in the development and progression of uveal nevi and melanoma.

BACKGROUND: Activating Gαq signalling mutations are considered an early event in the development of uveal melanoma. Whereas most tumours harbour a mutation in GNAQ or GNA11, CYSLTR2 (encoding G-protein coupled receptor CysLT2R) forms a rare alternative. The role of wild-type CysLT2R in uveal melanoma remains unknown. METHODS: We performed a digital PCR-based molecular analysis of benign choroidal nevi and primary uveal melanomas. Publicly available bulk and single cell sequencing data were mined to further study mutant and wild-type CYSLTR2 in primary and metastatic uveal melanoma. RESULTS: 1/16 nevi and 2/120 melanomas carried the CYSLTR2 mutation. The mutation was found in a subpopulation of the nevus, while being clonal in both melanomas. In the melanomas, secondary, subclonal CYSLTR2 alterations shifted the allelic balance towards the mutant. The resulting genetic heterogeneity was confirmed in distinct areas of both tumours. At the RNA level, further silencing of wild-type and preferential expression of mutant CYSLTR2 was identified, which was also observed in two CYSLTR2 mutant primary melanomas and one metastatic lesion from other cohorts. In CYSLTR2 wild-type melanomas, high expression of CYSLTR2 correlated to tumour inflammation, but expression originated from melanoma cells specifically. CONCLUSIONS: Our findings suggest that CYSLTR2 is involved in both early and late development of uveal melanoma. Whereas the CYSLTR2 p.L129Q mutation is likely to be the initiating oncogenic event, various mechanisms further increase the mutant allele abundance during tumour progression. This makes mutant CysLT2R an attractive therapeutic target in uveal melanoma.

Semi-quantification of lipids in human meibomian gland epithelial cells using dual staining microplate assays.

Two spectrophotometric microplate assays with dual staining for either fluorescent Nile red (NR) plus 4,6-diamidino-2-phenylindole (DAPI) or non-fluorescent Oil red O (ORO) plus Crystal violet (CV) were applied and optimised to evaluate the lipid producing capacity of immortalised human meibomian gland epithelial cells (iHMGEC). Cells were treated with rosiglitazone (Rosi, 10-50 µM), a known lipid producing inducer for iHMGEC, and were analysed for lipids using the NR-DAPI and ORO-CV microplate assays. The lipid producing capacity of iHMGEC after each treatment was determined by normalising lipid quantity (measured with NR or ORO) to cell number (measured with DAPI or CV). The dye concentrations of NR 1 µg/mL, DAPI 5 µg/mL, ORO 0.3% (v/v) and CV 0.5% (v/v), provided optimal linearity and coverage of signals over a range of cell densities (corresponding to 10-100% cell confluence). Both NR-DAPI and ORO-CV showed a dose-dependent effect of Rosi on lipid production in iHMGEC, consistent with the results reported previously using traditional microscopic imaging methods. The microplate assays offer a rapid, high throughput and objective measurement of the amount of lipids in iHMGEC (and potentially other lipid-producing cells) and can be used for screening the effects of biological agents or incubation changes on lipid production in cells in future studies.

HMG-CoA reductase expression in human eyelid tissue and in a human meibomian gland epithelial cell line.

PURPOSE: 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme of cholesterol production, has been found to contribute to lipid secretion from skin sebaceous glands and hair follicles. We assessed for HMGCR expression in human eyelid tissue and in immortalized human meibomian gland epithelial cells (HMGECs) using immunohistochemistry. METHODS: Full thickness human eyelid specimens in archival paraffin blocks were obtained. A section from each block was stained with hematoxylin and eosin and examined by an ocular pathologist for validation of tissue pathology. Immunohistochemistry was performed using rabbit anti-human HMGCR antibody on serial sections using the Ventana automated staining system. HMGCR expression was examined for in HMEGCs with fluorescence immunocytochemistry and confocal microscopy. RESULTS: Thirteen full thickness eyelid specimens met the inclusion criteria. All specimens contained meibomian glands, and 2 (15%) contained glands of Zeis, 3 (23%) pilosebaceous glands, 2 (15%), accessory lacrimal glands, and 2 (15%), glands of Moll, respectively. Immunohistochemistry showed HMGCR expression in meibocytes of meibomian glands and sebocytes of Zeis and pilosebaceous glands in all specimens. HMGCR expression was also evident in vascular endothelium. Immunofluorescence was positive for HMGCR expression on HMGEC cells. No labeling was seen for the negative Ig control. CONCLUSION: HMGCR was expressed in all eyelid sebaceous-type glands and in HMGECs, consistent with a role for cholesterol production in the genesis of tear film lipids. The observed expression also provides a rationale for using topical statins, inhibitors of HMGCR, as novel tear film lipid stabilizers in conditions such as blepharitis, where meibum production is aberrant.

Human choroidal melanocytes express functional Toll-like receptors (TLRs).

Toll-like receptors (TLRs) are a class of pattern recognition receptors that sense highly conserved pathogen associated antigenic determinants, triggering an innate immune response and subsequently instructing the adaptive immune system so that together, the pathogen can be eliminated. TLRs are widely distributed in human ocular tissues and cell types, and are active players in ocular inflammation. To date, the presence and function of TLRs on human choroidal melanocytes (HCMs), the most abundant choroidal cell type, have not been characterized. The current study investigated the in vitro and in situ expression and functional status of TLRs on HCMs. HCMs were isolated and cultured from post-mortem human donor eyes, and displayed characteristic melanocyte morphology and MART1 expression - a key melanocyte lineage marker up to passage 5 (P5). In vitro experiments used P1 to P4 HCMs from different donor eyes. Initial quantitative real-time PCR (qPCR) analysis revealed that HCMs (n = 3 donors) expressed specific mRNA transcripts for TLR1-10 and MYD88 (a key adaptor protein initiating the TLR signalling pathway). HCMs were stimulated with a set of synthetic TLR specific agonists and the secretion of pro-inflammatory cytokines, MCP-1 and IL-8, at 24 h measured by ELISA (n = 3 donors). The agonists Pam3CSK4 (TLR1/2), Poly I:C (TLR3), LPS (TLR4), Flagellin (TLR5), and FLS-1 (TLR2) induced a significant increase in the production of MCP-1 and IL-8, compared to untreated cells. Application of biotinylated Pam3CSK4 provided in vitro visualization of receptor-agonist interactions for TLR1/2. We confirmed that cultured HCMs (n = 3 donors) expressed TLR1-6 protein using immunocytochemistry and confocal microscopy. The expression and distribution of TLR 1-6 was also studied in human choroid and retinal pigment epithelium (RPE) sections (n = 3 eyes) using immunofluorescence and confocal microscopy. Strong TLR1-6 immunolabelling that co-localized with melanocyte-dense areas (and RPE) was consistently observed; intraluminal and blood vessel-related cells (including endothelial cells) also expressed several TLRs. Taken together these observations show for the first time that HCMs constitutively express a range of functional TLRs, and as such can contribute to choroidal responses during infection and inflammation.

Conjunctival MUC5AC+ goblet cell index: relationship with corneal nerves and dry eye.

PURPOSE: To evaluate the relative proportion of conjunctival MUC5AC+ and MUC5AC- goblet cells in a post-LASIK population and their association with dry eye indicators and corneal nerve morphology using a MUC5AC+ Goblet Cell Index. METHODS: Twenty subjects who had undergone LASIK > 12 months previously and 20 age-matched controls were recruited. Dry eye symptoms, tear breakup time, osmolarity, meniscus area and corneal nerve morphology were examined. Conjunctival impression cytology samples were collected from inferior-temporal bulbar conjunctiva using Millicell® inserts. Total goblet cell density was determined from positive cytokeratin-7 (CK7) immunolabelling; MUC5AC+ goblet cell density was determined from both CK7+- and MUC5AC+-immunolabelled cells. The ratio of MUC5AC+ to total density was defined as the "MUC5AC+ Goblet Cell Index". Differences in variables between groups and the associations between goblet cell variables and clinical assessments were examined. RESULTS: No significant differences in the total and MUC5AC+ goblet cell density and tear film parameters were found between groups, although greater ocular discomfort was reported in the post-LASIK group (P = 0.02). A higher MUC5AC+ Index was associated with worse/greater dry eye symptoms (ρ = 0.55, P = 0.01) and higher nerve tortuosity (ρ = 0.57, P = 0.01) in the post-LASIK group; lower nerve density and thickness was found in controls (ρ > -0.45, P < 0.05), but not associated with tear film parameters. CONCLUSIONS: The MUC5AC+ Goblet Cell Index provides an indicator of mucin secretion for assessing the goblet cell function in dry eye. In the post-LASIK participants, we found an increased MUC5AC+ Index associated with worse dry eye symptoms and adverse changes in corneal nerve morphology.

Investigation of corneal endothelial changes post selective laser trabeculoplasty.

IMPORTANCE: Transient corneal endothelial changes are routinely noted on slit-lamp examination immediately following selective laser trabeculoplasty (SLT). BACKGROUND: To determine the mechanism of transient corneal endothelial changes observed following SLT. DESIGN: University laboratory-based observational study. SAMPLES: Ten corneas from six human cadaveric donors. METHODS: Corneas were treated with SLT, direct laser or peroxide, or used as controls. Haematoxylin and eosin staining and immunolabelling for zonula occludens-1 (ZO-1) and beta-catenin were performed. MAIN OUTCOME MEASURES: Histological appearance; ZO-1 and beta-catenin immunostaining. RESULTS: There were no differences in histological features between SLT-treated and control corneas. Corneas treated with SLT or peroxide showed reduced and less regular ZO-1 immunofluorescence along cell membranes compared with ZO-1 expression in controls. These changes were generalized across the endothelium. There was no effect on the ZO-1 immunostaining after direct laser. There was no difference in beta-catenin immunostaining patterns between control, SLT and peroxide-treated corneas. CONCLUSIONS AND RELEVANCE: Altered ZO-1 immunostaining may represent disassembly of tight junctions between corneal endothelial cells. The similarity of our findings between SLT-treated and peroxide-treated corneas suggests that both conditions trigger changes at the level of endothelial tight junctions, perhaps triggered by liberation of free radicals as previously proposed.

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration.

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

Proteome changes induced by laser in diabetic retinopathy.

BACKGROUND: Diabetic macular oedema (DMO) is the commonest cause of vision loss in people with diabetes. Laser photocoagulation can be effective in the treatment of DMO; however, its mechanism of action is still poorly understood. A better understanding of these mechanisms may allow the development of therapeutic approaches that could avoid the deleterious adverse events associated with photocoagulation. METHODS: We have used proteomic techniques to identify the protein changes induced by threshold intensity retinal laser treatment in a rodent model of diabetic retinopathy. Retinae were obtained from diabetic Dark Agouti rats 8 weeks following laser treatment. Extracted proteins from lasered and non-lasered diabetic retinae were separated and compared using two-dimensional gel electrophoresis. RESULTS: Image analysis revealed 24 protein spots with decreased expression after laser treatment and 9 spots with increased expression. On lasered retinal gels, four spots were uniquely expressed, with eight unique spots on non-lasered gels. Twenty-two protein spots of interest were identified using matrix-assisted desorption ionization-mass spectrometry with database matching. Following laser, Wnt-5 beta, LEK-1, GADPH, claudin-12 and calretinin were significantly down-regulated in expression. CONCLUSIONS: The present study provides a proteomic insight into the underlying biological basis for the therapeutic effects of laser for DMO. We provide further evidence of the involvement of Wnt pathway signalling in the neural retina in DMO, and for up to 2 months following laser treatment. Changes in LEK-1 and claudin-12 may have effector roles, and changes in glyceraldehyde-3-phosphate dehydrogenase and calretinin may reflect the altered retinal microenvironment resulting from laser treatment.

Basal Linear Deposit: Normal Physiological Ageing or a Defining Lesion of Age-Related Macular Degeneration?

Objective: To determine if basal linear deposit (BLinD) is a specific lesion of age-related macular degeneration (AMD). Methods: The cohort was selected from a clinically and histopathologically validated archive (Sarks Archive) and consisted of 10 normal eyes (age 55-80 years) without any macular basal laminar deposit (BLamD) (Sarks Group I) and 16 normal aged eyes (age 57-88 years) with patchy BLamD (Sarks Group II). Only eyes with in vivo fundus assessment and corresponding high-resolution transmission electron microscopy (TEM) micrographs of the macula were included. Semithin sections and fellow-eye paraffin sections were additionally examined. BLinD was defined as a diffuse layer of electron-lucent vesicles external to the retinal pigment epithelium (RPE) basement membrane by TEM and was graded as follows: (i) Grade 0, absence of a continuous layer; (ii) Grade 1, a continuous layer up to three times the thickness of the RPE basement membrane (0.9 µm); (iii) Grade 2, a continuous layer greater than 0.9 µm. Bruch's membrane (BrM) hyalinisation and RPE abnormalities were determined by light microscopic examination of corresponding semithin and paraffin sections. Results: BLinD was identified in both normal (30%) and normal aged (62.5%) eyes. BLinD was thicker in normal aged eyes (p = 0.045; 95% CI 0.04-3.4). BLinD thickness positively correlated with both the degree of BrM hyalinisation (p = 0.049; 95% CI 0.05-2.69) and increasing microscopic RPE abnormalities (p = 0.022; 95% CI 0.188-2.422). RPE abnormalities were more likely to be observed in eyes with increased BrM hyalinisation (p = 0.044; 95% CI 0.61-4.319). Conclusions: BLinD is most likely an age-related deposit rather than a specific lesion of AMD. Its accumulation is associated with increasing BrM hyalinisation and microscopic RPE abnormalities, suggesting a relationship with dysregulated RPE metabolism and/or transport.

Tear levels of SFRP1 are significantly reduced in keratoconus patients.

PURPOSE: To measure secreted frizzled-related protein 1 (SFRP1) levels in human tears and to investigate tear SFRP1 as a potential biomarker for keratoconus (KC). METHODS: Tears were collected from control (n = 33) and KC patients (n = 33) using micropipette tubes. Total tear protein was measured using a FluoroProfile Protein Quantification kit. An in-house enzyme-linked immunosorbent assay (ELISA) was developed to measure SFRP1 in control and KC tears. Statistical analyses of age, gender, the association of SFRP1, and total tear protein with KC were conducted. RESULTS: Tear SFRP1 was significantly decreased in KC, compared to age-matched controls (3.41 ng/µl ± 3.12 versus 5.55 ng/µl ± 5.62, respectively; p = 0.039). Conversely, total tear protein was significantly increased in KC, compared to age-matched controls (12.38 µg/µl ± 4.76 versus 9.40 µg/µl ± 3.88, respectively; p = 0.038). The ratio of SFRP1/total tear protein was also found to be significantly decreased in the KC group (p = 0.007). No significant association between tear SFRP1 and total tear protein was detected. CONCLUSIONS: Tear SFRP1 was significantly decreased in age-matched KC versus control patients, and may be further reduced in moderate KC. Tear-SFRP1 levels alone do not provide an obvious biomarker for KC; however, our results provide further evidence that tear-protein profiles are altered in KC, and suggest the involvement of SFRPs in the pathogenesis of KC.

Corneal epithelial dendritic cells, tear neuropeptides and corneal nerves continue to be affected more than 12 months after LASIK.

PURPOSE: LASIK causes corneal nerve damage and may affect the neuro-immune crosstalk. This study examined the effects of LASIK on corneal epithelial dendritic cells (CEDC) density and morphology and explored their relationships with corneal nerves and tear neuropeptides. A grading system was developed to assess CEDC morphology. METHODS: Intra- and inter-observer repeatability of the CEDC morphology grading system was established using kappa (κ). In vivo confocal microscope images of the central cornea were captured from 20 participants who had undergone LASIK 12-16 months earlier and 20 controls (age 18-32 years, 55%F). CEDC density was counted manually, and CEDC morphology was assessed using a new grading system. CEDC sub-types (contacting nerves [CEDCc] and not contacting nerves [CEDCnc]) were also assessed. Differences in CEDC density and morphology were examined using mixed models and chi-squared test. Relationships between CEDC and corneal nerve parameters and tear substance P were explored using Spearman's correlation. RESULTS: Excellent intra- and inter-observer repeatability was demonstrated for the grading system (κ = 0.82-0.97). In post-LASIK participants, CEDC density was lower compared with controls (5 [0-34] vs. 21 [7-77] cells/mm2 ; p = 0.01), and the proportion of CEDC with thick dendrites was higher (55%-73% vs. 11%-21%, p < 0.003). Higher tear substance P levels were associated with higher CEDC density (rho = 0.48, p = 0.003). Fewer nerve interconnections were observed in participants in whom CEDC had dendrites (p = 0.03). CEDC sub-types followed a similar pattern to CEDC. CONCLUSIONS: The findings suggest that CEDC may remain altered more than 12 months post-LASIK. The association with substance P suggests a role for CEDC in corneal neurogenic inflammation.

Lapatinib dysregulates HER2 signaling and impairs the viability of human uveal melanoma cells.

Uveal melanoma (UM) is the principal type of intraocular malignancy in adults. Up to 50% of UM patients develop metastatic disease with very poor survival. There are few drugs available to treat the primary or metastatic UM. This study was undertaken to evaluate the anti-cancer effect of lapatinib and corroborate the potential of HER2 inhibition in the treatment of UM. The anti-UM activity of lapatinib was assessed using cell viability, cell death and cell cycle analysis, and its anti-metastatic actions were evaluated using would healing, invasion and colony formation assays. Immunoblotting was used to substantiate the actions of lapatinib on apoptotic and HER2 signaling. The anti-UM activity of lapatinib was further evaluated in a UM xenograft mouse model. Lapatinib decreased the viability of four UM cell lines (IC50: 3.67-6.53 µM). The antiproliferative activity of lapatinib was corroborated in three primary cell lines isolated from UM patient tumors. In UM cell lines, lapatinib promoted apoptosis and cell cycle arrest, and strongly inhibited cell migration, invasion and reproductive cell growth. Lapatinib dysregulated HER2-AKT/ERK/PI3K signalling leading to the altered expression of apoptotic factors and cell cycle mediators in UM cell lines. Importantly, lapatinib suppressed tumourigenesis in mice carrying UM cell xenografts. Together the present findings are consistent with the assertion that HER2 is a viable therapeutic target in UM. Lapatinib is active in primary and metastatic UM as a clinically approved HER2 inhibitor. The activity of lapatinib in UM patients could be evaluated in future clinical trials.

The multifunctional human ocular melanocortin system.

Immune privilege in the eye involves physical barriers, immune regulation and secreted proteins that together limit the damaging effects of intraocular immune responses and inflammation. The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) normally circulates in the aqueous humour of the anterior chamber and the vitreous fluid, secreted by iris and ciliary epithelium, and retinal pigment epithelium (RPE). α-MSH plays an important role in maintaining ocular immune privilege by helping the development of suppressor immune cells and by activating regulatory T-cells. α-MSH functions by binding to and activating melanocortin receptors (MC1R to MC5R) and receptor accessory proteins (MRAPs) that work in concert with antagonists, otherwise known as the melanocortin system. As well as controlling immune responses and inflammation, a broad range of biological functions is increasingly recognised to be orchestrated by the melanocortin system within ocular tissues. This includes maintaining corneal transparency and immune privilege by limiting corneal (lymph)angiogenesis, sustaining corneal epithelial integrity, protecting corneal endothelium and potentially enhancing corneal graft survival, regulating aqueous tear secretion with implications for dry eye disease, facilitating retinal homeostasis via maintaining blood-retinal barriers, providing neuroprotection in the retina, and controlling abnormal new vessel growth in the choroid and retina. The role of melanocortin signalling in uveal melanocyte melanogenesis however remains unclear compared to its established role in skin melanogenesis. The early application of a melanocortin agonist to downregulate systemic inflammation used adrenocorticotropic hormone (ACTH)-based repository cortisone injection (RCI), but adverse side effects including hypertension, edema, and weight gain, related to increased adrenal gland corticosteroid production, impacted clinical uptake. Compared to ACTH, melanocortin peptides that target MC1R, MC3R, MC4R and/or MC5R, but not adrenal gland MC2R, induce minimal corticosteroid production with fewer adverse systemic effects. Pharmacological advances in synthesising MCR-specific targeted peptides provide further opportunities for treating ocular (and systemic) inflammatory diseases. Following from these observations and a renewed clinical and pharmacological interest in the diverse biological roles of the melanocortin system, this review highlights the physiological and disease-related involvement of this system within human eye tissues. We also review the emerging benefits and versatility of melanocortin receptor targeted peptides as non-steroidal alternatives for inflammatory eye diseases such as non-infectious uveitis and dry eye disease, and translational applications in promoting ocular homeostasis, for example, in corneal transplantation and diabetic retinopathy.

A preliminary study of changes in tear film proteins in the feline eye following nictitating membrane removal.

OBJECTIVE: To investigate the influence of nictitating membrane (third eyelid) removal on selected proteins in feline tears. ANIMAL STUDIED: Domestic short-haired cats (7-17 months; 2.6-5.2 kg) were used. PROCEDURES: Eye-flush tears were collected periodically for up to 18 weeks from both eyes of animals with nictitating membranes removed, but nictitating gland left intact, (n = 4) or with nictitating membranes intact (n = 4). Tear comparisons were based on total protein content (TPC) using micro bicinchoninic acid assay, immunoglobulin A (IgA), and matrix-metalloproteinase (MMP)-9 measurements using sandwich enzyme-linked immunosorbent assay (ELISA) and tear gelatinase activity using gelatin zymography. Expression of MMP-2 and -9 in nictitating membranes removed at baseline (week 0) and eyes collected at 18 weeks were also investigated in histological sections using immunoperoxidase for visualization. RESULTS: Nictitating membrane removal did not significantly change TPC and MMP-9 in tears within the first 4 weeks. MMP-9 was not detected by ELISA in tears from eyes without nictitating membranes from week 5 onwards. IgA (%IgA of TPC) data varied between animals. Gelatin zymography showed increased MMP-2 and -9 activity in tears from eyes without nictitating membranes at week 1 and a decrease following week 2 post-surgery. MMP-2 and -9 were immunolocalised to conjunctival goblet cells of removed nictitating membranes and to the conjunctival epithelium, respectively. After 18 weeks, the distribution of MMPs in tissue was comparable between eyes with and without nictitating membranes. CONCLUSIONS: Based on this preliminary study, nictitating membrane removal appeared to cause long-term changes in expression of tear proteins, including reduced MMP-9 expression.

Human meibomian gland epithelial cell culture models: Current progress, challenges, and future directions.

The widely used immortalised human meibomian gland epithelia cell (iHMGEC) line has made possible extensive studies of the biology and pathophysiology of meibomian glands (MG). Tissue culture protocols for iHMGEC have been revised and modified to optimise the growth conditions for cell differentiation and lipid accumulation. iHMGEC proliferate in serum-free medium but require serum or other appropriate exogenous factors to differentiate. Several supplements can enhance differentiation and neutral lipid accumulation in iHMGEC grown in serum-containing medium. In serum-free medium, rosiglitazone, a peroxisome proliferator activator receptor-γ (PPARγ) agonist, is reported to induce iHMGEC differentiation, neutral lipid accumulation and expression of key biomarkers of differentiation. iHMGEC cultured in serum-containing medium under hypoxia or with azithromycin increases DNAse 2 activity, a biomarker of terminal differentiation in sebocytes. The production of lipids with composition similar to meibum has not been observed in vitro and this remains a major challenge for iHMGEC culture. Innovative methodologies such as 3D ex vivo culture of MG and generation of MG organoids from stem cells are important for further developing a model that more closely mimics the in vivo biology of human MG and to facilitate the next generation of studies of MG disease and dry eye.

The unfolded protein response and the biology of uveal melanoma.

Uveal melanoma (UM) is a highly metastatic ocular cancer that arises from the melanocytes of the uveal tract (the choroid, ciliary body and iris). Despite a growing understanding of UM biology, effective systemic treatments are currently lacking and the cancer has an extremely poor prognosis. Therefore, identifying novel agents that act by new tumorigenic mechanisms in UM is essential to address this unmet clinical need. Endoplasmic reticulum (ER) stress occurs when misfolded proteins accumulate in the organelle, and the unfolded protein response (UPR) is the cellular mechanism that is activated so that cells may adapt to the situation. Dysregulated UPR signalling has been detected in UM tumors and has been associated with an increase in immune evasion and metastatic activity. A number of established and novel oncology drugs act in part by modulating ER stress and the UPR. The induction of protein-folding stress and the UPR could be a novel approach for the development of new therapeutics in UM. Further studies are now warranted to understand the mechanisms and consequences of UPR signalling in UM.

The multi-kinase inhibitor afatinib serves as a novel candidate for the treatment of human uveal melanoma.

PURPOSE: Uveal melanoma (UM) is the most common intraocular malignancy in adults with a poor prognosis and a high recurrence rate. Currently there is no effective treatment for UM. Multi-kinase inhibitors targeting dysregulated pro-tumorigenic signalling pathways have revolutionised anti-cancer treatment but, as yet, their efficacy in UM has not been established. Here, we identified the multi-kinase inhibitor afatinib as a highly effective agent that exerts anti-UM effects in in vitro, ex vivo and in vivo models. METHODS: We assessed the anti-cancer effects of afatinib using cell viability, cell death and cell cycle assays in in vitro and ex vivo UM models. The signaling pathways involved in the anti-UM effects of afatinib were evaluated by Western blotting. The in vivo activity of afatinib was evaluated in UM xenograft models using tumour mass measurement, PET scan, immunohistochemical staining and TUNEL assays. RESULTS: We found that afatinib reduced cell viability and activated apoptosis and cell cycle arrest in multiple established UM cell lines and in patient tumour-derived primary cell lines. Afatinib impaired cell migration and enhanced reproductive death in these UM cell models. Afatinib-induced cell death was accompanied by activation of STAT1 expression and downregulation of Bcl-xL and cyclin D1 expression, which control cell survival and cell cycle progression. Afatinib attenuated HER2-AKT/ERK/PI3K signalling in UM cell lines. Consistent with these observations, we found that afatinib suppressed tumour growth in UM xenografted mice. CONCLUSION: Our data indicate that afatinib activates UM cell death and targets the HER2-mediated cascade, which modulates STAT1-Bcl-xL/cyclin D1 signalling. Thus, targeting HER2 with agents like afatinib may be a novel therapeutic strategy to treat UM and to prevent metastasis.

Metabolism Dysregulation in Retinal Diseases and Related Therapies.

The human retina, which is part of the central nervous system, has exceptionally high energy demands that requires an efficient metabolism of glucose, lipids, and amino acids. Dysregulation of retinal metabolism disrupts local energy supply and redox balance, contributing to the pathogenesis of diverse retinal diseases, including age-related macular degeneration, diabetic retinopathy, inherited retinal degenerations, and Macular Telangiectasia. A better understanding of the contribution of dysregulated metabolism to retinal diseases may provide better therapeutic targets than we currently have.

Motilin Receptor Expression Found in the Human Main and Accessory Lacrimal Glands.

INTRODUCTION: In this study, we investigated the presence of motilin receptors (MR) in adnexal tissue including the human main lacrimal gland. METHOD: 17 adnexal human specimens comprising of 11 isolated human main lacrimal gland specimens, four full-thickness human eyelid excisions and two exenterations containing full-thickness eyelid and portions of the main lacrimal gland were immunolabelled with a rabbit polyclonal human MR antibody. RESULTS: Our results demonstrated that all main lacrimal gland specimens (13/13, 100%) were positive for MR expression with a predominance (10/13 (77%) of grade 1+ punctate distribution. Motilin receptors were not found in eccrine glands, cutaneous sebaceous glands, glands of Zeis or glands of Moll (0/6, 0%). We also confirmed MR expression in the accessory lacrimal gland tissue. CONCLUSION: In summary, we discovered the MR receptor in the lacrimal and accessory lacrimal gland - the significance of which, in the lacrimal gland, remains unclear - but motilin may play a role in the muscarinic control of aqueous tear secretion.