ACE2 and TMPRSS2 are expressed on the human ocular surface, suggesting susceptibility to SARS-CoV-2 infection.

PURPOSE: Conjunctival signs and symptoms are observed in a subset of patients with COVID-19, and SARS-CoV-2 has been detected in tears, raising concerns regarding the eye both as a portal of entry and carrier of the virus. The purpose of this study was to determine whether ocular surface cells possess the key factors required for cellular susceptibility to SARS-CoV-2 entry/infection. METHODS: We analyzed human post-mortem eyes as well as surgical specimens for the expression of ACE2 (the receptor for SARS-CoV-2) and TMPRSS2, a cell surface-associated protease that facilitates viral entry following binding of the viral spike protein to ACE2. RESULTS: Across all eye specimens, immunohistochemical analysis revealed expression of ACE2 in the conjunctiva, limbus, and cornea, with especially prominent staining in the superficial conjunctival and corneal epithelial surface. Surgical conjunctival specimens also showed expression of ACE2 in the conjunctival epithelium, especially prominent in the superficial epithelium, as well as weak or focal expression in the substantia propria. All eye and conjunctival specimens also expressed TMPRSS2. Finally, Western blot analysis of protein lysates from human corneal epithelium obtained during refractive surgery confirmed expression of ACE2 and TMPRSS2. CONCLUSIONS: Together, these results suggest that ocular surface cells including conjunctiva are susceptible to infection by SARS-CoV-2, and could therefore serve as a portal of entry as well as a reservoir for person-to-person transmission of this virus. This highlights the importance of safety practices including face masks and ocular contact precautions in preventing the spread of COVID-19 disease.

The presence of kynurenine aminotransferases in the human cornea: Evidence from bioinformatics analysis of gene expression and immunohistochemical staining.

PURPOSE: Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), a compound of significant biological activity. The aim of this study is to investigate the presence and distribution of KAT immunoreactivity in the healthy human cornea. METHODS: Data on gene expression in human eye structures were extracted from public microarray experiments using Genevestigator software. Immunohistochemistry was conducted using polyclonal antibodies against KAT I, II, and III on sections of eight enucleated eyes from patients with choroidal melanoma. RESULTS: Bioinformatics analysis showed that all four KAT isoforms were actively transcribed in the cornea and the conjunctiva. Immunohistochemical analysis revealed the presence of KAT I, II, and III in all examined corneal sections. The corneal endothelium showed the strongest reactivity for all three KAT isoforms. There was a slight positive staining of the corneal stroma for KAT I and II. KAT III immunoreactivity was found only in the stroma of the limbal region. In the corneal epithelium, the expression of all three KAT isoforms showed a specific pattern of the stain with fine squatter granules throughout the cytoplasm. This reactivity was more pronounced in the basal cell layers. The intermediate cell layers showed only faint immunoreactivity, and occasionally, there was no staining. KAT I, II, and III were also present in the adjacent limbal conjunctiva. CONCLUSIONS: The results indicate that kynurenine can be metabolized to KYNA in the corneal epithelium, stroma, and endothelium.

Role of glutathione peroxidase 4 in conjunctival epithelial cells.

PURPOSE: The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in conjunctival epithelial cells. METHODS: An immortalized human conjunctival epithelial cell line was used. Cells were transfected with catalase, GPx1, GPx4, SOD1, SOD2, or control siRNA. Knockdown was confirmed by RT-PCR and immunoblotting. The cytotoxicity induced by knockdown of these antioxidant enzymes was examined by assay of LDH activity. Furthermore, evaluations of lipid peroxidation, cellular levels of reactive oxygen species, cell proliferation, and apoptosis were conducted in cells treated with GPx4 or control siRNA. In oxidative stress study, cells treated with GPx4 or control siRNA were applied with hydrogen peroxide or ferric sulfide, and their cytotoxicity was evaluated by assay of LDH activity. RESULTS: Small interfering RNA of catalase, GPx1, GPx4, SOD1, and SOD2 siRNA remarkably inhibited the mRNA and protein expression of each gene. Knockdown of GPx4 and SOD1 but not catalase, GPx1, and SOD2 significantly induced cytotoxicity. Glutathione peroxidase 4 knockdown increased lipid oxidation and reactive oxygen species. The proliferation of GPx4 siRNA-treated cells was reduced compared with control siRNA-treated cells. Moreover, cell death in GPx4 siRNA-treated cells was characterized by positive staining for annexin V. In an oxidation stress study, GPx4 siRNA knockdown enhanced the cytotoxicity induced by hydrogen peroxide or ferric sulfide. CONCLUSION: These results suggest that GPx4 is essential for maintaining oxidative homeostasis and keeping defense against oxidative stress in conjunctival epithelial cells.

Conjunctival matrix metalloproteinases and their inhibitors in glaucoma patients.

PURPOSE: Chronic conjunctival inflammation, caused by various reasons, for example long-term use of topical drugs and/or their preservatives, affects the outcome of glaucoma surgery by interfering with wound healing. Matrix metalloproteinases (MMPs) remodel extracellular matrix (ECM) and are involved in the wound healing process. This study was designed to evaluate the conjunctival expression of MMPs and their tissue inhibitors (TIMPs) in the normal eye, primary open-angle glaucoma (POAG) and exfoliation glaucoma (ExG) and whether there is an association between staining intensities and deep sclerectomy outcome. METHODS: Immunohistochemical procedures were performed on conjunctival samples which were obtained from POAG (n=11) and ExG (n=14) patients as well as normal (n=7) subjects. Antibodies against MMPs (MMP-1, -2, -3 and -9) and TIMPs (TIMP-1, -2 and -3) were used. RESULTS: In conjunctival stroma, expression levels of MMP-2 (p=0.047), MMP-3 (p=0.009), MMP-9 (p<0.001), TIMP-1 (p=0.003), TIMP-2 (p<0.001) and TIMP-3 (p<0.001) in ExG and MMP-9 (p=0.008), TIMP-2 (p=0.02) and TIMP-3 (p=0.002) in POAG were significantly increased compared to control. We further found correlations between expression of MMP-1 and MMP-3 and the length of pilocarpine treatment. CONCLUSION: The expression of MMPs and TIMPs is increased in the conjunctiva of POAG and ExG patients having a long history of topical antiglaucoma drops. Antiglaucoma agents and/or their preservatives alter the remodelling balance of ECM in conjunctiva of POAG and ExG eyes. The balance between MMPs and TIMPs may play a crucial role in the conjunctival wound healing process and the outcome of glaucoma surgery.

Matrix metalloproteinase 9 and transglutaminase 2 expression at the ocular surface in patients with different forms of dry eye disease.

OBJECTIVE: To evaluate the expression of matrix metalloproteinase 9 (MMP9) and transglutaminase 2 (TG2) in different forms of dry eye. DESIGN: Case control study. PARTICIPANTS: Seventy-five female subjects divided into 3 groups: group 1, 15 healthy controls; group 2, 30 subjects with Sjögren syndrome (SS); and group 3, 30 subjects with Meibomian gland dysfunction (MGD). METHODS: A clinical assessment was carried out and impression cytologic specimens were processed for immunoperoxidase staining for MMP9 and TG2 and real-time polymerase chain reaction analyses were carried out for MMP9, TG2, interleukin-6, interferon-γ, B-cell lymphoma 2, and caspase 3. To study MMP9 and TG2 expression after anti-inflammatory treatment, patients were divided into 2 subgroups, one treated with saline and the other treated with saline plus topical corticosteroid eye drops (0.5% loteprednol etabonate) 4 times daily for 15 days. For statistical analysis, Student t test, Mann-Whitney U test, and Spearman's correlation coefficient were used as appropriate. MAIN OUTCOME MEASURES: Conjunctival expression of MMP9 and TG2. RESULTS: MMP9 and TG2 expression were higher in both patient groups than in controls (P < 0.0001). Group 2 patients showed higher expression than group 3 (P < 0.0001). The Spearman's correlation coefficient showed in group 2 a positive correlation between MMP9 and TG2 expression (ρ = 0.437; P = 0.01), but no correlation in group 3 (ρ = 0.143; P = 0.45). Corticosteroid treatment significantly reduced MMP9 and TG2 expression in both groups, ameliorating symptoms and signs. A much higher percentage reduction was observed in SS. CONCLUSIONS: The pathogenic mechanisms of the 2 forms of dry eye give an account for the different MMP9 and TG2 expressions in the 2 groups of patients. The higher expression in SS is determined by the direct autoimmune insult to the ocular surface epithelia, whereas in MGD patients, with an epithelial damage due to an unbalanced tear secretion, the molecules expression is significantly lower, although higher than in controls. The corticosteroid treatment induced a reduction of both molecules, although higher in SS than in MGD, because of its direct inhibitory effect on inflammation.

Contributions of ocular surface components to matrix-metalloproteinases (MMP)-2 and MMP-9 in feline tears following corneal epithelial wounding.

PURPOSE: This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding. METHODS: Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs. RESULTS: The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining. CONCLUSIONS: Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.

Overexpression of peroxiredoxin 2 in pterygium. A proteomic approach.

Pterygium is one of the most frequent pathologies in ophthalmology, and is a benign, fibrovascular lesion originating from the bulbar conjunctiva. It is composed of an epithelium and highly vascular, subepithelial, loose connective tissue. The etiology of pterygium is not clearly understood; the most widely recognized originating factor is ultraviolet radiation. It has been proposed that pterygium and neoplasia have common features, raising the possibility that pterygium is a neoplastic-like growth disorder. In this study, proteomic analysis was performed to show that peroxiredoxin 2 is overexpressed in pterygia compared to healthy conjunctivas. Twelve pterygium specimens were obtained together with healthy conjunctival tissue from the same eyes. Total proteins of pterygia and healthy conjunctivas were analyzed in SDS-PAGE. This analysis showed protein bands expressed exclusively in pterygium samples at the range of 20-25 kDa. After this, 2D electrophoresis was performed for the separation of total proteins; differential spots expressed in pterygium were excised and sequenced. Mass spectrometry (MS) data were searched in the NCBInr and EST databases using the MASCOT program. The spot was identified as peroxiredoxin 2. Real-time PCR, western blot and immunohistochemistry showed that peroxiredoxin 2 was increased in pterygium compared to healthy conjunctiva. Although, these results suggest that overexpression of peroxiredoxin 2 in pterygium could protect the cell against oxidative stress-induced apoptosis, further studies are required to establish the functional role of peroxiredoxin 2 in pterygium to determine its role in peroxidation and apoptosis in this pathology.

Immunohistochemical analysis of angiotensin converting enzyme in Sardinian pterygium.

Pterygium is a common ocular surface disorder characterized by excessive cell proliferation, inflammation, fibrosis, angiogenesis and extracellular matrix remodeling. The Angiotensin converting enzyme (ACE or ACE I) is the major component of the Renin-angiotensin system (RAS) converting the inactive decapeptide Angiotensin I (Ang I) to the active octapeptide Angiotensin II (Ang II). Besides this 'classical role', it can act as transcriptional regulator in response to external stimuli that may lead to cell damage and tissue remodeling. Due to this role, it can be internalized into the nuclear compartment to act as transcriptional factor for proteins involved in the inflammatory response. The aim of the present study was to determine ACE expression and localization in pterygium and culture pterygium cells by immunohistochemistry. Our results are the first to demonstrate nuclear immunolocalization of ACE, more so in pterygium compared to conjunctiva epithelial cells in histological sections. ACE was not detected in the nuclei of subcultivated pterygium epithelial cells. The nuclear localization of ACE may be correlated with an anti-inflammatory path mediated by activation of its transcriptional role.

Activation of MAPK signaling pathway and NF-kappaB activation in pterygium and ipsilateral pterygium-free conjunctival specimens.

PURPOSE: To evaluate mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways in pterygium and pterygium-free conjunctivas. METHODS: Primary pterygia (n = 21), ipsilateral superior-temporal bulbar conjunctivas (n = 8), and healthy conjunctival (n = 5) biopsy specimens were analyzed. Total and phosphorylated (phospho) levels of extracellular-regulated 1/2 (ERK1/2), p38, and c-jun N-terminal (JNK) MAPKs and NF-κB inhibitor-alpha (IκΒ-α) were analyzed by immunobead-based assay. Tissue phospho-, total protein, and activation values determined by phospho/total ratios were compared. Correlation among those values and clinical parameters were determined. Average-linkage hierarchical cluster analysis identified patients with similar protein activation values. The k-nearest neighbor classifier predicted the origin of specimens based on protein levels. RESULTS: Pterygium samples had significantly lower total JNK and IκΒ-α levels than did healthy conjunctivas. Decreased total JNK and IκΒ-α and increased phospho-IκΒ-α levels and phospho/total ratio of JNK and IκΒ-α were present in ipsilateral conjunctivas compared with healthy conjunctivas. Protein levels were correlated among them in pterygium, ipsilateral, and healthy conjunctivas and with sun exposure, pterygium grade, and pterygium measurements. Cluster analysis of activation values and ratios in pterygium and ipsilateral-conjunctiva revealed different groups of patients with similar values. Prediction accuracy was 70% to 80% for the classifiers phospho- and total protein levels and phospho/total ratio. CONCLUSIONS: Pterygium and pterygium-free ipsilateral conjunctivas had alterations in MAPK and NF-κB pathways not present in healthy conjunctivas. The high prediction accuracy based on phospho- and total protein levels and phospho/total ratio of ERK1/2, p38, JNK, and IκB-α suggests these molecules as potential biomarkers of inflammation in pterygia.

Increased conjunctival expression of protease activated receptor 2 (PAR-2) in seasonal allergic conjunctivitis: a role for abnormal conjunctival epithelial permeability in disease pathogenesis?

AIMS: Aeroallergen exposure to the conjunctival epithelium in seasonal allergic conjunctivitis (SAC) may induce a cellular stress response that disrupts the barrier properties of the conjunctival epithelium, resulting in allergic disease. Whether such changes occur in SAC is unknown. Epithelial permeability is known to be increased when protease activated receptor 2 (PAR-2) is activated. We evaluated the expression of PAR-2 in patients with SAC-in-season (SACS) and compared it with control non-atopic subjects or those with out-of-season allergic conjunctivitis (OSAC). METHODS: Six SACS, eight normal and four OSAC specimens were examined immunohistochemically for PAR-2 and quantified in a masked fashion for the percentage of epithelia stained for each marker using Image-J software. Conjunctival epithelial heights were measured in all groups to confirm the presence of allergic eye disease. RESULTS: Mean percentage staining of PAR-2 was significantly greater in SACS that in normal specimens (73.4 ± 15.4% vs 32.8 ± 30.0%, p=0.038) or in OSAC (73.4 ± 15.4% vs 1.4 ± 2.2%, p=0.01). Mean conjunctival epithelial height was significantly raised in SACS (63.8 ± 9.0 µm) versus controls (44.7 ± 11.2 µm) (p=0.003, unpaired t test). CONCLUSIONS: Conjunctival epithelial PAR-2 is significantly upregulated in SAC. This supports the view that disruption of the barrier properties of the conjunctival epithelium is an important event in SAC pathogenesis.

Study of alkaline phosphatase activity and DNA content of pterygium tissue showing its degenerative character.

A prospective, non-randomised, interventional case series study was designed among 127 specimens obtained from 100 consecutive patients presenting with primary pterygium attending the outpatient department of a teaching hospital in Kolkata, West Bengal between September 2009 and August 2010. After harvesting the pterygium it was transported in 10% formol saline for staining, fixing and embedding prior to evaluation of tissue alkaline phosphatase level and estimation of DNA content in nucleus of cells in pterygium tissue. The main outcome was to measure alkaline phosphatase activity and DNA content in pterygium tissue and normal bulbar conjunctiva along with histopathological staining and to see if any correlation exists between alkaline phosphatase activity and DNA content of pterygium tissue in different stages of evolution as suggested by histological staining. The elastic proliferation and hyaline degeneration are prominent in well developed pterygium. The alkaline phosphatase activity in all cases of pterygium is maximum and the DNA content of the cells of the pterygium tissue is diminished as compared to normal bulbar conjunctiva. Histopathological changes as evident from epithelial cell changes from cylindrical to flat cells with cyst formation in basal layers, the disappearance of nuclei in the submucosa coupled with increase in hyaline content of the tissue all point towards degenerative changes. Biochemically too there is a decrease in DNA content in the nuclei of the cells in pterygium, histochemically there is an increase in the alkaline phosphatase activity, all these changes point to pterygium being a degenerative tissue.

Topical cyclosporine in thyroid orbitopathy-related dry eye: clinical findings, conjunctival epithelial apoptosis, and MMP-9 expression.

OBJECTIVES: To evaluate the effects of topical cyclosporine A (CsA) 0.05% (Restasis) on the signs and symptoms of dry eye, on apoptosis, and on MMP-9 expression in conjunctiva epithelial cells in thyroid orbitopathy (TO)-related dry eye patients. METHODS: Prospective, clinical study. Twenty-four eyes of 12 consecutive TO patients with dry eye findings instilled CsA twice daily for 2 months. Ocular surface disease index, Schirmer tear test, tear breakup time (TBUT), conjunctival apoptosis index, and conjunctival MMP-9 expression were evaluated before and after 2 months treatment. Conjunctival biopsies were harvested from all eyes at baseline and after 2 months treatment. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) assay. MMP-9 expression was determined by immunohistochemistry. RESULTS: After 2 months of topical CsA treatment, the mean OSDI score was significantly decreased from 58.08 +/- 6.28 to 36.41 +/- 11.75 (P = 0.001). At baseline, the mean Schirmer tear test score was 8.92 +/- 5.52 mm. It was increased to 11.25 +/- 4.71 mm after treatment (P > 0.05). The mean TBUT increased significantly from 3.92 +/- 2.18 sec to 9.16 +/- 3.34 sec (P = 0.001). The mean percentage of apoptosis index at baseline was 72.10 +/- 35.82%. This was significantly decreased to 53.29 +/- 34.46% after treatment (P = 0.008). The mean percentage of MMP-9 expression of the conjunctival epithelial cells was significantly decreased from 48.12 +/- 28.58% to 26.66 +/- 25.13% following treatment (P = 0.005). CONCLUSIONS: Topical CsA treatment appears to improve the signs and symptoms of dry eye and inhibits apoptosis and MMP-9 expression in conjunctival epithelial cells in TO-related dry eye patients after 2 months of treatment.

Effect of latanoprost on the expression of matrix metalloproteinases and tissue inhibitor of metalloproteinase 1 on the ocular surface.

OBJECTIVE: To investigate the effect of topical latanoprost on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase 1 (TIMP-1) on the ocular surface. METHODS: Tears were collected from 39 patients with glaucoma who used latanoprost, 0.005%, eyedrops (Xalatan) and 28 healthy volunteers. The MMP-9 concentration was measured. Conjunctival epithelial cells were collected from 10 eyes of 10 patients before and 1 to 3 months after starting to take topical latanoprost, 0.005%, and MMP-1, MMP-9, and TIMP-1 messenger RNA (mRNA) expression was analyzed. Both eyes of 48 mice were treated once a day with latanoprost, 0.005%, timolol gel, 0.5%, eyedrops, vehicle of Xalatan, or phosphate-buffered saline, and MMP-9 and TIMP-1 mRNA expression was analyzed. RESULTS: The median MMP-9 concentration in latanoprost-treated cases was 91.2 ng/mL (in controls, 19.7 ng/mL; P < .001). In latanoprost-treated cases, the relative ratio of MMP-9 to glyceraldehyde 3-phosphate dehydrogenase mRNA was significantly increased from 6.42 to 21.3 (P = .04, paired t test) and the relative amount of TIMP-1 was significantly decreased from 154 to 105 (P = .009). The relative amount of MMP-1 to GAPDH mRNA before and after latanoprost use was not significantly different (P = .16). In mice, MMP-9 expression was increased and TIMP-1 expression was decreased on the ocular surface at 8 weeks after latanoprost use. CONCLUSION: The topical use of latanoprost increases MMP-1 and MMP-9 and decreases TIMP-1 on the ocular surface. CLINICAL RELEVANCE: The use of topical latanoprost might not be recommended in patients with keratoconus or after laser-assisted in situ keratomileusis.

Conjunctival expression of matrix metalloproteinase and proinflammatory cytokine genes after trichiasis surgery.

PURPOSE. Trachoma, the leading infectious cause of blindness, is a chronic inflammatory scarring condition. Blindness follows the development of trichiasis, which is treated surgically. Unfortunately, it frequently recurs, compromising the treatment. In this study, gene expression analysis was used to examine factors that may be involved in the inflammation and tissue remodeling after surgery. METHODS. Subjects were examined before and at 1 and 4 years after surgery. Conjunctival swab samples were collected for bacterial culture, Chlamydia trachomatis PCR, and RNA isolation at 1 year. Quantitative real-time PCR was performed to measure the expression of tumor necrosis factor-alpha (TNF), interleukin-1beta (IL1B), matrix metalloproteinase-1 (MMP1), MMP-2, MMP-9, tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), TIMP-2, and hypoxanthine phosphoribosyl transferase-1 (HPRT1). RESULTS. Two hundred forty individuals with trachomatous trichiasis were recruited. One year after surgery, recurrent trichiasis was associated with a reduced MMP-1/TIMP-1 ratio (P = 0.029). IL1B expression was elevated in the presence of either conjunctival bacterial infection (P = 0.011) or inflammation (P = 0.002). TNF expression was greater in the Mandinka ethnic group (P < 0.0001), and it was increased when clinical inflammation was associated with nonchlamydial bacterial infection (P = 0.012). MMP-9 expression increased when conjunctival inflammation was associated with bacterial infection (P = 0.007). CONCLUSIONS. Recurrent trichiasis was associated with a reduced MMP-1 to TIMP-1 ratio, which may favor the accumulation of fibrotic tissue. Nonchlamydial bacterial infection may induce factors that contribute to conjunctival tissue remodeling and recurrent trichiasis in trachoma. Prospective studies are needed to assess the potential importance of these and other factors in progressive disease.

Effects of mitomycin C on the expression of chymase and mast cells in the conjunctival scar of a monkey trabeculectomy model.

PURPOSE: To determine the effects of mitomycin C (MMC) on the expression of chymase and mast cells in the conjunctival scar after trabeculectomy. METHODS: Ten eyes of five monkeys were used. Three eyes underwent trabeculectomy with MMC (MMC-treated), four eyes had trabeculectomy without MMC (placebo-treated), and three eyes served as control eyes. Intraocular pressure was measured before and three weeks after surgery. The scores of the degree of conjunctival adhesion were evaluated. Immunohistochemistry was used to analyze the densities of proliferative cell nuclear antigen-positive cells, chymase-positive cells, and mast cells. The ratio of collagen fiber areas to conjunctival and scleral lesions was analyzed by Mallory-Azan staining. RESULTS: After trabeculectomy, the intraocular pressure reduction of MMC-treated eyes was significantly different from placebo-treated and control eyes (p=0.032, 0.035). The adhesion score of MMC-treated eyes was also significantly lower than that of placebo-treated eyes (p=0.034). Densities of proliferative cell nuclear antigen-positive cells, chymase-positive cells, and areas of collagen fiber in conjunctival and scleral lesions were significantly decreased in MMC-treated eyes, compared with placebo-treated eyes (p=0.034, 0.034, 0.049, respectively). There was a tendency for the density of mast cells to be suppressed in MMC-treated eyes (p=0.157). CONCLUSIONS: Chymase might be involved in one of the mechanisms by which MMC suppresses scar formation after trabeculectomy.

Acidic mammalian chitinase in dry eye conditions.

PURPOSE: An acidic mammalian chitinase (AMCase) seems to be implicated in allergic asthma and allergic ocular pathologies. The aim of this work was to investigate the role of AMCase during Sjögren's Syndrome (SS) and Meibomian Gland Dysfunction (MGD) dry eye diseases. METHODS: Six patients with MGD dry eye (20-58 years, median 40) and six patients with dry eye associated to SS (32-60 years, median 47) were enrolled in this study. AMCase activity was measured in tears and AMCase mRNA expression was evaluated by real-time polymerase chain reaction from RNA extracted from epithelial cells of the conjunctiva. Six healthy adult subjects of the same age (34-44 years, median 39) were also studied as the control group. RESULTS: AMCase activity was significantly increased in patients affected by MGD dry eye (18.54 +/- 1.5 nmol/ml/h) and SS dry eye (8.94 +/- 1.0 nmol/ml/h) respectively, compared to healthy controls (1.6 +/- 0.2 nmol/ml/h). AMCase activity was higher in the tears of subjects with MGD dry eye (P < 0.001). AMCase mRNA was detected in conjunctival epithelial cells and the expression was significantly higher in MGD dry eye than SS dry eye. A significant correlation between AMCase activity in the tears and mRNA in conjunctival epithelial cells was found. CONCLUSION: AMCase may be an important marker in the pathogenesis of dry eye, suggesting the potential role of AMCase as a therapeutic target in these frequent pathologies.

Production and activity of matrix metalloproteinase-9 on the ocular surface increase in dysfunctional tear syndrome.

PURPOSE: To evaluate production and activity of metalloproteinase (MMP)-9 on the ocular surface of patients with dysfunctional tear syndrome (DTS) and determine any correlation between MMP-9 activity and clinical parameters. METHODS: Forty-six patients with newly diagnosed DTS and 18 control subjects were recruited. Complete ocular surface examinations were performed. Tear MMP-9 activity was assessed with an MMP-9 activity assay in 1 microL of unstimulated tear fluid. Using conjunctival epithelial cells from 19 patients with DTS and 16 controls, levels of MMP-9 and its regulating cytokine mRNA transcripts were evaluated by semiquantitative real-time PCR. RESULTS: Each of four DTS severity-based groups had significantly higher mean MMP-9 activities than did the control group, which was 8.39 +/- 4.70 ng/mL. The DTS4 group had the highest MMP-9 activity (381.24 +/- 142.83 ng/mL), for which the mean was significantly higher than that of other DTS groups. In addition, patients with DTS had significantly higher levels of IL-1beta, IL-6, TNF-alpha, and TGF-beta1 mRNA transcripts in their conjunctival epithelia than did the control subjects. Tear MMP-9 activities showed significant correlation with symptom severity scores, decreased low-contrast visual acuity, fluorescein tear break-up time, corneal and conjunctival fluorescein staining, topographic surface regularity index (SRI), and percentage area of abnormal superficial corneal epithelia by confocal microscopy. CONCLUSIONS: Tear MMP-9 activity was significantly higher in patients with DTS. This activity was associated with increased mRNA expression of MMP-9 and its regulating genes and correlated strongly with clinical parameters. MMP-9 appears to be a potentially useful biomarker for diagnosing, classifying, and monitoring DTS.

Otago Glaucoma Surgery Outcome Study: the pattern of expression of MMPs and TIMPs in bleb capsules surrounding Molteno implants.

PURPOSE: To determine whether MMPs and TIMPs are present in the bleb wall of Molteno implants. METHODS: An observational case series consisting of ocular specimens from 10 human eyes obtained postmortem from patients who had undergone placement of Molteno implants for glaucoma. Immunohistochemistry was performed on the specimens to determine the distribution of MMP1, -2, and -3 and TIMP1, -2, and -3 in each bleb. The immunohistochemical staining was correlated with the histologic features observed in hematoxylin and eosin (H&E)-stained sections of the bleb wall. RESULTS: The specimens demonstrated extensive cytoplasmic staining for MMP1, -2, and -3 in fibroblasts within the bleb wall and in degenerating collagen near inner bleb wall fibroblasts undergoing apoptosis. In addition, there was staining for the presence of TIMP2 but not -1 or -3. CONCLUSIONS: MMPs are present in the bleb walls of Molteno implants. TIMP2 is expressed in most bleb capsules. The observations from this study support the hypothesis that bleb capsules undergo a cycle of collagen breakdown and renewal throughout the life of the bleb as members of the MMP family were localized in the bleb wall.

Role of cPKCalpha and nPKCepsilon in EGF-stimulated goblet cell proliferation.

PURPOSE: The authors determined the role of the protein kinase C (PKC) isoforms cPKCalpha and nPKCepsilon in EGF-stimulated proliferation of cultured rat and human conjunctival goblet cells. METHODS: Rat and human conjunctivas were minced, and goblet cells were allowed to grow. Passage 1 cells were serum starved for 24 to 48 hours and were incubated with the PKC inhibitors calphostin C and Gö 6983 (10(-10)-10(-7) M) for 20 minutes before stimulation with EGF (10(-7) M) for 24 hours. The presence and localization of PKC isoforms in cultured rat goblet cells were determined by Western blot analysis and immunofluorescence microscopy, respectively. Cultured rat goblet cells were serum starved and incubated with adenoviruses containing genes for dominant-negative cPKCalpha (Ad DNPKCalpha, 10(4) pfu), dominant-negative nPKCepsilon (Ad DNPKCepsilon, 10(4) pfu), and wild-type cPKCalpha (Ad WTPKCalpha, 10(7) pfu), and proliferation was measured. RESULTS: In rat goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C, whereas Gö 6983 inhibited proliferation by 53%+/-15%. In human goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C. PKCalpha, -betaI, -betaII, -delta, -epsilon, -iota/lambda, -theta, -gamma, and -zeta were found in cultured rat goblet cells. Ad DNPKCalpha and Ad DNPKCepsilon inhibited EGF-stimulated proliferation in rat goblet cells by 78%+/-6% and 92%+/-8%, respectively. Incubation with Ad WTPKCalpha alone significantly increased proliferation. CONCLUSIONS: cPKCalpha and nPKCepsilon play key roles in conjunctival goblet cell proliferation.

Increased mast cell numbers in the conjunctiva of glaucoma patients: a possible indicator of preoperative glaucoma surgery inflammation.

PURPOSE: Inflammation is a risk factor for scarring after trabeculectomy surgery. Mast cells are important mediators of inflammation and scarring in allergic eye disease. This exploratory project investigated the presence of mast cells in the conjunctiva of glaucoma patients having trabeculectomy surgery. METHODS: Conjunctival biopsies from glaucoma patients belonging to specific groups: medically treated glaucoma (M, n=6), repeat glaucoma surgery (S, n=8), and uveitic glaucoma (U, n=7). The control group (C, n=8) was retinal detachment patients undergoing repair surgery for the first time. Immunohistochemistry techniques stained for the presence of the intracellular mast cell enzyme tryptase. RESULTS: The median mast cell tryptase-positive counts for all glaucoma groups (M, S, and U) ranged from 0.102-0.113 cells/mm2 compared to 0.064 cells/mm2 for group C. This was statistically significant comparing group S to group C (P=0.0063), but not when comparing groups U or M to group C. The mast cell tryptase-positive counts did not significantly differ among the groups. CONCLUSIONS: Mast cell numbers were significantly increased in glaucoma patients who have previously undergone surgery (group S). Mast cell activity may contribute to the scarring process and the increased risk of excessive conjunctival scarring after trabeculectomy surgery. Further investigation needs to be performed to evaluate this potential role.