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Purpose: This study aimed to investigate the effects of subconjunctival injection of aflibercept, a soluble protein decoy for VEGFR-1 and VEGFR-2, on corneal angiogenesis and VEGFR-expressing CD11b + cells in a mouse model of suture-induced corneal neovascularization., Methods: Corneal neovascularization was induced in BALB/c mice by placing three sutures on the cornea. Immediately after surgery, either 200 µg aflibercept (5 µL) or an equal volume of phosphate-buffered saline (PBS) was administered into the subconjunctival space. Seven days after later, corneal new vessels were quantified through clinical examination and measurement of the CD31-stained area in corneal flat mounts. The levels of pro-angiogenic and inflammatory markers in the cornea were evaluated using RT-qPCR. The percentages of VEGFR-2 + CD11b + cells and VEGFR-3 + CD11b + cells were analyzed in the cornea, blood, and draining cervical lymph nodes (DLNs) using flow cytometry., Results: Subconjunctival injection of aflibercept significantly reduced the growth of corneal new vessels compared to subconjunctival PBS injection. The mRNA levels of Cd31, vascular growth factors (Vegfc and Angpt1), and pro-angiogenic/inflammatory markers (Tek/Tie2, Mrc1, Mrc2, and Il6) in the cornea were downregulated by subconjunctival aflibercept. Also, the percentage of VEGFR-3 + CD11b + cells in the cornea, blood, and DLNs was decreased by aflibercept, whereas that of VEGFR-2 + CD11b + cells was unaffected., Conclusion: Subconjunctival aflibercept administration inhibits inflammatory angiogenesis in the cornea and reduces the numbers of cornea-infiltrating and circulating VEGFR-3 + CD11b + cells., Competing Interests: Declarations. Ethical approval: The experimental protocol was approved from the Institutional Animal Care and Use Committee of Seoul National University Hospital Biomedical Research Institute (Seoul, Korea) and followed the guidelines stated by ARVO for ophthalmic and vision research. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose., (© 2024. The Author(s).)

Background: Benzalkonium chloride (BAK), the most commonly used preservative in anti-glaucoma eye drops, inflicts damage to the ocular surface. A novel anti-glaucoma formulation that avoids the use of BAK has been developed. The aim of this study was to evaluate the cytotoxicity of this formulation and to compare it with an ophthalmic solution containing BAK., Methods: Two different latanoprost eye drops were used: one ophthalmic solution (LSc) containing BAK 0.02% and one ophthalmic nanoemulsion (LNe) with a soft preservative (potassium sorbate 0.18%). Human epithelial conjunctival cells were incubated for 15, 30, and 60 min with either LSc or LNe. The cytotoxicity was determined by MTT assay. Cell death was measured by flow cytometry using annexin V-FITC and propidium iodide., Results: The values of cell viability and proliferation obtained from cells exposed to LNe were between 80 and 90% relative to the control group, whereas values obtained from cells exposed to LSc were around 30% at all study times (p < 0.05 at 15 and 30 min; p < 0.01 at 60 min). The percentage of viable cells decreased significantly when cells were incubated with LSc compared with cells incubated with LNe at all the study times, while the percentage of cells in late apoptosis/necrosis increased significantly in cells exposed to LSc compared to LNe., Conclusions: The new latanoprost nanoemulsion is significantly less cytotoxic on human conjunctival cells than LSc. These results suggest that the new formulation might be gentler on the eye surface than currently available BAK-preserved latanoprost solutions., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: The aim of this study is to evaluate ocular surface morphological and functional changes in patients with neurofibromatosis type 1 (NF1)., Methods: Twenty-eight patients with NF1 and 14 healthy subjects were included in this study. All participants underwent a medical history collection, a complete ophthalmological examination including slit lamp exam and assessment of best-corrected visual acuity (BCVA), corneal sensitivity, and lacrimal function (Schirmer test and fluorescein tear break-up time test). Corneal nerves' morphology and endothelial cells density were evaluated by in vivo corneal confocal microscopy (IVCM). Tear and conjunctiva epithelium samples were collected to evaluate nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) tear levels and conjunctival expression of their receptors TrkA and p75NTR., Results: Patients with NF1 showed a significant decrease of FTBUT when compared with healthy subjects (p < 0.001). Corneal sensitivity was ≤ 50 mm in 46% of NF1 patients. IVCM showed a significant increase of corneal nerve branching and of corneal endothelial cells density. No significant difference was observed between the two groups on NGF and BDNF tear levels and conjunctival expression of their receptors., Conclusion: This study demonstrated the presence of ocular surface changes in NF-1 patients including decrease of tear stability and of corneal sensitivity. Patients with NF1 also showed changes of corneal endothelial cells' density.

Purpose: This study aimed to evaluate the differences in the expressions of fractalkine in normal bulbar conjunctiva and primary pterygium tissues., Methods: The study included 48 patients who had been operated on for primary pterygium. Histopathologically, the presence of epithelial atypia, epithelial hyperplasia, goblet cell hyperplasia, epithelial lymphocytic exocytosis, stromal inflammation, mast cell count, and stromal vascularity were evaluated in the primary pterygium tissues. An immunohistochemical fractalkine stain was applied to the primary pterygium tissue samples and normal bulbar conjunctival tissue samples., Results: Primary pterygium and normal bulbar conjunctival tissue samples were histopathologically analyzed. Epithelial atypia, epithelial hyperplasia, epithelial lymphocytic exocytosis, stromal inflammation, stromal vascularity, and mast cell count were found to be significantly higher in the primary pterygium (p = 0.001, p = 0.002, p = 0.024, p = 0.007, p = 0.024, and p = 0.013, respectively). When evaluated in terms of fractalkine expression, the epithelial, vascular endothelial, and inflammatory cells were significantly higher in the primary pterygium (p ≤ 0.001, p = 0.002, p = 0.001, respectively). Moreover, compared to the normal bulbar conjunctiva, Ki-67 expression was significantly higher in the primary pterygium tissue samples., Conclusion: Fractalkine might play a key role in the etiopathogenesis of pterygium. Fractalkine may be important in developing new treatment approaches.

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.

Purpose: Mooren's ulcer (MU) is a peripheral corneal ulceration of presumed autoimmune etiology. NLRP3 inflammasome has been shown to be involved in a variety of autoimmune and auto-inflammatory diseases. However, the role of NLRP3 inflammasome in MU has not been investigated. Here, we evaluate the expression of NLRP3 inflammasome and its downstream inflammatory factors in human MU., Methods: Conjunctival biopsy specimens were obtained from seven patients with MU and six healthy donors. The removed conjunctivas were histopathologically evaluated for NLRP3 inflammasome component expression using antibodies directed against NLRP3, Caspase-1 (CASP1), and Interleukin-1β (IL-1β). Quantitative real-time PCR was used to measure the mRNA expression of NLRP3 and IL-1β, and the protein expressions of NLRP3, pro-CASP1, CASP1, and IL-1β were detected by Western blotting., Results: NLRP3 and IL-1β mRNA expression showed higher levels in the MU group than in healthy controls. Western-blot and immunofluorescence analysis also showed that basal expression of NLRP3 inflammasome components (NLRP3, CAPS1, and IL-1β) was elevated in patients with MU compared with healthy controls. Most importantly, we found that the cleavaged form of CASP1 and IL-1β was significantly increased in MU patients compared with healthy donors, which indicates that the upregulation of NLRP3 inflammasome was probably responsible for the enhanced IL-1β production in MU patients., Conclusions: This study demonstrated that the expression of the NLRP3-CASP1-IL-1β signaling pathway was markedly increased in the conjunctival lesions of patients with MU, suggesting the involvement of NLRP3 inflammasome in the onset and development of the inflammation in MU.

Purpose: To evaluate ocular surface (OS) parameters recovery in intolerant contact lens (CL) wearers after a period of discontinuation., Methods: This is a retrospective analysis of data from 87 intolerant CL wearers who had discontinued their use for an average period of 12 weeks because of associated discomfort and failure to successfully refit. Data were collected from clinical charts. Data from 50 matched healthy volunteers served as controls. Clinical tests included subjective discomfort symptoms questionnaire (Ocular Surface Disease Index, OSDI), Schirmer test, break-up time (BUT), corneal esthesiometry and corneo-conjunctival staining. Laboratory tests included scraping and imprint cytology. Tear protein analysis included dosage of total tear protein (TP), lysozyme-C (LYS-C), lactoferrin (LACTO), zinc-α2-glycoprotein (ZAG-2), IgA heavy chain bands (Ig-A), and serum albumin (ALB). Data were correlated to wear parameters., Results: All values were significantly worse in intolerant CL wearers group (p always <0.001). In particular, lower values compared to controls were found for BUT, corneal esthesiometry, goblet cell density, LYS-C, LACTO, ZAG-2, and TP. On the contrary, higher values compared to controls were found for OSDI, staining, imprint cytology, scraping cytology, ALB, IgA-heavy chain. The IgA/LYS-C ratio calculated as an index of the increased activity of the IgA-producing cell was found significantly higher in the intolerant group and in correlation with discomfort symptoms., Conclusions: Ocular surface parameters were altered in intolerant CL wearers, even after a prolonged discontinuation period. Our data suggest that OS recovery necessary to successfully refit lenses may need a discontinuation time longer than 3 months.

Purpose: To evaluate the effectiveness and safety of collagen crosslinking for leaking cystic blebs., Methods: A prospective study was conducted on subjects with cystic bleb leaks without indications for urgent surgical intervention. Collagen crosslinking with riboflavin 0.1 % and ultraviolet A irradiation for 30 min was applied to the cystic bleb surface in five patients. Subjects were followed up for at least 20 weeks (mean: 35.5 ± 11.5 weeks) to monitor the length of time to the cessation of bleb leak, as well as visual acuity, intraocular pressure, and the presence of adverse effects arising from the treatment., Results: Results showed that a single session of collagen crosslinking was effective in stopping cystic bleb leak. In two of the five subjects, the bleb leak subsided 1 week post-treatment, in one patient after 2 weeks, and in the two more complicated cases, after 4 weeks. This effect was maintained for a mean period of 33.5 ± 10.2 weeks after bleb leak cessation. Treatment was effective even in patients with friable conjunctiva following multiple surgical interventions and severe leak, although a longer period was needed for resolution of the bleb leak. The intraocular pressure and visual acuity were largely stable before and after treatment. No side effects or complications arose from this treatment., Conclusions: We recommend a trial of collagen crosslinking on leaking cystic blebs without indications for immediate surgical interventions. It is a relatively easy, non-invasive, pain-free, and potential repeatable procedure for treating leaking cystic blebs, with the aim of strengthening the underlying pathological conjunctiva.

Purpose: Increased levels of oxidative stress have been seen in animal models of dry eye and in the conjunctival epithelial cells of patients with Sjögren's syndrome. The aims of this study were to compare the levels of oxidative stress in patients with dry eye and patients without dry eye and to evaluate the effects of treatment with preservative-free eye drops containing hyaluronic acid 0.15 % and vitamin B12 on oxidative stress and dry eye symptoms., Methods: Three cohorts of patients who were to undergo planned cataract surgery were enrolled: patients with dry eye randomized to either no treatment (n = 29) or treatment (n = 32) with hyaluronic acid/vitamin B12 eye drops, and patients without dry eye (n = 42). Patients were assessed by Schirmer's type I test, fluorescein clearance test (FCT), Break Up Time (BUT), and Ocular Surface Disease Index (OSDI). Lipid peroxidation, a marker of oxidative stress, was assessed by LP-CHOLOX test., Results: Compared with patients without dry eye, patients with dry eye had significantly increased levels of oxidative stress, higher OSDI and FCT scores, and significantly lower Schirmer's test and BUT scores. Treatment with eye drops containing hyaluronic acid 0.15 % and vitamin B12 was associated with significantly reduced levels of oxidative stress and OSDI and FCT scores and significantly increased Schirmer's test and BUT scores., Conclusions: These findings indicate that oxidative stress is associated with dry eye and that hyaluronic acid/vitamin B12 eye drops may attenuate oxidative stress and inflammation, improving dry eye symptoms. Further study in controlled clinical trials is warranted.

Background: CCL20, the single chemokine ligand for CCR6, contributes to recruiting CCR6-expressing memory B cells, memory T cells, Th17 cells and dendritic cells, and is involved in regulating immune responses, homeostasis, and inflammation in mucosal tissues., Methods: CCL20 messenger RNA (mRNA) expression was analyzed in the conjunctival epithelium in an in vivo study of patients with vernal keratoconjunctivitis (VKC group) and healthy volunteers (control group) using impression cytology. In vitro analysis of CCL20 mRNA was performed using cultured conjunctival epithelial cells (CECs). Real-time polymerase chain reaction was used to assess IL-8 and eotaxin-2 mRNA expression for comparison with CCL20 mRNA expression., Results: In the control group, CCL20 mRNA expression was present in all conjunctival locations. However, CCL20 mRNA expression was significantly higher in the upper palpebral conjunctiva in the severe VKC group than in the mild VKC and control groups (p < 0.05, Steel test). In vitro stimulation of CECs with lipopolysaccharide (LPS) significantly increased CCL20 expression in a concentration-dependent manner that was significantly correlated with expression of IL-8 (p < 0.001, Spearman's rank correlation coefficient), but not eotaxin-2., Conclusion: We conclude that CCL 20 mRNA expression in the conjunctival epithelium plays a crucial role in regulating homeostasis at the ocular surface and in exacerbation of VKC.

Purpose: To analyze, using in vivo laser scanning confocal microscopy (LSCM), the conjunctival features in glaucomatous patients receiving prostaglandin analogues (PGA)., Methods: Eighty eyes of 80 consecutive glaucomatous patients naive for therapy were enrolled; 30 eyes of 30 healthy subjects served as a control. Patients were randomized to: preservative-free (PF) and preserved latanoprost (groups 1 and 2, respectively), PF and preserved timolol (groups 3 and 4), and controls to vehicle of latanoprost or physiological buffered saline solution (groups 5 and 6). All subjects underwent LSCM of bulbar conjunctiva at baseline and 3 months after initiating therapy. The main outcomes were: mean density (MMD: cysts/mm(2)) and mean area (MMA: cysts/mm(2)) of epithelial microcysts. The relations between MMA and MMD with intraocular pressure (IOP), age, and mean defect (MD), were analyzed., Results: At baseline, microcysts were found in all subjects. At month three, MMD did not change in all groups (p > 0.05). MMA significantly increased only in group 1 from 2,158.81 ± 524.09 to 3,877.77 ± 867.31, and in group 2 from 2,019.71 ± 541.03 to 5,560.39 ± 1,176.14, with values significantly higher in group 2 (p < 0.001). Significant relations were not found between MMD and MMA with IOP, MD, and age (p > 0.05)., Conclusions: PGA increased MMA in therapy-naive glaucomatous patients, indicating a possible enhancement of the trans-conjunctival aqueous humor outflow. Therefore, conjunctiva seems an additional target tissue to evaluate the hydrodynamic pathways in glaucoma and modifications induced by medical therapy.

Background: To evaluate if conjunctival epithelial cells' expression of HLA-DR and ICAM-1 could be helpful as early topical markers of inflammation in Graves' orbitopathy (GO)., Methods: The ocular examination evaluated a clinical activity score (CAS) by assessment of clinical features, (e.g., eyelid or conjunctival inflammation, lid width, lid closure, proptosis, ocular motility). Conjunctival epithelial cell specimens for flow-cytometric evaluations of ICAM-I and HLADR expression were collected by impression cytology from ten eyes with active GO (CAS  ≥  4 and duration  ≤  12 months), from 15 eyes with Graves' disease (GD) without active GO (CAS 0-2) and from 15 normal specimens without any ocular disorders., Results: The percentage of HLA-DR + conjunctival epithelial cells was significantly elevated in patients with active GO comparing to GD without active GO and healthy controls, 10.7 % (8.5-17.7) and 7.78 % (3.92-10.1) (p < 0.05) vs. control 4.89 % (3.5-5.5) (p < 0.005), respectively. The expression of ICAM - 1+ conjunctival epithelial cells was greater only in patients with GO vs. controls, 5.5 % (4.8-7.03) and 1.46 % (0.69-2.51) (p < 0.005), respectively., Conclusion: The percentage of HLA-DR⁺ and ICAM-1⁺ conjunctival epithelial cells in patients with the active GO may serve as a topical inflammation marker in Graves' orbitopathy.

Purpose: The objective of this study was to determine whether cells from the conjunctiva could be reprogrammed into induced pluripotent stem (iPS) cells, providing an alternative source of stem cells., Methods: We employed a doxycycline-induced reprogrammable mouse strain to generate iPS cells from conjunctiva. The identity of the stem cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assays. Immunocytochemistry and teratoma assays are established means for scoring stem cell pluripotency. The reprogramming efficiencies of conjunctival cells and ear fibroblasts were compared., Results: We confirmed the identity of the stem cells and demonstrated expression of pluripotency markers (OCT4, SOX2, NANOG, and SSEA1), as tested by RT-PCR and immunofluorescence assays. In addition, derived iPS cells differentiated successfully into embryoid bodies, and showed teratoma formation when injected into immunodeficient mice. Reprogramming conjunctival tissue is as efficient as reprogramming ear fibroblasts. Conjunctiva-iPS exhibited classic features of embryonic stem (ES) cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, capacity to differentiate in vitro, and the ability to form all three germ layers in vivo., Conclusion: The present study demonstrated that conjunctival cells, which are readily obtained during the course of many routine conjunctival biopsies and ophthalmic procedures, can be another reliable source of iPS cells.

Background: Previously we have shown that acute exposure to thimerosal (Thi) can induce oxidative stress and DNA damage in a human conjunctival cell line. However, the long-term effect of Thi on Chang conjunctival cells is not clear. Therefore, the aim of this study was to further investigate the fate of the cells after acute exposure to Thi., Method: Cells were first exposed to various concentrations of Thi (0.00001 % ∼ 0.001 %) for 30 min, and then cells were assessed after a 24-h recovery period. Morphologic changes were observed under a light microscope and cell viability was evaluated. Cell apoptosis, cell cycle distribution and mitochondrial membrane potential (MMP) (rhodamine 123 assay) were detected by flow cytometry analysis. Poly (ADP-ribose) polymerase (PARP), activation of caspase-3 and microtubule-associated protein light chain 3 (LC-3) were examined by western blot analysis., Results: DNA strand breaks were significantly increased in a dose-dependent manner with 30 min exposure to Thi, although no significant cell death was detected. However, after 24-h recovery, the ratio of apoptotic cells was significantly increased to 0.0005 % and 0.001 % in Thi treated groups (p < 0.001 compared to the control group). Apoptosis was confirmed by the cleavage of PARP and caspase-3 activation. In addition, G2/M cell cycle arrest and decrease of MMP were recorded. Finally, the LC-3 results indicated the occurrence of autophagy in Thi-treated cells., Conclusion: Acute exposure to Thi can induce DNA damage, and eventually can lead to cell death, probably through the caspase-dependent apoptosis pathway, while autophagy might also be involved.

Background: To analyze the effects of supplemental epidermal growth factor (EGF) and the roles of inflammatory cytokines (interleukin [IL]-6) in an ex vivo dry-eye model under hyperosmotic stress using a multilayered culture of human conjunctival epithelial cells (HCECs)., Methods: Multilayered cultures of HCECs were exposed to hyperosmotic stress (400 mOsm/L) for 24 h in addition to 0.5 ng/mL EGF (low-EGF group) or 25 ng/mL EGF (high-EGF group). Apoptosis was analyzed using the TUNEL assay. Cell proliferation was measured using the [3H]-thymidine incorporation assay. The expression of IL-6, EGF, EGF receptor (EGFR), and phosphorylated extracellular signal-regulated kinase (p-ERK) was measured by western blot analysis. The secretion of IL-6 was measured using ELISA. Western blot analysis was also performed using antibodies against cleaved caspase-3., Results: The percentage of apoptotic cells was lower in the high-EGF group (6.7%) than in the low-EGF group (10.3%). The high-EGF group demonstrated increased proliferation (323.7 counts/min in the low-EGF group vs 649.1 counts/min in the high-EGF group). EGF induced higher phosphor-EGFR expression and upregulated p-ERK in HCECs. In addition, EGF significantly decreased the secretion of IL-6 and cleaved caspase-3 in HCECs., Conclusions: The level of IL-6 was increased in the ex vivo HCEC dry-eye model that was under hyperosmotic stress. Supplemental EGF reduces the level of IL-6, decreases apoptosis, and increases proliferation. These findings indicate that EGF has potential as a therapeutic agent for the treatment of dry eyes.

Background: Many contact lens wearers suffer from dry eye syndrome. Previous studies show significant changes in the protein composition of the tears depending on the lens-cleaning solution used. Therefore, the aim of this study was to detect the influence of different lens solutions on conjunctival cells, cells also involved in tear film composition., Methods: Conjunctival epithelium cells (HCjE; IOBA-NHC) were exposed to medium containing Complete® Multipurpose Solution Easy Rub® Formula (Complete) (AMO) or Opti-Free® Express Multipurpose Disinfecting Solution (Opti-Free) (Alcon) in different concentrations (0.1, 0.5, 1, 2.5 %) for 12 and 24 h. Apoptosis and necrosis using FACS and protein profiles of the cells using SELDI-TOF-MS and MALDI-TOF-MS were measured. Multivariate statistics were calculated to detect the most significant changes., Results: Complex protein profiles were measured with SELDI-TOF-MS and MALDI-TOF-MS. Significant differences of protein profiles between control and treatment cells were detected after 12 and 24 h, although cells incubated with Complete showed significantly fewer changes than cells incubated with Opti-Free, also showing concentration-dependent changes of some significantly changed proteins, e.g., protein at 6,736 Da (p > 0.002). Cells incubated with Complete showed significantly less apoptosis or necrosis in comparison to control cells (p < 0.05), whereas cells incubated with Opti-Free showed significantly more (p < 0.05)., Conclusions: We were able to demonstrate that Complete shows very little effect on the protein profiles of conjunctival cells in comparison to Opti-Free, where the cells showed very large protein profile changes, and apoptosis and necrosis of the cells was increased. These results are in concordance to clinical studies showing that the use of Complete solution made the tear film proteins similar to those of people not wearing contact lenses. Therefore, we believe that Complete is less aggressive and should provoke fewer side-effects, such as dry eye syndrome, for the users.

Background: This study was conducted to evaluate whether polyunsaturated fatty acids (PUFA) such as γ-linolenic acid (GLA) and eicosapentaenoic acid (EPA), as found in the diet, may affect the lipid composition of conjunctival epithelium and whether these modifications affect prostaglandin (PG) production after inflammatory stimulation., Methods: Chang and IOBA-NHC conjunctival human cells were treated with GLA and/or EPA at 5, 10, 20, 30, 40, or 50 μg/ml for 72 h and then were stimulated with interferon-gamma (IFN-γ) for 48 h. Changes in the composition of neutral lipids and phospholipids were monitored by gas chromatography. PGE1 and PGE2 levels were measured by enzyme immunoassay., Results: PUFA supplementations in the culture medium induced incorporation of these fatty acids and of their metabolites in neutral lipids and phospholipids of the conjunctival cells. The fatty acid composition of neutral lipids and phospholipids was not affected by stimulation with IFN-γ. The production of PGE1 and PGE2 was affected by GLA supplementation whereas it was not modified by EPA supplementation. A combined supplementation of EPA and GLA did not change the production of PGE1 but decreased the production of PGE2., Conclusions: These results suggest that modulation of fatty acid composition and PG production by PUFA supplementation is possible in the conjunctival epithelium, which is an important site of inflammation in dry eye syndrome.

Purpose: Epidemiological studies suggest that dietary n-3 polyunsaturated fatty acids (PUFAs) may protect against dry eye. This study aimed to evaluate whether a dietary deficiency in n-3 PUFAs may increase the severity of the pathology in a scopolamine-induced model of dry eye in the rat., Methods: Lewis rats of three consecutive generations were bred under a balanced diet or a diet deprived of n-3 PUFAs. Dry eye was experimentally induced by continuous scopolamine delivery in female animals from the third generation of both groups. After 10 days of treatment, the clinical signs of ocular dryness were evaluated in vivo using fluorescein staining. MHC II and the rat mucin rMuc5AC were immunostained on ocular sphere cryosections. The transcript levels of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were quantified in the exorbital lacrimal glands (LG) and in the conjunctiva using reverse transcription followed by polymerase chain reaction. Lipids were extracted from the exorbital LG for fatty acid analysis of the phospholipids using gas chromatography., Results: When compared to control animals, the scopolamine treatment induced an increase in the cornea fluorescein staining score (from 0.5 ± 0.0 to 2.5 ± 1.0 arbitrary units (AU) for the balanced diet and from 1.2 ± 0.8 to 2.6 ± 0.5 AU for the n-3 PUFA-deficient diet); a decrease in rMuc5AC immunostaining in the conjunctival epithelium (-34% for the balanced diet and -23% for the n-3 PUFA-deficient diet); an increase in the LG transcript levels of TNF-α for the balanced diet and of TNF-α and IFN-γ for the deficient diet; an increase in the conjunctival transcript levels of IL-1β and IL-6 for the deficient diet; an increase in arachidonic acid (AA) and in the ∆5-desaturase index (ratio of AA to dihomo-gamma-linolenic acid) in the exorbital LG for both diets. When compared to the balanced diet, the n-3 PUFA-deficient diet induced an increase in the LG transcript levels of IL-6 for the control animals and of TNF-α for the control and dry eye animals as well as an increase in the conjunctival transcript levels of IL-6 for the dry eye animals. There was no significant diet difference in fluorescein staining, rMuc5AC, and MHC II immunostaining scores., Conclusions: Our data suggest that an n-3 PUFA deficiency does not increase the severity of dry eye in a rat model of dry eye.

Background: This study was conducted to evaluate the efficacy of dietary n-6 and n-3 polyunsaturated fatty acids (PUFAs) in dry eye in a rat model., Methods: Female Lewis rats were fed with diets containing (1) gamma-linolenic acid (GLA), (2) eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA), or (3) GLA + EPA + DHA, for 2 months before the induction of dry eye using a continuous delivery of scopolamine and during scopolamine treatment. Two, 10 and 28 days after dry-eye induction, clinical signs of corneal dryness were evaluated in vivo using fluorescein staining. MHC II expression and mucin rMuc5AC production in the conjunctival epithelium were evaluated by immunostaining. Lipids and prostaglandins (PGs) E(1) and E(2) were analysed from the exorbital lacrimal gland (LG)., Results: Dietary PUFAs minimised the occurrence of corneal keratitis 28 days after induction of dry eye. The decrease in mucin production observed on the conjunctival epithelium was partially prevented by EPA + DHA supplementation after 2 days of scopolamine treatment, as well as by GLA and GLA + EPA + DHA diets after 10 days of treatment. The overexpression of MHC II in the conjunctival epithelium caused by dry eye induction was significantly reduced only with the GLA + EPA + DHA diet after 28 days of treatment. Dietary PUFAs were incorporated into phospholipids of the exorbital LG. Induction of dry eye was associated with a significant increase in PGE(1) and PGE(2) levels in the exorbital LG, which was inhibited by dietary EPA + DHA at 10 days (for PGE(2)) and 28 days (for PGE(1))., Conclusions: Dietary GLA, EPA and DHA significantly interfered with lipid homeostasis in the exorbital LG and partially prevented the course of dry eye. In particular, our results demonstrate the efficacy of the combination of n-6 and n-3 PUFAs.

Background: At the time of posterior subtenon infusion of triamcinolone acetonide (STI-TA) in patients with diabetic macular edema (DME), drug reflux of TA has sometimes been observed from the conjunctival incision site. We investigated the influence of this reflux on regression of DME and postoperative intraocular pressure (IOP)., Methods: STI-TA was performed on one hundred and twenty-four eyes of 88 consecutive patients with DME. Eligible eyes were divided into two groups: those with observed drug reflux of TA and those without observed drug reflux of TA. Visual acuity (VA), foveal thickness (FT) and IOP were monitored in each eye for up to 12 weeks after STI-TA., Results: STI-TA with drug reflux was observed in ten individual eyes of seven patients. These patients were significantly younger than those patients without observed drug reflux. After STI-TA, both improvement of VA and regression of FT in reflux(+) eyes were less than in reflux(-) eyes. Postoperative IOP elevation in reflux(+) eyes was much higher, and four of the ten eyes needed anti-glaucoma therapy. This was in contrast to three of the 118 eyes without drug reflux that required anti-glaucoma therapy., Conclusions: At the time of STI-TA in DME, drug reflux of TA is a risk factor not only for insufficient reduction of edema, but also for postoperative IOP elevation.

Introduction: The toxicity of conventional formulations of amphotericin B deoxycholate (AmB(DOC)) limits its clinical applications. To reduce the toxicity of AmB(DOC), lipid formulations of amphotericin B (AmB) have been developed and clinically applied. In the present study, we evaluated the ocular toxicity and pharmacokinetics of subconjunctival injection of liposomal AmB., Materials and Methods: Subconjunctival injection of either AmB(DOC) (containing 1.5 mg of AmB and 1.2 mg of deoxycholate), deoxycholate (1.2 mg), or liposomal amphotericin B (L-AmB) (containing 1.5 mg of AmB and lipids) was given to white New Zealand rabbits. After 24 hours, toxicities of the drugs were evaluated by slit lamp and histologically. To evaluate the pharmacokinetics of subconjunctival injection of L-AmB, the concentration of the drug in the cornea was evaluated at 4, 12, 24, and 48 hours after subconjunctival injection of L-AmB, with or without corneal epithelial removal., Results: Subconjunctival injection of AmB(DOC) or deoxycholate alone induced severe corneal and conjunctival edema, with necrosis and infiltration of inflammatory cells. In contrast, subconjunctival injection of L-AmB induced only mild inflammation near the injection site. The concentration of AmB injected in eyes with intact corneal epithelium was 4.93-2.49, 0.63-0.31, 0.15-0.07 microg/g at 4, 12, and 24 hours respectively after the injection of L-AmB. When injected in eyes after corneal epithelial removal, the concentration of AmB was 19.7-9.87, 2.49-1.25, and 1.25-0.63 microg/g at 4, 12, and 24 hours after injection respectively, Conclusions: Subconjunctival injection of L-AmB has reduced ocular toxicities and gives satisfactory concentrations in corneal stroma compared with conventional AmB(DOC). Subconjunctival injection of L-AmB will be a choice of treatment for mycotic keratitis.

Background: Probiotics have been shown to improve allergic inflammation. The aim of this study was to evaluate the efficacy of Lactobacillus Acidophilus eye-drops in controlling signs and symptoms of vernal keratoconjunctivitis (VKC)., Methods: Seven patients (mean age 11.8 +/- 4.3; five M, two F) with mild to moderate VKC were included in the study. Lactobacillus Acidophilus diluted in saline solution (2 x 10(8) CFU/ml) was administrated as eye-drops four times daily for 4 weeks in both eyes. Clinical signs (conjunctival hyperemia, chemosis, secretion, Trantas dots, superficial punctuate keratitis) and symptoms (itching, photophobia, burning, tearing) were evaluated and scored from 0 to 3 at baseline, after 2 and 4 weeks of treatment. Total sign (TSS) and symptom (TSyS) scores were calculated. Conjunctival impression cytology was performed in three patients at baseline and after 4 weeks of treatment, in order to evaluate the expression of ICAM-1 and TLR-4., Results: In the six out of seven patients who completed the study, symptoms were significantly improved after both 2 weeks (TSyS: baseline 6.7 +/- 0.9 vs 4.1 +/- 1.2; p = 0.017) and 4 weeks (TSyS: baseline 6.7 +/- 0.9 vs 3.6 +/- 1.2, p = 0.011) of treatment. A significant improvement of clinical signs was observed after 4 weeks of treatment (TSS: baseline 7.5 +/- 1.6 vs 3.9 +/- 1.7, p = 0.034) but not after 2 weeks of treatment (TSS: baseline 7.5 +/- 1.6 vs 5.3 +/- 1.5; NS). In particular, photophobia was significantly reduced (2 +/- 0.6 vs 1 +/- 0.3; p = 0.023) at 2 weeks, while at 4 weeks the scores for itching (1.8 +/- 0.3 vs 1 +/- 0.3), tearing (1.6 +/- 0.4 vs 0.8 +/- 0.2), conjunctival hyperemia (2.3 +/- 0.2 vs 1.4 +/- 0.5) and chemosis (1.2 +/- 0.4 vs 0.4 +/- 0.4) were significantly lower compared to baseline. A down-regulation of ICAM-1 and TLR-4 was observed in two patients showing clinical improvement after 4 weeks of treatment., Conclusion: Our open pilot study showed that 1-month treatment with probiotic eye-drops improves signs and symptoms in patients with VKC. Additional double-blind controlled clinical trials with a larger sample of patients are needed to confirm the effects of topical Lactobacilli on VKC patients.

Background: Inflammation can cause a local increase in temperature. Digital infrared thermal imaging (DITI) has been used to monitor the temperature distribution of human skin. Graves' ophthalmopathy (GO) is an autoimmune disease, and patients are treated with immunosuppressive agents if the ophthalmopathy is at an inflammatory state. The aim of the present study was to elucidate the feasibility of DITI in determining inflammatory state and follow-up effect of methylprednisolone pulse therapy in patients with GO., Methods: Digital infrared thermal imaging was used to measure local temperatures of lateral orbit (reference point), upper eyelid, caruncle, medial conjunctiva, lateral conjunctiva, lower eyelid, and cornea, and to make thermal density plots in 14 patients (28 eyes) with GO including inflammatory signs (mean clinical activity score 2.5), and 16 normal controls (32 eyes). We also performed methylprednisolone pulse therapy in 11 patients (22 eyes) with active GO, and measured clinical activity score and local temperatures before and after treatment. The focal change in temperature after treatment and the correlation between temperature variation and change in clinical activity score were then analyzed. Thermal density plots were also compared., Results: Local temperatures of the caruncle, medial conjunctiva, lateral conjunctiva, and lower eyelid of the patients were significantly higher than those of normal controls. In the 11 GO patients treated with methylprednisolone pulse therapy, the temperatures of the caruncle, medial conjunctiva, and lower eyelid were significantly decreased after treatment. Temperature variation significantly and positively correlated with a change in clinical activity score (correlation coefficient = 0.8, n = 22, p = 0.000). The temperature decreased after treatment in patients who were responsive to methylprednisolone pulse therapy, and the thermal density plot was close to that of normal controls., Conclusions: Digital infrared thermal imaging might be helpful in evaluating the inflammatory state of GO and the follow-up effect of methylprednisolone pulse therapy.

Background: Our study aims to identify anatomical characteristics of glaucoma filtering blebs by means of slit lamp-adapted optical coherence tomography (SL-OCT) and to identify new parameters for the functional prognosis of the filter in the early post-operative period., Methods: Patients with primary open-angle glaucoma, aged 18 years and older, scheduled for primary trabeculectomy at the Department of Ophthalmology, Radboud University Nijmegen Medical Centre, were considered for our study. All patients underwent standardized trabeculectomy with intra-operative application of mitomycin C. The filtering blebs were evaluated clinically and with SL-OCT on day 1 and 1, 2, 4 and 12 weeks following surgery. The resulting data were analysed and weighed against surgical success. To better understand the SL-OCT data a small comparative histologic study was performed., Results: The study included 20 eyes of 20 patients. After completion of our study, 15 eyes of 15 patients (mean age+/-SD 67 +/- 16 years) were eligible for data analysis and 5 eyes missed at least one follow-up visit. Filtering surgery was considered successful (intraocular pressure < or = 21 mmHg without antiglaucomatous medication) in 11 of 15 eyes. SL-OCT frequently demonstrated multiple hypo-reflective layers within Tenon's capsule ("striping" phenomenon) in the first post-operative week. Presumably, these layers corresponded with drainage channels in the histological specimen. These channels were present in functional filters but not in the failures. In addition, the visualisation of the sclera below the filtering zone was better defined in failures compared with successful filtering blebs ("shading" phenomenon). We observed no differences in the volume and clinical aspect of the blebs in the successful group compared with the unsuccessful group., Conclusions: Successful filtering blebs show characteristic optical properties on SL-OCT. These phenomena suggest a diffusely enhanced fluid content and the presence of intra-bleb drainage channels in functional filtering blebs.

Sex steroid hormones are essential for a healthy ocular surface and the androgen receptor impairment found in patients with complete androgen insensitivity syndrome (CAIS) has been described to cause meibomian gland dysfunction and functional dry eye for lipid tear film layer instability. However, it has not been reported if the mucous layer is also affected. A 37-year-old CAIS patient with persistent symptoms of dry eye underwent ophthalmological examination and was evaluated for qualitative and quantitative tear function tests and conjunctival cytology. Samples obtained from the conjunctival epithelium were stained for histology and immunohistochemistry and compared with three age-matched female controls. Western blot and relative real-time RT-PCR for MUC1 and MUC5AC were also performed on these samples. Immunohistochemistry, Western blot and relative real-time RT-PCR showed a decrease in the expression of MUC1 and MUC5AC in CAIS. Changes in the tear film mucous layer were accompanied by a reduction in the tear film break up time test. This is the first report describing mucous layer alteration associated with androgen receptor impairment. Decreased mucin levels contribute in explaining the tear film instability in CAIS and should be considered an additional cause of dry eye in sex steroid hormone pathology.

Background: The purpose of the study was to compare toxic effects and responses to histamine and IFN gamma associated with the use of some widely used anti-allergic eye drops commercially available today., Methods: For dynamic studies, the Wong-Kilbourne cell line was stimulated for 24 h with histamine or IFN gamma in the presence or absence of anti-allergic eye drops. Supernatants of histamine-stimulated cells were evaluated for the production of IL-6 and IL-8 by ELISA, while the expression of ICAM-1 was evaluated by flow cytometry on IFN gamma-stimulated cells. Toxicological assays were performed using cold light cytofluorometry: viability and apoptosis as well as reactive oxygen species (ROS) and O2(.)- production were assessed using neutral red, Hoechst/propidium iodide, H(2)-DCFDA and hydroethidine tests, respectively., Results: Antihistamines reduced IL-6 release and presented dose-dependent inhibitory effects on IL-8 production. None of the eye drops decreased the basal or IFN gamma-stimulated expression of ICAM-1. Conversely, eye drops preserved with benzalkonium chloride (BAC) induced even higher ICAM-1 expression levels on IFN gamma-stimulated cells than did IFN gamma alone, whereas unpreserved drugs had no effect. Toxicological assays confirmed the pivotal role of BAC in proportionally reducing cell viability while increasing apoptosis and oxidative stress., Conclusions: The ability of topical ocular anti-H(1) drugs to significantly reduce the production of IL-6 and IL-8 argues that they may help treat the inflammatory processes occurring in allergic ocular surface disorders. Nevertheless, preserved ophthalmic formulations may enhance epithelial conjunctival expression of ICAM-1 in the presence of a low inflammatory stimulus, such as IFN gamma, and displayed toxic as well as pro-oxidative effects on these cells. Therefore, BAC used as preservative might in part interfere with the potential anti-inflammatory properties of the active compound by modulating the immuno-inflammatory response of epithelial conjunctival cells.

Background: We wished to investigate the toxicity of four immunosuppressant and antimetabolic drugs, which are known to influence postoperative wound healing, on three different human ocular cell lines., Methods: Acute toxicity to cyclosporin A, azathioprine, mitomicyn C and daunorubicin was assessed in Chang cells by monitoring their uptake of propidium iodide during a 3-h period. Chronic toxicity was assessed by monitoring the proliferation and viability of subconfluent cultures of Chang cells, human corneal endothelial cells (HCECs) and retinal pigmented epithelial (RPE) cells after continuous exposure to the drugs for 7 days., Results: Acute toxicity testing revealed no obvious effects. However, the chronic toxicity tests disclosed a narrow concentration range over which cell proliferation decreased dramatically but calcein metabolism was sustained. Although the three lines reacted similarly to each agent, HCECs were the most vulnerable to daunorubicin and mitomycin. At a daunorubicin concentration of 0.05 microg/ml, a 75% decrease in calcein metabolism (P < 0.001) and a > or = 95% cell loss (P < 0.001) were observed. At a mitomycin concentration of 0.01 mug/ml, cell density decreased by 61% (P < 0.001) without a change in calcein metabolism, but at 0.1 microg/ml, the latter parameter decreased to 12% (P = 0.00014). At this concentration the proliferation of Chang and RPE cells decreased by more than 50%, whilst calcein metabolism was largely sustained. Cyclosporin inhibited cell proliferation moderately at lower concentrations (< 5 microg/ml; P=0.05) and substantially at higher ones, with a corresponding decline in calcein metabolism. Azathioprine induced a profound decrease in both parameters at concentrations above 5 microg/ml., Conclusion: Daunorubicin, cyclosporin and azathioprine could be used to inhibit excessive intraocular scarring after glaucoma and vitreoretinal surgery without overly reducing cell viability. The attributes of immunosuppressants lie in their combined antiproliferative and immunomodulatory effects.

Background: To determine the expression of bactericidal/permeability-increasing protein (BPI), a novel antimicrobial molecule, in the main lacrimal gland and its content in tears of young healthy subjects., Methods: BPI concentration of tears was measured in 42 healthy volunteers, 13 men and 29 women, with ages ranging from 22 to 30 (mean 24.7+/-2.1) years by a time-resolved fluoroimmunoassay (TR-FIA). Immunohistochemical analysis was made to localize BPI in lacrimal gland and conjunctiva of eight autopsied subjects, two men and six women, with the age range from 44 to 87 (mean 72.3+/-14.9) years., Result: The mean concentration of BPI in tears was 27.8+/-29.5 microg/l, and it decreased with an increase in tear flow rate (P<0.0001). There was no statistically significant difference in BPI content of tears between the genders. BPI was immunohistochemically seen in outer basal epithelial cells of intralobular and excretory ducts, squamous and basal cells of conjunctiva as well as faintly in myoepithelial cell layer of acini. The presence of BPI in the lacrimal gland and in the tear fluid was verified by Western blotting., Conclusions: The results indicate that outer basal epithelial cells of lacrimal gland ducts contain BPI, which occurs in a relatively high concentration in tears. BPI may have a substantial antibacterial role in human tears.

Purpose: To evaluate the potential of the vitamin E compound alpha-tocotrienol as antifibrotic agent in vitro., Methods: Using human Tenon's capsule fibroblast cultures, the antiproliferative and cytotoxic effects of the different vitamin E forms alpha-tocopherol, alpha-tocopheryl acetate, alpha-tocopheryl succinate and alpha-tocotrienol were compared with those of mitomycin C. To mimic subconjunctival and regular oral application in vivo, exposure time of serum-stimulated and serum-restimulated fibroblasts (SF and RF, respectively) to vitamin E forms was set at 6 days. Cultures were only exposed for 5 min to mitomycin C due to its known acute toxicity and to mimic the short-time intraoperative administration. Proliferation (expressed as % of control) was determined by DNA content quantification on days 2, 4 and 6, whereas cytotoxicity was assessed by cell morphology and glucose 6-phosphate dehydrogenase (G6PD) release after 24 h., Results: alpha-Tocopherol and alpha-tocopheryl acetate stimulated growth of SF, but not RF. Reduction of fibroblast content by alpha-tocopheryl succinate was accompanied by increased G6PD release and necrosis. Contrary to alpha-tocopheryl succinate, 50 microM or repeatedly 20 microM of alpha-tocotrienol significantly inhibited proliferation without causing cellular toxicity (maximal effect: 46.8%). RF were more sensitive to this effect than SF. Mitomycin C 100-400 microg/ml showed a stronger antiproliferative effect than alpha-tocotrienol (maximal effect: 13.8%). Morphologic characteristics of apoptosis were more commonly found under treatment with mitomycin C., Conclusions: Of the vitamin E forms tested, only alpha-tocotrienol significantly inhibited growth at non-toxic concentrations. In this in vitro study, antiproliferative effects of mitomycin C were stronger than those of alpha-tocotrienol.

Maf encodes a transcription factor protein containing a typical basic leucine zipper domain structure, a motif for protein dimerization and DNA binding. We examined the expression of maf-B mRNA in the epithelium around the eyelid closure. Expression of maf-B mRNA was examined in C57Bl6 mice at the embryonic stages in 12.5 days of gestation (E12.5) and E18 using in situ hybridization with 35S-labeled antisense riboprobes. In embryos studied 12.5 days postconception, a message specific for maf-B was not detected around the developing eyelid. In contrast, maf-B was strongly expressed in the epithelium of the eyelid closure at E18. Expression of maf-B was strongly noted in the suprabasal differentiating cells derived from the basal layer of the conjunctiva and epidermis. In contrast, basal cells in the eyelid closure and in the epidermis, as well as keratinizing cells, did not express maf-B. These data indicate that maf-B mRNA is expressed during development of the eyelid closure.

Introduction: Components of the mucosal immune system (MALT) have been identified in the conjunctiva (as CALT) and the lacrimal drainage system (as LDALT). Their structural and functional relation with the established immune protection by the lacrimal gland is unclear., Material and Methods: Macroscopically normal and complete tissues of the conjunctiva, lacrimal drainage system and lacrimal gland from human body donors were investigated by analysis of translucent whole mounts, and using histology, immunohistology as well as scanning and transmission electron microscopy., Results: A typical diffuse lymphoid tissue, composed of effector cells of the immune system (T-lymphocytes and IgA producing plasma cells) under an epithelium that contains the IgA transporter SC, is not isolated in the conjunctiva and lacrimal drainage system. It is anatomically continuous from the lacrimal gland along its excretory ducts into the conjunctiva and from there via the lacrimal canaliculi into the lacrimal drainage system. Lymphoid follicles occur in a majority (about 60%) and with bilateral symmetry. The topography of CALT corresponds to the position of the cornea in the closed eye., Conclusion: These results show that the MALT of the lacrimal gland, conjunctiva and lacrimal drainage system constitute an anatomical and functional unit for immune protection of the ocular surface. Therefore it should be integrated as an "eye-associated lymphoid tissue" (EALT) into the MALT system of the body. EALT can detect ocular surface antigens by the lymphoid follicles and can supply other organs and the ocular surface including the lacrimal gland with specific effector cells via the regulated recirculation of lymphoid cells.

Background: We examined the effects of interferon-gamma (IFN-gamma) on protein production of extracellular matrix (ECM) components in cultured human subconjunctival fibroblasts or those stimulated by exogenous transforming growth factor beta1 (TGFbeta1). IFN-gamma reportedly up-regulates Smad7, an inhibitory mediator of TGFbeta-Smad signaling, and blocks TGFbeta effects., Methods: Proliferation and migration as well as the ultrastructure of these cells were examined in the presence and absence of IFN-gamma. Cell migration was examined using an in vitro wound healing model in monolayer fibroblast cultures., Results: The results showed that IFN-gamma reduced ECM production in normal subconjunctival fibroblasts, as well as in those treated with TGFbeta1, below the control levels. IFN-gamma had no effect on cell proliferation and fibroblast ultrastructure. On the other hand, IFN-gamma delayed defect closure in monolayer cell sheets in a dose-dependent manner. Immunohistochemistry also revealed that the addition of IFN-gamma attenuated the translocation of Smads2/4 into the nuclei of TGFbeta1-treated subconjunctival fibroblasts., Conclusion: These findings suggest that IFN-gamma may be clinically effective in attenuating excessive ECM accumulation in conjunctiva after ocular surgery and in the presence of inflammatory ocular surface disorder. IFN-gamma modulates the Smads2/4 pathway of TGFbeta1 signal transduction toward the up-regulation of ECM components.

Purpose: The purpose of this study was to assess morphologically the interaction between zinc and vitamin A in their effect on the ocular surface., Methods: Three-week-old Wistar Kyoto rats were divided into five groups: group A(+) Zn(+) was fed a diet containing both vitamin A and Zn; group A(-) was fed a vitamin A-deficient diet; group A(-) Zn(+) was given a vitamin A-deficient diet and deionized distilled water with Zn; group Zn(-) and group Zn(-) A(+) were fed a Zn-deficient diet, whereby group Zn(-) A(+) received an intraperitoneal injection of vitamin A. Corneas and conjunctivas of each group were examined by electron microscopy., Results: The corneas and conjunctivas of group A(-) demonstrated a decrease in the number of microvilli in the epithelium. The corneas and conjunctivas of group A(-) Zn(+) had a decrease in microvilli but more microvilli than those of group A(-). The corneas of group A(-) showed keratinization, but those of group A(-) Zn(+) showed no keratinization. In group Zn(-) the microvilli on the conjunctiva and cornea were sparse. Group Zn(-) A(+) had a decrease in microvilli but more microvilli than group Zn(-). The conjunctivas of group Zn(-) A(+) had more goblet cells with granules than did those of group Zn(-)., Conclusion: These results suggest that a synergistic interaction exists between vitamin A and zinc for the maintenance of the corneal and conjunctival epithelium.

Background: To our knowledge there have been no studies on intermediate filament (IF) expression in conjunctival melanocytic tumors to date. Melanocytic lesions occurring at various body sites are known to express, in addition to the predominant IF protein vimentin, the epithelial-specific cytokeratins (CKs) and the neuronal IF peripherin. The present study was therefore carried out to assess the expression of IF proteins in conjunctival melanocytic lesions., Methods: Paraffin-embedded material from 34 tumors - 16 conjunctival nevi, nine specimens of primary acquired melanosis (PAM; eight with and one without atypia), and nine conjunctival melanomas - was assessed after the application of a panel of antibodies directed against diverse IF proteins, including vimentin, CKs and peripherin., Results: The most significant finding of this study was that all the tumors investigated expressed vimentin exclusively. While simple-epithelium CKs were found in epithelial cysts of nevi and in pseudoglandular portions adjacent to the melanoma, they were not identified in the tumor cells themselves. Similarly, peripherin and neurofilaments were not detected within any of the tumor cells., Conclusion: The IF expression pattern of conjunctival melanocytic lesions differs from that seen in melanocytic tumors of other body sites (including uveal melanomas), in that it includes neither CKs nor peripherin.

The molecular architecture of the human ocular mucus is not yet completely understood. Recently, TFF peptides (formerly known as trefoil factors or P-domain peptides) could be identified as new constituents of this delicate mucus. Members of the TFF-peptide family are typical secretory products of mucous epithelia and three are known in humans and designated as TFF1, TFF2 and TFF3. They enhance cell migratory processes (motogenic effect), they show anti-apoptotic effects and are inflammatory modulators Both TFF1 and TFF3 expression could be monitored by the reverse transcription-polymerase chain reaction (RT-PCR) in the human conjunctiva; in contrast, TFF2 transcripts were not detectable. Using immunohistochemistry, TFF1 and TFF3 peptides were found in varying concentrations solely in secretory vesicles of conjunctival goblet cells. This localisation matches precisely that of the secretory mucin MUC5AC. Thus, conjunctival TFF1 and TFF3 have to be considered as typical mucin-associated peptides probably modulating the rheological properties of the ocular mucus and the tear fluid. Future investigations are in progress to elucidate the role of TFF-peptides during pathological conditions of the eye as well as their diagnostic and therapeutic potential.

Purpose: Disturbances of the ocular tear film layer and dry eye symptoms are common complications following retinal surgery and ocular tumour therapy. Examined were the histopathological changes of the conjunctiva following posterior segment surgery and plaque radiotherapy., Methods: Biopsy specimens of the superior bulbar conjunctiva were obtained during cataract surgery between 2 weeks and 7 years following vitrectomy (n=92) or plaque radiotherapy for uveal melanoma (n=20) and from control subjects without previous ocular surgery (n=29). These were examined using conventional histology (HE, PAS, Van Gieson) and immunochemistry [APAAP, using antibodies directed against MUC1, MUC5AC, syndecan-1 and tenascin-C (TN-C)]. The histopathological changes were graded and statistical analysis was performed using Wilcoxon and Kruskal-Wallis rank sum tests., Results: Conjunctival specimens of patients following vitrectomy or plaque radiotherapy for uveal melanoma demonstrated increased epithelial stratification, a significant decrease in the number of PAS- and MUC5AC-positive goblet cells, and distributional changes in expression of MUC1, syndecan- and TN-C within conjunctival epithelium or stroma. These alterations - in particular the goblet cell reduction and stromal fibrosis - were most prominent in those patients who had undergone radiotherapy., Conclusions: Posterior segment surgery can lead to morphological alterations of the conjunctiva and distributional changes in ocular mucins, which may cause dry eye symptoms.

Purpose: To examine immunohistochemically whether extracellular matrix (ECM) of the filtering bleb and of cultured human subconjunctival fibroblasts contains latent TGF beta binding protein-1 (LTBP-1) and TGF beta., Methods: An enucleated human eye that had undergone trabeculectomy and cultured human subconjunctival fibroblasts were processed for light microscopic immunohistochemistry. Antibodies against LTBP-1, collagen types, fibrillin-1 and TGF beta s were used. TGF beta 1 was located by detecting beta 1-latency associated peptide (LAP)., Results: LTBP-1, beta 1-LAP and fibrillin-1 were all located in the subepithelial ECM as well as in the basal epithelial cells of the conjunctiva over the filtering bleb. TGF beta 2 and beta 3 were immunolocated to epithelium and/or fibroblasts/keratocytes. ECM deposited in confluent fibroblast cultures was positive for beta 1-LAP, LTBP-1 and fibrillin-1, whereas sparse cells were negative., Conclusions: LTBP-1, beta 1-LAP and fibrillin-1 are co-localized to the ECM of the filtering bleb and of cultured conjunctival fibroblasts. Both conjunctival epithelium and fibroblasts are considered to be the source of TGF beta in healing bleb. ECM secreted by in vivo and in vitro subconjunctival fibroblasts may works as a scavenger or repository of TGF beta.

Background: Chronic conjunctival inflammatory diseases may depend upon various mechanisms. Discriminating allergy from nonspecific inflammation has become of striking importance for diagnosis and treatment. We investigated conjunctival inflammatory response by comparing two objective biological tools, tear IgE and HLA-DR expression by conjunctival epithelium, as indirect indicators of activation of the Th2 and Th1 subsets, respectively., Methods: Eighty-two patients with chronic conjunctivitis underwent tear IgE measurement by an ELISA technique and quantitation of HLA-DR expression in impression cytology specimens. Forty-two had direct or indirect clinical indications of allergic mechanisms, 26 had chronic conjunctivitis without any sign of allergy, and 14 suffered from isolated nonallergic dry eyes., Results: Patients clinically considered as allergic only showed positive IgE in 47 of 84 eyes (56%), whereas 21% and 25% of eyes with nonspecific conjunctivitis and dry eyes respectively were also positive. IgE levels were significantly higher in the allergic group than in the other two groups. HLA-DR positivity in epithelial cells was found in 28.5%, 48% and 50% of eyes, respectively. HLA-DR expression by epithelial cells was negatively correlated with tear IgE, as most specimens positive to one criterion were negative to the other one (49 eyes DR+, IgE-; 47 eyes DR-, IgE+; only 9 eyes positive to both criteria; chi-square: P = 0.0001)., Conclusion: As IgE synthesis and HLA-DR induction may represent indirect indicators of the activation of the Th2 and Th1 subsets, association of these two simple tests could be interesting for the routine assessment of the mechanisms of inflammatory ocular surface diseases.

Background: Multidrug resistance (MDR) describes the phenomenon of cross-resistance between different cytostatic agents which are structurally and functionally dissimilar. Two recently discovered proteins, lung resistance protein (LRP) and the multidrug resistance-related protein (MRP) have been implicated in the development of MDR. Since resistance to chemotherapeutic agents is a common problem in filtration surgery, especially in cases of complicated glaucoma, we decided to investigate the presence of MRP and LRP in surgically removed Tenon specimens from glaucoma patients., Methods: The presence of MRP and LRP in surgically removed Tenon tissue (n=15) was analyzed by immunohistochemistry. The expression by cultured Tenon fibroblasts was assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) and fluorocytometry., Results: LRP expression was detected in 8 of 10 Tenon specimens. Positive staining for MRP was obtained in 5 of 10 specimens. Negative controls with non-immune mouse IgG did not display any specific staining. RT-PCR and fluorocytometry revealed constitutive expression of MRP and LRP, at the RNA and protein level respectively, that was unaltered by pretreatment of the cells with mitomycin C or 5-fluorouracil., Conclusion: Our results demonstrate, that besides P-glycoprotein, other components of the MDR-system are present in conjunctival fibroblasts. Future developments in the use of chemotherapeutic agents in association with of filtration surgery need to take account of the presence of these counteracting mechanisms.

Background: Since postoperative hypotony has been a frequent complication of glaucomatous filtration surgery with adjunctive use of mitomycin C (MMC), the question arises of whether there may be another application method which can minimize this side effect. The purpose of this study was to establish the morphologic side effects of different application methods., Methods: MMC 0.2 mg/ml was applied to the episclera of nine eyes of six pigmental rabbits at random via collagen shield (CS), soft contact lens (CL), or lyophilisate (20 microg; LY) for 5 min. Two eyes (controls) had a subconjunctival injection of BSS only. Another control eye was left untreated (no injection). No trabeculectomy was performed. One hour later the amounts of MMC in the conjunctiva and aqueous were analyzed by reverse-phase high-pressure liquid chromatography. Ciliary bodies were dissected from the enucleated eyes, embedded and investigated by transmission electron microscopy (TEM). Cell height of the nonpigmented ciliary epithelium was morphometrically assessed by means of computer-assisted image analysis., Results: The light-microscopic analysis of the sectioned cell area revealed reduction of the cell height of the non-pigmented ciliary epithelium (NPCE) after application with soft contact lens (fourfold) and collagen shield (2.5-fold) but not with lyophilisate compared to the untreated eye. The following ultrastructural changes were seen: loss of apical microvilli (CS, CL, LY), disintegrating melanin granules within NPCE (CS), lysis of entire areas with NPCE cells (CS), myelin figures within mitochondria (LY), intracellular vacuoles (CS, CL), lysis of myelinated nerves (CS), myelin figures in mitochondria of endothelial cells (LY), and lysis of stromal fibrocytes (CS). In the control eyes (injection of BSS) none of these ultrastructural changes were detected in the cylindrical NPCE cells. The concentration of mitomycin in the aqueous humor after topical application of MMC on the episclera for 5 min were all below the detection limit (<10 ng/ml). The concentration of MMC in the conjunctiva ranged from 2.1 to 3.7 microg/g., Conclusion: Severe morphologic alterations were seen at the electron-microscopic level after application of MMC 0.2 mg/ml with a collagen shield and with a soft contact lens. They were mildest with lyophilisate and absent in the BSS controls. A new administration device is needed if trabeculectomy is to be performed successfully using MMC in human glaucomatous eyes.

TFF-peptides (formerly P-domain peptides, trefoil factors) form a new family of mucin-associated peptides mainly in the gastrointestinal tract. TFF3 is a typical secretory product of intestinal goblet cells and occurs also in the respiratory tract. Here, polyclonal antisera specific for TFF3 were used in Western blot analysis and immunofluorescence to determine the presence and distribution of TFF3 in the porcine conjunctiva, which is the primary source for ocular mucins. Significant accumulation of TFF3 was detected in conjunctival goblet cells but not in the lacrimal glands. This peptide, together with ocular mucins, may play a role in the rheological function of the tear film.

Primary cultures of human epithelial cells from normal conjunctiva were developed and characterized to determine whether they retained epithelial characteristics. Conjunctival explants were obtained from the upper fornix of healthy donors and cultured in supplemented DMEM/F-12 medium for 5 days. The epithelial outgrowth was maintained for an additional 10 days. Primary cultures were then processed for light microscopy, transmission and scanning electron microscopy (TEM, SEM), and immunocytochemistry. They exhibited typical features of conjunctival epithelium on light microscopy (polygonal morphology, intimate cohesion, production of mucins), TEM (abundant desmosomes, keratin bundles, granules, microvilli), SEM (polygonal shape, microvilli, intimate cohesion), and immunocytochemistry (positivity for the receptor of epidermal growth factor, desmosomal proteins, and cytokeratins). In conclusion, primary cultures developed from normal human conjunctiva maintained the epithelial characteristics in vitro. Because the conjunctiva is a major component of the anterior ocular surface, we propose this in vitro system as suitable for physiopathologic and toxicologic studies.

Background: The etiology and pathogenesis of keratoconus remain unclear, and therefore we decided to study the distribution of different isoforms of tenascin (Tn) and fibronectin (Fn) in normal human corneas and in those obtained from penetrating keratoplasty for keratoconus and corneal scarring., Methods: Frozen sections of human cornea and conjunctiva were stained by immunohistochemical methods with a panel of monoclonal antibodies (MAbs) against different isoforms of Tn and Fn., Results: In the normal human eye, Tn was found in the limbal and conjunctival basement membrane region, in the conjunctival blood vessels and at the junction of the cornea and sclera, but no immunoreaction was seen in the normal cornea. In the corneas from the keratoconus patients, a clear immunoreaction for Tn was seen in the defects of Bowman's membrane as well as in the distorted stroma beneath the defects. In some of the keratoconus corneas, basement membrane adjacent to the defects also showed reactivity for Tn, and in clinically and histologically scarred keratoconus corneas the scars expressed Tn. In the scarred corneas, only blood vessels in the posterior portion of the cornea showed immunoreactivity for Tn, while no Tn was noted in the scar area or in Bowman's membrane. No major differences were noticed in the reactivity of different MAbs against Tn isoforms. Fn, extradomain A Fn (EDA-Fn) and oncofetal Fn (onc-Fn) were found in the basement membrane of the central cornea of the normal eye. In keratoconus corneas, the defects and clinical and histological scars bound MAbs against Fn, EDA-Fn and onc-Fn, but in the scarred corneas no enhancement in the expression of Fns was noted. Extradomain B cellular Fn (EDB-Fn) was not expressed in any of the eyes studied., Conclusions: The results suggest that the anterior portion of the cornea is involved in the pathogenesis of keratoconus. Furthermore, it seems that the expression of Tn and Fns in the clinically scarred keratoconus corneas is due to a process in which both repairing and scar-forming mechanisms operate at the same time. However, the origin of the defects in Bowman's membrane seen in keratoconus still remains unclear. They may be minor scars due to the disease or primary defects in the process leading to keratoconus.

Cyclosporine is an important tool for the therapy of immunological diseases of the cornea and conjunctiva, as well as the treatment of patients with high-risk corneal grafts. However, potentially severe systemic side effects are disadvantageous. The purpose of our study was to determine if topical ocular application leads to about the same concentrations in ocular tissues as systemic application. Therefore, the concentration of cyclosporine in conjunctiva, aqueous humor and blood was measured by radioimmunoassay in six patients with systemic administration of cyclosporine and ten patients who received one drop of topical cyclosporine 2% four times prior to cataract surgery. All patients with systemic application showed measurable concentrations of cyclosporine in blood, as did four patients in the group receiving topical cyclosporine. There was no significant difference between both groups concerning cyclosporine concentration in aqueous humor. The level of cyclosporine in the conjunctiva was significantly higher after topical application (P < 0.02). In conclusion, therapy with cyclosporine eye drops results in levels in the conjunctiva and aqueous humor that are comparable to or even higher than those after systemic application if the last application was no more than 18h prior to the measurement. Therefore, topical ocular application of cyclosporine, which reduces or eliminates the drug's systemic side effects, can be used to induce local immunosuppressive activity, particularly in the treatment of superficial immunological diseases and after limbal allograft transplantation.

Background: Primary open-angle glaucoma is commonly treated with long-term hypotensive medical therapy. When this approach becomes inadequate, therapy proceeds with surgery. The present study investigates morphological changes in the conjunctival and subconjunctival tissues induced by short- and long-term topical medical therapy of primary open-angle glaucoma., Methods: Comparisons were made between biopsy specimens from glaucomatous patients, who received specific eyedrop therapy (timolol and pilocarpine) for various periods of time, and control patients with no conjunctival pathology or topical treatment. Histological, immunohistochemical and ultrastructural parameters were investigated., Results: The morphometric analysis of histological sections and immunohistochemistry (anti-fibronectin antibody) in medium- and long-term therapy patients showed: (a) significant increases in the thickness and number of epithelial cell layers; (b) significant increases in the fibroblast density in both subepithelial and deep connective tissue; and (c) a more compact connective tissue, richer in collagen fibers arranged in whirls, with some inflammatory elements. These findings were confirmed by the ultrastructural analysis. In the same patients, the other immunohistochemical parameters investigated (anti-HLA-DR, anti-CD1a, anti-CD4, anti-CD8, anti-IL2 and C3b antibodies) revealed a tendency to chronic inflammation. Following specific surgery, this tendency manifested itself in a diffuse immune response, especially in those patients who underwent medium- and long-term medical therapy., Conclusion: According to these results, antiglaucomatous surgery should be rehabilitated and considered as an alternative to long-term medical therapy in the first-instance treatment of primary open-angle glaucoma.

Background: Topical medication applied chronically for the treatment of glaucoma changes the cellular profile of the conjunctiva. We wanted to determine the role of preservatives, which are usually combined with the drugs, on this effect., Methods: We applied metipranolol 0.3% and pilocarpine 2% with and without benzalkonium chloride 0.01% and cetrimonium chloride 0.004%, respectively. Twenty-four rabbits, divided into four groups, were treated for 3 months. The complete globes and the adherent bulbar conjunctiva were examined histopathologically and immunohistochemically., Results: With special stains for collagen, a slight increase of the thickness of subepithelial collagen of the conjunctiva was present in both groups treated with medication and preservative compared with eyes treated with medication alone. This effect was also true for special antibodies for collagen type IV and alpha-smooth muscle actin in the eyes treated with pilocarpine, but not in the eyes treated with metipranolol., Conclusion: The results suggest that preservatives may have an additional adverse effect on the conjunctiva in addition to the effects of the medications alone.

The aim of this study was to evaluate the potential role of certain soluble factors in conjunctival scar tissue formation of pemphigoid patients. Epibulbar conjunctival biopsy specimens were taken from patients with acute ulcerative (n = 4), subacute (n = 8) and chronic (n = 8) mucous membrane pemphigoid and from twelve age-matched healthy individuals. The tissues were embedded in glycol methacrylate and analysed by immunohistochemical methods. Interleukin-2 (IL-2), interferon-gamma, transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), tumour necrosis factor-alpha and proliferating cells (as identified with the antibody Ki-67) were found in both pemphigoid patients and normal controls. Interleukin-4 was not found with this method in either normal or diseased conjunctiva. Significant differences between normal and diseased conjunctiva were found for TGF-beta and for proliferating cells, which were both increased in the acute disease group. More intense staining was found in the subacute disease group for IL-2, bFGF and PDGF. Our findings showed that a variety of cytokines were present in normal and diseased bulbar conjunctiva. Acute conjunctival disease in mucous membrane pemphigoid may indicate active scar tissue formation, implied by an increase in TGF-beta and the presence of proliferating fibroblasts.