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Purpose: To investigate the molecular mechanism of pathological keratinization in the chronic phase of ocular surface (OS) diseases., Methods: In this study, a comprehensive gene expression analysis was performed using oligonucleotide microarrays on OS epithelial cells obtained from three patients with pathological keratinization (Stevens-Johnson syndrome [n = 1 patient], ocular cicatricial pemphigoid [n = 1 patient], and anterior staphyloma [n = 1 patient]). The controls were three patients with conjunctivochalasis. The expression in some transcripts was confirmed using quantitative real-time PCR., Results: Compared to the controls, 3118 genes were significantly upregulated by a factor of 2 or more than one-half in the pathological keratinized epithelial cells (analysis of variance P < 0.05). Genes involved in keratinization, lipid metabolism, and oxidoreductase were upregulated, while genes involved in cellular response, as well as known transcription factors (TFs), were downregulated. Those genes were further analyzed with respect to TFs and retinoic acid (RA) through gene ontology analysis and known reports. The expression of TFs MYBL2, FOXM1, and SREBF2, was upregulated, and the TF ELF3 was significantly downregulated. The expression of AKR1B15, RDH12, and CRABP2 (i.e., genes related to RA, which is known to suppress keratinization) was increased more than twentyfold, whereas the expression of genes RARB and RARRES3 was decreased by 1/50. CRABP2, RARB, and RARRES3 expression changes were also confirmed by qRT-PCR., Conclusions: In pathological keratinized ocular surfaces, common transcript changes, including abnormalities in vitamin A metabolism, are involved in the mechanism of pathological keratinization.

Purpose: The present study was designed to investigate the role of myofibroblast transdifferentiation and the conjunctival renin-angiotensin system (RAS) in the pathogenesis of graft-versus-host disease (GVHD)-associated conjunctival fibrosis., Methods: A mouse model of major histocompatibility-matched allogeneic transplantation was used to induce GVHD, with male B10.D2 mice as donors and female BALB/c mice as recipients. Male BALB/c to female BALB/c syngeneic transplantation was used as control. Y chromosome staining in the spleen cells obtained from female recipient mice was used to confirm engraftment. The phenol red thread test and fluorescein staining were used to quantify tears and corneal keratopathy. Eyes were harvested at 4 and 8 weeks after the transplant for alpha-smooth muscle actin (α-SMA), angiotensinogen, and angiotensin-converting enzyme (ACE) immunostaining. Conjunctiva was harvested for gene expression quantification of α-SMA, angiotensinogen, and ACE., Results: More than 80% of the spleen cells in the recipient mice were chromosome Y positive, thus conforming successful engraftment. A significant decrease in tear secretion and a marked increase in corneal keratopathy score after allogeneic transplantation indicated the onset of ocular GVHD in these mice. A significant increase in α-SMA gene expression and the presence of a large number of α-SMA-positive cells was noted in the bulbar orbital conjunctiva of mice after allogeneic transplantation. Allogenic transplantation also caused a significant increase in the gene expression and protein expression of angiotensinogen and ACE in the subconjunctival eyelid area., Conclusions: Results of the present study demonstrate that GVHD-associated conjunctival fibrosis is accompanied by myofibroblast formation and activation of the local conjunctival RAS.

Purpose: Conjunctivochalasis (CCH) is a common ocular disease and has received extensive attention recently. However, its exact pathogenesis remains largely unknown. Owing to the high morbidity of CCH in older people, this study aimed to investigate whether cellular senescence contributes to CCH progression and the underlying mechanism., Methods: Loose conjunctival tissues from CCH patients (n = 13) and normal conjunctival tissues from age-matched persons (n = 12) were obtained and the fibroblasts were separately induced and obtained. Cellular senescence, and the expression of senescence-associated genes (p53 and p21) and p38 in CCH conjunctival tissues and normal controls, were determined by senescence-associated β-galactosidase (SA-β-Gal) staining and quantitative (q)RT-PCR, respectively. To explore the effects of p38 on cellular senescence in CCH fibroblasts, small interfering RNA (siRNA) targeting p38 (siP38) and p38-specific inhibitor SB203580 was performed in CCH fibroblasts. Then, cellular senescence, cell viability, reactive oxygen species (ROS) production, and gene expression were detected according to the corresponding methods., Results: CCH conjunctival tissues had significantly more senescent cells, evidenced by more SA-β-Gal-positive cells, and higher expression of senescence-associated genes (p53 and p21) and p38. CCH fibroblasts transfected with siP38 or treated with SB203580 had obviously reduced numbers of senescent cells, decreased ROS production, and increased cell viability, as well as reduced expression of senescence-associated genes. Meanwhile, blocking p38 signaling decreased the expression of p53 and p21., Conclusions: Therefore, these findings indicate that cellular senescence might be a causative factor for CCH. P38 signaling might play an important role in the progress of cellular senescence in CCH fibroblasts via manipulation of p53/p21 signaling.

Purpose: Chemokines play a role in the progression and metastatic spread of both cutaneous and uveal melanomas. The aim of this study was to examine the prognostic value of expression of chemokine receptors CCR7, CXCR4, and CCR10 in conjunctival melanocytic lesions., Methods: In total, 44 conjunctival nevi, 21 cases of primary acquired melanosis (PAM) with atypia and 35 conjunctival melanomas, were included. After immunohistochemical staining for CCR7, CXCR4, and CCR10 the immunoreactive score (IRS) was determined. The findings were correlated for association with melanoma and development of metastasis. For mechanistic evaluation, we used a mouse melanoma metastasis model using two human conjunctival melanoma cell lines, CM2005.1 and CRMM1., Results: All tested chemokines showed a significantly higher expression in conjunctival melanoma than conjunctival nevi. There was a statistically significant difference between the IRS in nevi and PAM with atypia for nuclear IRS in CCR10 (P = 0.03) and both nuclear and cytoplasmic IRS in CXCR4 (P < 0.01 and P = 0.03, respectively); this was also true evaluating the groups PAM with atypia and melanoma all together (P < 0.01). Furthermore, a trend for lower IRS was seen in cases of melanoma without metastasis, with a suggestive pattern of a higher IRS in cases that did develop metastases, supported for CXCR4 using the mouse melanoma metastasis model., Conclusions: Expression of specific chemokines changes during the progression and metastatic spread of conjunctival melanocytic lesions. Differential chemokine profiles may hold prognostic value for patients with conjunctival melanomas and might be considered as a therapeutic target.

Purpose: To investigate the roles and pathways of microRNAs 143 and 145 in transforming growth factor (TGF)-β1-induced human subconjunctival fibrosis., Methods: Human tenon's capsule fibroblasts (HTFs) were obtained from a healthy eye. After treating cultured HTFs with TGF-β1, the expression of microRNAs 143 and 145 was evaluated using polymerase chain reaction. To identify the pathways of TGF-β1-induced microRNA 143/145 expression, HTFs were treated with specific inhibitors of p38MAPK, PI3K/Akt, JNK, ERK, and with siRNAs for SMAD2 and SMAD4. Mutagenesis studies were performed to evaluate the role of the CArG box and SMAD-binding element (SBE). To investigate the role of microRNA 143/145 in TGF-β1-induced myofibroblast transdifferentiation, microRNA 143/145 mimics and microRNA 143/145 inhibitors were applied to the HTFs., Results: Array analysis revealed that TGF-β1 induced the expression of microRNA 143/145 in a dose- and time-dependent manner. When inhibitors and siRNAs for p38MAPK, PI3K/Akt, ERK, and JNK were applied, the TGF-β1-induced expression of microRNA 143/145 was inhibited; however, SMAD2 and SMAD4 inhibition did not affect the TGF-β1-induced expression of these microRNAs. In the mutagenesis studies, both the CArG box and SBE were associated with TGF-β1-induced expression of microRNA 143/145. Mimics of microRNA 143/145 induced increased myofibroblast formation, whereas their inhibitors had the opposite effect., Conclusions: TGF-β1-induced human subconjunctival fibrosis was mediated by the expression of microRNA 143/145, mainly via SMAD-independent pathways. Inhibition of TGF-β1-induced microRNA 143/145 expression in HTFs might represent a novel strategy to prevent subconjunctival fibrosis.

Purpose: To investigate the effects and toxicities of gelatin hydrogel (GH) loading mitomycin C (MMC) on IOP and conjunctival scarring in a canine model of glaucoma surgery in comparison with conventional MMC application., Methods: Glaucoma surgery models were made in six beagles. An MMC-loaded GH was implanted under the conjunctiva of one eye (GH-MMC group) and 0.04% MMC-soaked sponges were placed under the conjunctiva of the other eye (MMC group) for 5 minutes. Intraocular pressures and bleb features were then assessed for 4 weeks postoperative, followed by histological evaluation. The ratio of conjunctival area to scleral area, the densities of collagen and the numbers of fibroblasts, vessels, and proliferative cell nuclear antigen (PCNA)-positive cells were then quantified., Results: In both groups, IOP reduction and bleb formation were maintained in a similar manner for 4 weeks postoperative. No significant difference in the ratio of conjunctival area to scleral area was found between the two groups. Collagen density and the numbers of fibroblasts and vessels were significantly lower in the MMC-treated group than in the GH-MMC-treated group. No significant difference in PCNA-positive cells was found between the two groups., Conclusions: Implantation of MMC-loaded GH ameliorated toxicity to conjunctiva compared with the 5-minute placement of MMC, whereas its effect on IOP reduction and bleb formation was similar. These results suggest that using GH for the application of MMC is a safer method than the conventional application of MMC in glaucoma filtration surgery.

Aging of the ocular surface and corneal tissues, major components of the visual system, causes major eye disease and results in substantial cost in medical and social terms. These diseases include the highly prevalent dry eye disease that affects the ocular surface and its glands, leading to tear film alterations, discomfort, and decreased vision. Studies show that 14.4% of the population in the United States older than 50 years have dry eye disease and demonstrate that it is particularly prevalent among women. Annual medical costs per patient with dry eye in the United States are estimated at $783 per year, with an overall medical cost adjusted to prevalence of $3.84 billion per year. Societal costs, which include loss of productivity, are estimated per patient at $11,302 per year, with overall costs adjusted to prevalence of $55.4 billion per year. Because there are few effective treatments for the disease, more research on its etiology and mechanisms is warranted and needed. Increased public education about risk factors for the disease is also required. Another major age-related eye disease of the cornea that leads to vision impairment and potentially blindness if left untreated is Fuchs' endothelial corneal dystrophy. This disease leads to loss of the endothelial cells on the internal side of the cornea that are responsible for keeping the cornea in the proper hydration state to ensure its transparency to light. The mechanism of cell loss is unknown, and the only treatment available to date is surgical transplantation of the cornea or inner part of the cornea. These medically costly procedures require donor corneas, eye banking, and medical follow-up, with accrued costs. Fuchs' endothelial corneal dystrophy is a major cause of corneal transplantation in the United States; therefore, research support is needed to determine the mechanism of this age-related disease, to develop medical, nonsurgical methods for treatment.

Purpose: This study investigated the effects of dietary α-lipoic acid (α-LA) against ultraviolet B (UVB)-induced corneal and conjunctival degeneration in a mouse model., Methods: Female CBA mice were randomly divided into five study groups, including blank control, UVB without α-LA, and UVB with dietary α-LA at 1, 10, and 100 mg/kg body weight. Following UVB exposure, corneal surfaces were assessed along with immunohistochemistry for nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), malondialdehyde (MDA) accumulation, and P63⁺ basal cell distribution. Matrix metalloproteinase (MMP)-2 and MMP-9 activities were determined by gelatin zymography. ELISA assay was performed to confirm the findings of immunohistochemistry for NF-κB, COX-2, and MDA, along with the levels of TNF-α and IL-6. Tear production and goblet cell density were determined after tear strip assay and periodic acid Schiff staining, respectively., Results: The results showed that UVB irradiation caused corneal surface damage, polymorphonuclear leukocyte infiltration, and loss of P63⁺ basal cells. Dietary α-LA ameliorated the UVB-induced corneal damage while simultaneously reducing MDA accumulation and maintaining P63⁺ basal cell survival. NF-κB-p65, COX-2, TNF-α, IL-6, and MMP-9 activity were all reduced by dietary α-LA. In addition, α-LA helped to reverse aqueous tear reduction, conjunctival squamous epithelium metaplasia, and goblet cell loss after UVB exposure., Conclusions: Dietary α-LA can prevent UVB-induced corneal damage and can be used as a prophylactic agent prior to excessive UVB exposure.

Purpose: For the successful topical administration of drugs or vaccines to treat ocular surface diseases, efficient and well-tolerated delivery systems/carriers for conjunctival delivery are crucial in the development of new treatment strategies. The present study investigated the efficiency of internalization of bacterial ghosts (BGs) produced from probiotic Escherichia coli Nissle 1917 (EcN) by human conjunctival epithelial (HCjE) cell line, the EcN BGs cytotoxicity for HCjE cells, and in vivo uptake of EcN BGs by conjunctival guinea pig epithelial cells., Methods: The uptake of EcN BGs by HCjE cells was analyzed by laser scanning microscopy and flow cytometry. Immunohistochemistry was used to localize the EcN BGs in the guinea pig conjunctival tissue. Cytotoxicity of EcN BGs on HCjE cells was evaluated by measurement of LDH., Results: Laser scanning microscopy and flow cytometry revealed that EcN BGs internalization by HCjE cells was time- and dose dependent. No cytotoxic effect on HCjE cells was observed after EcN BGs inoculation for 30 and 120 minutes, as well as 24 hours. In addition, the uptake of EcN BGs was detected in the conjunctival cells after in vivo administration of EcN BGs into the eye of the guinea pig., Conclusions: The findings that EcN BGs are nontoxic and effectively internalized in vitro by human and in vivo by guinea pig conjunctival cells comprise an important contribution to the future use of BGs as a system for conjunctival delivery of drugs and vaccines, either to treat or prevent ocular surface diseases.

Purpose: Conjunctivochalasis (CCh) is an age-related inflammatory ocular surface disease manifesting redundant, loose conjunctiva folds. The pathogenic role of Pentraxin 3 (PTX3) in controlling upregulation of matrix metalloproteinase 1 (MMP-1) and MMP-3 in CCh remains undefined., Methods: Cytolocation of PTX3 and apoptosis were compared by immunostaining and terminal deoxyribonucleotidyl transferase-mediated FITC-linked dUTP nick-end DNA labeling (TUNEL) assay between normal and CCh specimens containing the conjunctiva and the Tenon. Second to third cultures of normal and CCh fibroblasts were treated with or without Aprotinin, Batimastat, or N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH), followed by transfection with or without PTX3 siRNA, and TNF-α or IL-1β. Cell lysates and culture media were collected to assess apoptosis measured by the Cell Death Detection ELISA and expression of PTX3, MMP-1, and MMP-3 transcripts and proteins by quantitative RT-PCR and Western blot, respectively., Results: PTX3 immunostaining was negative in normal specimens, but strongly positive in the subconjunctival stroma of CCh specimens. More apoptotic cells were found in CCh samples than in normal specimens. Expression of PTX3 transcripts and protein was not constitutive in resting normal fibroblasts but was in resting CCh fibroblasts and was upregulated by IL-1β in both cell lysates and culture media of both fibroblasts. PTX3 siRNA further upregulated MMP-1 and MMP-3 transcripts in resting normal fibroblasts, but synergistically with IL-1β upregulated the expression of MMP-1 and MMP-3 transcripts only in CCh fibroblasts, with activation of MMP-1 more so than MMP-3. PTX3 siRNA knockdown also promoted cell death characterized by apoptosis and necrosis, and such cell death could be rescued by inhibitors against serine proteinase, MMP1, or MMP3., Conclusions: Perturbation of PTX3 expression might partake in apoptosis and pathogenesis of CCh by upregulating expression of MMP-1 and MMP-3, and activation of MMP-1 and MMP-3.

Purpose: To investigate the effects of gelatin hydrogel (GH) containing a chymase inhibitor (CI) on intraocular pressure (IOP) and conjunctival scarring in a canine model of glaucoma surgery., Methods: Glaucoma surgery models were made in beagles. As the first experiment, GH was implanted. IOP was measured for 4 weeks, followed by histologic evaluation. As the second experiment, GH containing a CI or GH alone was implanted. IOP and bleb features were evaluated for 12 weeks. The densities of proliferative cell nuclear antigen (PCNA)-positive cells, fibroblasts, and mast cells were quantified. The mRNA levels of transforming growth factor-β (TGF-β) and chymase were determined by real-time PCR., Results: In the first experiment, IOP was significantly lower in the eyes treated with GH than that in the control eyes. The conjunctival area normalized by the scleral area was reduced in the treated eyes. In the second experiment, IOP reduction was maintained for 12 weeks in the eyes treated with GH containing a CI, but not in the eyes treated with GH alone. In addition, the bleb score was larger, whereas the adhesion score and densities of PCNA-positive cells, fibroblasts, mast cells, and chymase-positive cells were lower in the eyes treated with GH containing a CI. The mRNA levels of TGF-β and chymase were significantly decreased in the eyes treated with GH containing a CI., Conclusions: Implanting GH alone maintained IOP reduction, whereas GH containing a CI enhanced the IOP-reducing effect by suppressing cell proliferation. This drug delivery system might be useful for maintaining filtering blebs for a longer duration after glaucoma surgery.

Purpose. To investigate the status of oxidative stress and histopathologic alterations in patients with conjunctivochalasis and compare the findings with those in healthy control subjects. Methods. Eleven patients (n = 20 eyes) with Yokoi grade 3 conjunctivochalasis and 11 health control subjects (n = 22 eyes) were prospectively recruited. ELISA for tear hexanoyl-lysine (HEL) and inflammatory cytokines, tear film break-up time tests, Schirmer test measurements, and fluorescein and rose bengal vital staining were performed. Conjunctival specimens obtained during surgery for conjunctivochalasis and cataract underwent immunohistochemical staining for HEL+8-OHdG, MMP-3, and MMP-9, and positively stained cells were counted. Transmission electron microscopy was also performed, with staining for elastic fibers in the conjunctival stroma. Results. The mean tear stability and vital staining scores were significantly worse in the conjunctivochalasis patients than in the control subjects. The tear HEL and tear cytokine levels showed significantly higher values in eyes with conjunctivochalasis. IL-1beta and IL-6 levels showed a significant correlation with corneal epithelial damage. IL-1beta and TNFalpha showed a significant correlation with 8-OHdG-stained cell counts. Specimens from patients with conjunctivochalasis revealed a significantly higher number of cells positively stained for HEL, 8-OHdG, MMP-3, and MMP-9 than did specimens from age- and sex-matched control subjects. Transmission electron microscopy showed decreased intercellular cohesiveness, with the conjunctival stroma showing an accumulation of elastic fibers. Conclusions. Lipid and DNA oxidative stress were present in the conjunctiva. Increased tear inflammation seemed to coexist with loss of conjunctival epithelial cohesiveness and increased collagenolytic activity, which may explain the conjunctival laxity observed in patients with conjunctivochalasis.

Purpose: To evaluate goblet cell density (GCD) on conjunctiva and cornea in patients with ocular chemical burns by in vivo laser scanning confocal microscopy (LSCM) and impression cytology (IC) and to explore the correlation between two methods., Methods: Fifty-four patients (58 eyes) with chemical burn were enrolled in the study. LSCM was applied to identify the goblet cells on conjunctiva and cornea under in vivo conditions, and GCD was analyzed with the customized software. Impression cytology was then performed, and the biopsy specimens were stained to visualize goblet cells in vitro and to measure the density. Statistical software was used to analyze the correlation between GCD taken by two methods., Results: Conjunctival goblet cells could be discriminated in 55 eyes and 57 eyes by in vivo LSCM and IC. They could be identified on the cornea in nine eyes and eight eyes by two methods. The positive rate of two methods had no significant difference. GCDs on conjunctiva measured by in vivo LSCM and IC were 136 +/- 79 cells/mm(2) and 121 +/- 66 cells/mm(2). Median GCDs on cornea detected by two methods were 30 cells/mm(2) and 23 cells/mm(2), respectively. A significant positive correlation was found between the GCDs on conjunctiva measured by these two methods as well as the GCDs on cornea., Conclusions: GCD decreased in patients with chemical burns. A positive correlation was found between GCD measured by in vivo LSCM and IC after chemical burns. In vivo LSCM was a promising device to study goblet cells in vivo under pathologic conditions.

Purpose: To investigate the differentiation of conjunctival epithelium and tear film function in pingueculae., Methods: Twelve patients (12 eyes) who underwent simple excision for pingueculae were enrolled in the study. Immunostaining for K14, K19, K10, MUC5AC, Pax6, P63, Ki67, and K16 was performed on the pinguecula epithelium and normal conjunctival epithelium. Schirmer I test results and tear film break-up time (TFBUT) were evaluated just before and 1 month after surgery., Results: Abnormal epidermal differentiation of pinguecula tissue, as evidenced by a decline in or absence of Pax6 expression, was accompanied by a decline in or absence of K19 keratin and MUC5AC, and an increase in K10 and K14 keratin. Furthermore, the pinguecula epithelium was actively proliferating, as evidenced by positive expression of Ki67, P63, and K16 keratin. The Schirmer I test results did not indicate any significant differences before and after surgery. However, the TFBUT was significantly prolonged 1 month after surgery compared with before surgery., Conclusions: Pinguecula is a condition of abnormal epithelial differentiation. It is characterized by squamous proliferation and metaplasia, resulting in instability of tear film with normal basic tear secretion.

Purpose: To explore the inhibitive effect of SB-431542 (an ALK5 inhibitor) on scar formation after glaucoma surgery and to identify the potential pharmacologic target(s)., Methods: Twenty-four New Zealand rabbits underwent filtration surgery on the right eye and were divided into a control group and three experimental groups (n=6). Human Tenon's fibroblast monolayer was scraped to generate a single gap, and then the control medium with SB-431542 only or containing 10 microg/L TGF-beta1 and SB-431542 (1-20 microM) was added. The cells were pretreated with SB-431542 or in control medium for 30 minutes before induction with 10 microg/L TGF-beta1 or 1 microg/L TGF-beta2. The expression of alpha-SM-actin, CTGF, and Col I, as well as changes in the Smad, ERK, P38, and AKT signaling pathways were detected., Results: In comparison with the control rabbits, the IOPs in the experimental groups remained at lower levels until day 25 (P<0.05) after the surgery. Histologic profiles showed that there was only a mild deposition of collagen in the subconjunctival space in the experimental groups. The cell growth and migration were inhibited effectively by SB-431542, regardless of whether TGF-beta was present in the culture system. SB-431542 abrogated TGF-beta-induced upregulation of alpha-SM-actin, CTGF, and Col I. It effectively inhibited the phosphorylation of Smad2 stimulated by TGF-beta but not that of the components of the MAPK pathways., Conclusions: SB-431542 inhibits scar formation after glaucoma filtration surgery. The mechanism may be that SB-431542 interferes in the phosphorylation of Smad2, thus abrogating TGF-beta-induced fibroblast transdifferentiation and then decreasing Col I synthesis.

Purpose: The study was conducted to analyze, by in vivo confocal microscopy (IVCM), the conjunctival epithelial characteristics in untreated ocular hypertension (OH) and in topically treated primary open-angle glaucoma (POAG)., Methods: The study included 30 eyes affected with untreated OH, 96 eyes with POAG receiving medical therapy, and 15 healthy control eyes. The main outcome measures were the mean density and the mean area of conjunctival epithelium microcysts. The relations among the microscopic parameters intraocular pressure (IOP), and age in both hypertensive and glaucomatous eyes and between mean defect (MD) of visual fields and the time on therapy in patients with glaucoma were analyzed., Results: There was no evidence of conjunctival microcysts in any of the healthy eyes examined; conversely, conjunctival microcysts were found in all ocular hypertensive eyes (mean microcyst density of 19.7 +/- 3.5 cysts/mm(2) and mean total microcyst area of 4063.6 +/- 921.2 mum(2)). All patients with POAG showed conjunctival microcysts (mean density of 28.7 +/- 2.7 cysts/mm(2) and a mean total microcyst area of 6564.2 +/- 671.4 mum(2)). No significant differences were found between OH and POAG subjects for microcyst parameters and no significant relations were found in either OH or POAG eyes for microcyst density, area, IOP, MD, and time on therapy., Conclusions: The results of the study show that conjunctival microcysts are features present in all hypertensive and glaucomatous eyes. Based on these findings, conjunctiva could be an additional potential target tissue available for the investigation by a noninvasive in vivo approach of glaucoma-induced pathologic modifications.

Purpose: Postoperative subconjunctival wound healing remains the commonest cause of late bleb failure after glaucoma filtration surgery. This study was undertaken to investigate whether the human monoclonal antibody that neutralizes transforming growth factor-beta2 (CAT-152; lerdelimumab) could be used as a postoperative agent to prevent scarring after glaucoma surgery and compared it with 5-fluorouracil (5-FU), to benchmark its potential clinical benefit., Methods: In a randomized, controlled, masked-observer study, after modified glaucoma surgery, 48 rabbits were randomly allocated to receive a postoperative course of seven subconjunctival injections of CAT-152 (1 mg/mL), 5-FU (50 mg/mL), or no treatment. Bleb characteristics, the presence of subconjunctival drainage, and local reaction to treatment were assessed. Animals were killed on days 10, 21, and 30. Immunohistochemistry, histologic staining and electron microscopy were performed to demonstrate the mechanism of CAT-152-mediated effects on the extracellular matrix., Results: CAT-152 significantly improved surgical outcome (log rank test, P < 0.001) and reduced subconjunctival collagen deposition (P < 0.01) compared with 5-FU and control. Median bleb survival was increased in the CAT-152 group (23.5 days) compared with the 5-FU (20 days) and control (16 days) treatment groups. CAT-152 treatment improved bleb morphology (P < 0.05) and was well tolerated. 5-FU prolonged the duration of corneal epitheliopathy (P < 0.01)., Conclusions: Postoperative administration of CAT-152 significantly improved surgical outcome, reduced subconjunctival scarring, and minimized the risk of corneal side effects compared with the anti-scarring agent 5-FU. These findings suggest that CAT-152 may offer therapeutic benefit as a postoperative agent to prevent subconjunctival scarring after glaucoma filtration surgery.

Purpose: Increased stromal accumulation of macrophages and submucosal fibrosis due to excessive accumulation of collagens are central histologic features in ocular cicatricial pemphigoid (OCP). Interleukin (IL)-4 plays an important role in both the inflammatory and fibrotic events in several human and experimental diseases. In the present study, the possible role of IL-4 in the pathogenesis of OCP was investigated., Methods: Biopsy specimens from the conjunctivae of 10 patients with OCP and 5 normal subjects were studied for the expression of IL-4 by immunohistochemistry. The expression level of IL-4 was also examined in conjunctival fibroblasts of normal control subjects and patients with OCP. The effects of IL-4 in the induction of inflammatory and fibrogenic molecules was studied in IL-4-treated conjunctival fibroblasts, and the expression levels of macrophage colony-stimulating factor (m-CSF), heat shock protein (HSP)-47 and type I collagen was determined by quantitative real-time PCR. The level of IL-4 was also measured by enzyme-linked immunosorbent assay (ELISA) in serum samples obtained from patients with OCP during active stage and remission and were compared with the levels in control sera., Results: Compared with the weak expression of IL-4 in the normal conjunctival sections, an increased expression of IL-4 was noted in conjunctival sections of patients with OCP. A similar increase in the expression of IL-4 was also detected in fibroblasts isolated from conjunctiva of patients with OCP, compared with control fibroblasts. Real-time PCR and ELISA detected a significantly increased level of m-CSF, at both the mRNA and protein levels in IL-4-stimulated cells. Similarly, IL-4 treatment resulted in the induction of type I collagen and collagen-binding HSP47 by conjunctival fibroblasts, as detected by real-time PCR. However, no apparent changes in the levels of IL-4 were detected by ELISA in serum samples of patients with OCP and control subjects., Conclusions: Increased conjunctival expression of IL-4 may play an important role in the regulation of local accumulation of macrophages (by inducing m-CSF), and matrix accumulation (by inducing HSP47 and collagen) during conjunctival scarring in patients with OCP. IL-4, therefore, may augment or enhance both conjunctival inflammatory and subsequent fibrotic responses in patients with OCP.

Purpose: Local proliferation of macrophages has been reported to augment the inflammatory response in various human and experimental diseases. Macrophage accumulation in the submucosa is also an important feature in the pathogenesis of ocular cicatricial pemphigoid (OCP). In the present study, the role of local proliferation of macrophages in conjunctiva affected by OCP and the relationship between local proliferation of macrophages and expression of macrophage-colony-stimulating factor (m-CSF) in such conjunctiva were examined., Methods: Biopsy specimens from the conjunctiva of 10 untreated patients with active OCP and from 5 normal subjects were studied for the expression of m-CSF, macrophages, and proliferating cell nuclear antigen (PCNA), a cell cycle protein, by immunohistochemistry. Dual staining for CD68 (a cell surface marker for macrophages) and PCNA was also performed to identify proliferating macrophages. In addition, fibroblasts isolated from conjunctiva of normal individuals and from patients with OCP were studied for the expression of m-CSF by immunostaining and real-time PCR. To identify the factors that induce m-CSF in conjunctival fibroblasts, the fibroblasts were incubated with different concentrations of interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha, and the levels of m-CSF mRNA were determined by real-time PCR and the amount of m-CSF produced was determined by enzyme-linked immunosorbent assay (ELISA)., Results: Normal conjunctiva showed weak expression of m-CSF in the conjunctival epithelial cells and stroma. Conjunctival expression of m-CSF protein was significantly (P < 0.0001) increased in conjunctival biopsy specimens from patients with OCP. m-CSF was detected in the infiltrating macrophages, stromal cells (presumably fibroblasts), and conjunctival epithelial cells. Compared with normal control conjunctival tissue, a 1.2-fold increase in the expression of mRNA for m-CSF was detected by real-time PCR in the conjunctival tissue obtained from patients with OCP. Increased expression of m-CSF correlated significantly (P < 0.0004) with an increased stromal accumulation of macrophages in conjunctival biopsy specimens of patients with OCP. A number of these accumulated macrophages (CD68-positive) were found to be proliferating (PCNA-positive). In addition, fibroblasts isolated and cultured from conjunctiva of patients with OCP showed significantly increased (1.7-fold) expression of m-CSF compared with normal conjunctival fibroblasts. When conjunctival fibroblasts were treated with IL-1alpha or TNF-alpha, real-time PCR and ELISA detected an increased level of m-CSF., Conclusions: An increased expression of m-CSF was observed in conjunctiva from patients with active OCP. There was a positive correlation between expression of m-CSF and accumulation of macrophages in conjunctival biopsy sections obtained from patients with OCP. Increased expression of m-CSF, mainly by conjunctival fibroblasts and infiltrating inflammatory cells, may play an important role in the regulation of local proliferation of macrophages in OCP. In the conjunctiva of patients with OCP, this process could augment or enhance the local inflammatory response and tissue injury consequent to it.

Purpose: In severe ocular surface diseases, pathologic keratinization of the ordinarily nonkeratinized corneal and conjunctival mucosal epithelia results in severe visual loss. The expression in conjunctivalized corneas of various proteins known to play important roles in the physiological keratinization process in human epidermis was examined to better understand the mechanism of keratinization., Methods: Conjunctiva covering the cornea was examined in 12 eyes with ocular surface disease in the chronic cicatricial phase. These comprised four Stevens-Johnson syndrome, four ocular cicatricial pemphigoid, and four chemical injuries. Normal conjunctivas from four age-matched individuals served as controls. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate transglutaminase 1 gene expression and immunohistochemistry to study the expression of transglutaminase 1 protein along with other keratinization-related proteins (involucrin, loricrin, filaggrin, and cytokeratins 1 and 10) and cytokeratin pairs 4/13 and 3/12., Results: Semiquantitative RT-PCR showed that transglutaminase 1 mRNA expression was upregulated in keratinized conjunctiva compared with normal. Also, in this tissue, immunohistochemistry demonstrated elevated levels of transglutaminase 1, involucrin, filaggrin, and the cytokeratin pair 1/10. Levels of loricrin and cytokeratin pairs 4/13 and 3/12, however, remained the same., Conclusions: Various keratinization-related proteins, transglutaminase 1 included, are most likely involved in the pathogenesis of cicatrizing ocular surface diseases.

Purpose: Overexpression and increased activities of matrix metalloproteinases (MMPs) have recently been reported in cultured conjunctival fibroblasts from patients with conjunctivochalasis. The role of inflammatory cytokines in modulating expression of MMPs, their tissue inhibitors (TIMPs), and urokinase plasminogen activator (uPA) as potential contributors to the pathogenesis of conjunctivochalasis was investigated., Methods: Interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha) was added at 10 ng/ml to a serum-free medium. Expression of transcripts and proteins of MMPs, TIMPs, and uPA by cultured normal human conjunctival and conjunctivochalasis fibroblasts was determined by Northern hybridization, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. Gelatin and casein zymographies were performed in serum-free conditioned media with and without the respective enzyme inhibitors., Results: Without challenging the cells, conjunctivochalasis fibroblasts showed mRNA and protein overexpression of MMP-1 and MMP-3 compared with normal conjunctival fibroblasts, which showed minor or no expression of these enzymes. IL-1beta markedly and TNF-alpha to lesser extent increased mRNA and protein expression of MMP-1 and MMP-3 in conjunctivochalasis fibroblasts from 2 subjects when compared with normal conjunctival fibroblasts from 2 subjects and with their nonstimulated counterparts. In conjunctivochalasis fibroblasts and normal conjunctival fibroblasts, TNF-alpha, but not IL-1beta, induced a gelatinolytic activity of MMP-9, which was further confirmed by Western blot analysis and ELISA. Expression of MMP-2, TIMP-1, and TIMP-2 mRNA and protein was not influenced by IL-1beta or TNF-alpha, and no difference was found in the gelatinolytic activity of MMP-2 between both cell types., Conclusions: Inflammatory cytokines such as IL-1beta and TNF-alpha, which can potentially be derived from the ocular surface and tears, may be responsible for increased expression of MMPs in cultured conjunctivochalasis fibroblasts. Ocular inflammation might be one important denominator in the pathogenesis of conjunctivochalasis.

Purpose: To establish objectively measurable characteristics of the conjunctival vasculature that correspond with the judgment of erythema by human observers., Methods: Color images of bulbar conjunctiva from 21 subjects were digitally analyzed to extract the following variables characteristic of the scene: vessel width (W), number of vessels (V), proportion of area occupied by vessels (PA), relative redness both in vessels (RRV) and in the whole image (RRI), red-green difference both in vessels (RGV) and in the whole image (RGI), red-blue difference both in vessels (RBV) and in the whole image (RBI), and red hue value (RHV). These data were compared with subjective judgments by a panel of seven trained observers who independently rated erythema in the same images, using a 0 to 4 scale with decimal interpolation between grades., Results: Correlation analysis indicated significant associations (P<0.05) between the mean response of the human observers and all the objective variables except RHV. Associations with the morphometric variables PA (R2 = 0.93) and V(R2 = 0.90) were markedly stronger than for the best colorimetric variable RBV (R2 = 0.62)., Conclusions: Judgments of erythema made by human observers do not rely primarily on color but can be closely approximated by a univariate, linear model involving only the proportion of the scene occupied by vessels. Under the conditions of this study, grading of erythema by trained observers can be considered to constitute measurement to at least an interval level.

Purpose: To determine whether conjunctivochalasis, denoting redundant, loose, nonedematous inferior bulbar conjunctiva, is associated with increased expression and activity of matrix metalloproteinases (MMPS) over their tissue inhibitors (TIMPs)., Methods: Expression of transcripts and proteins of MMPs, TIMPs, and urokinase plasminogen activator (uPA) by cultured normal human conjunctival and conjunctivochalasis fibroblasts was determined by Northern hybridization, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis, respectively. Gelatin and casein zymography and quantitative collagenase activity assay were performed in the serum-free conditioned media., Results: Compared with normal conjunctival fibroblasts from six subjects, conjunctivochalasis fibroblasts from eight patients showed markedly increased transcript expression of MMP-1 (5- to 32-fold) and MMP-3 (4 to 30-fold), whereas that of MMP-2, TIMP-1, TIMP-2, and uPA was similar between the two groups. Protein levels were increased in the serum-free conditioned media of conjunctivochalasis fibroblasts for MMP-1 (3.5- to 7.6-fold) and MMP-3 (2.3- to 13-fold), determined by ELISA and Western blot analysis. There was increased caseinolytic activity of MMP-3 and collagenolytic activity of MMP-1 (2.2-fold) by conjunctivochalasis fibroblasts, whereas no difference was noted between these two types of fibroblasts in the protein and gelatinolytic activity of MMP-2 or expression of TIMP-1 and TIMP-2 proteins, although that of TIMP-1 transcript was slightly higher in some conjunctivochalasis fibroblasts. No expression of MMP-9 was detected., Conclusions: Overexpression of MMP-1 and MMP-3 mRNA by conjunctivochalasis fibroblasts is correlated with their increased protein levels and proteolytic activities. Collectively, these data help explain how conjunctivochalasis manifests excessive degradation of the conjunctival matrix and Tenon's capsule.

Purpose: Currently available anti-scarring regimens for glaucoma filtration surgery have potentially blinding complications and thus the need for alternative and safer agents. The effects of a new antibody to transforming growth factor (TGF)-beta2 on in vitro and in vivo conjunctival scarring and after glaucoma filtration surgery were investigated., Methods: The activity of a novel recombinant monoclonal neutralizing antibody (mAb) to human TGF-2 (rhAnti-TGF-beta2 mAb) was studied in conjunctival fibroblast-mediated proliferation, migration, and collagen contraction. Its safety in subconjunctival administration was assessed in vivo, and, in a rabbit model of glaucoma filtration surgery, its effects on conjunctival scarring and filtration surgery outcome were investigated., Results: The rhAnti-TGF-beta2 mAb effectively inhibited TGF-beta2-mediated conjunctival scarring activity in vitro, at 50% inhibitory concentrations (IC50) of less than 1 nM. It significantly improved glaucoma filtration surgery outcome in an animal model of aggressive conjunctival scarring compared with control (P = 0.0291) and was clinically safe, nontoxic, and well tolerated after subconjunctival administration., Conclusions: Subconjunctival rhAnti-TGF-beta2 mAb treatment significantly affects surgical outcome and effectively reduces conjunctival scarring both in vitro and in vivo. It appears safe for subconjunctival administration and when compared with mitomycin-C treatment histologically, much less destructive to local tissue. rhAnti-TGF-beta2 mAb may have potential as a new anti-scarring agent for use in glaucoma filtration surgery.

Purpose: To compare the effects of the three human isoforms of transforming growth factor (TGF)-beta in vivo using a mouse model of conjunctival scarring, both in normal eyes and after treatment with MMC, with a view to delineating the role of this growth factor in glaucoma filtration surgery., Methods: Application of recombinant human TGF-beta was assessed in a prospective, randomized study of mouse conjunctival scarring, in which subconjunctival TGF-beta1, -beta2, and -beta3 (all 10(-9) M) were compared with control (phosphate-buffered saline [PBS] carrier) and mitomycin C (MMC; 0.4 mg/ml) treatment at 6 hours, and 1, 3, and 7 days after surgery (six eyes/treatment/time point). Effects of TGF-beta2 on eyes previously treated with MMC were also assessed. Histologic studies of enucleated eyes were performed to analyze development of the scarring response, extracellular matrix deposition, and the inflammatory cell profile., Results: All three isoforms of TGF-beta behaved in a similar manner in vivo, being associated with a rapid-onset and exaggerated scarring response compared with control and MMC treatment. TGF-beta-treated eyes showed evidence of an earlier peak in inflammatory cell activity (P < 0.05) and increased collagen type III deposition (P < 0.05). TGF-beta2 treatment significantly stimulated scarring after MMC application (P < 0.05)., Conclusions: TGF-beta1, -beta2, and -beta3 appear to have similar actions in vivo and stimulate the conjunctival scarring response. Application of TGF-beta2 modified the effects of MMC. All TGF-beta isoforms may be potent modulators of the conjunctival scarring response. These studies indicate that TGF-beta2 may naturally modify the antiscarring effects of antimetabolites such as MMC in glaucoma filtration surgery.

Purpose: Recent studies have suggested the involvement of nerve growth factor (NGF) in the conjunctival inflammatory process and in corneal epithelium proliferation and differentiation. To verify the hypothesis that NGF could locally modulate the inflammatory and reparative processes, the authors evaluated the expression of NGF high-affinity receptor on the ocular surface in normal and pathologic conditions., Methods: Ten conjunctival biopsies (obtained from three healthy subjects, five patients affected by vernal keratoconjunctivitis [VKC], and two patients with cicatricial pemphigoid [CP]) and five corneal specimens obtained from the Eye Bank of Veneto (Italy) were evaluated. All specimens were histologically stained, and immunohistochemistry was performed to identify the NGF high-affinity receptor (TrkA)., Results: All tissues expressed immunoreactivity for NGF receptors. In conjunctival specimens of healthy subjects, basal epithelial cells strongly expressed immunoreactivity and, in the stroma, rare cells were immunopositive for TrkA. No significant difference in immunoreactivity was observed in the conjunctival epithelium between healthy subjects and patients with inflammatory conjunctival diseases, whereas there were more immunopositive cells observed in the conjunctival stroma of VKC and CP patients than in the controls. The immunoreactivity in the cornea was confined to basal epithelial cells and endothelium., Conclusions: The NGF receptor is present on the human ocular surface. The authors' data support the possibility that NGF modulates ocular inflammation and corneal epithelial proliferation and differentiation through its receptors.

We investigated the cellular profile of inflammatory infiltrate during development of a guinea pig conjunctival basophil hypersensitivity lesion at 1, 6, 24, 48, and 72 hr following injection of keyhole limpet hemocyanin (KLH). Neutrophils reached maximal accumulation at 6 hr. Eosinophils and basophils reached maximal accumulation at 24 hr. Basophil numbers remained high at 48 hr and were lower at 72 hr. The number of stromal monocytes did not vary significantly from PBS-injected tissues at any time. Mast cell number decreased at 24 hr following antigen challenge. Morphologic evidence for intense secretory activity was seen in cells at the stromal reaction site. Secretion channels and evidence of intense degranulation was seen from electron microscopy of neutrophils, eosinophils, and basophils. Inflammatory cells migrate through the basement membrane into the conjunctival epithelium. After maximal accumulation there was a decrease in the number of inflammatory cell types in the epithelium.

The present histological studies have demonstrated that histamine causes dose-dependent eosinophil infiltration into the conjunctiva. A highly directional movement toward the conjunctival epithelium was observed, and the presence of large numbers of degranulating eosinophils appeared to result in epithelial cell damage and goblet cell discharge. Blockade of H2-receptors by systemic cimetidine pretreatment significantly inhibited the eosinophil infiltration elicited by an intermediate histamine dose, whereas the H1-receptor blockade produced by systemic pyrilamine pretreatment markedly reduced the response to all histamine doses. The pyrilamine-insensitive residual eosinophil infiltrate was not affected by administering a cimetidine-pyrilamine combination. In animals presensitized to ovalbumin, antigen challenge evoked extensive and directional emigration of eosinophils toward the conjunctival epithelium with resultant exfoliation and depletion of goblet cell populations. In conjunctival immediate hypersensitivity, neither cimetidine nor pyrilamine alone produced an inhibitory effect, but a cimetidine-pyrilamine combination caused a significant reduction in the number of infiltrating eosinophils and prevented epithelial damage and goblet cell depletion. These results suggest that histamine may participate in the recruitment of eosinophils during immediate hypersensitivity reactions. The differential effect of pyrilamine on the eosinophil infiltration evoked by histamine or immediate hypersensitivity may, perhaps, reflect the importance of increased microvascular permeability in facilitating eosinophil emigration.

Goblet cell counts have been used to evaluate the suitability of conjunctiva as a source of ocular surface epithelial cells. However, since tear mucin content can be determined without tissue excision, it seemed that the concentration of those compounds might be a useful indicator of conjunctival vitality. To test the extent to which aqueous tear composition reflects conjunctival goblet cell frequency, goblet cell frequency and aqueous tear mucin content were measured after alkali injury in rabbits. Mild alkali injury (0.1 N NaOH for 30 sec) caused a transient but substantial decrease in goblet cells (to 25% of normal at day 7) with a return to normal by six weeks. Tear mucin content was decreased to a lesser degree, from a normal value of 6.4 +/- 0.47 nmol oligosaccharide per microliter (n = 10) to a minimal value of 4.7 +/- 0.64 (n = 7) (73% of normal) at day 7, returning to normal 4 weeks after injury. Thus, the direction of the change was the same, but the magnitudes were quite different. These results suggest that conjunctival goblet cell frequency is not accurately reflected by aqueous tear mucin content, and therefore, that tear mucin content cannot be used directly as an indicator of conjunctival health.

Although interactions of Toxoplasma gondii with host cells have been studied extensively in vitro, relatively little is known about the initial interactions of Toxoplasma with mucosal surfaces in vivo. We therefore studied the onset of a Toxoplasma infection in guinea pig conjunctiva. Toxoplasma were inoculated onto the conjunctival epithelium. The tissue was fixed 15 min to 48 hr after inoculation and examined by electron microscopy. Guinea pigs similarly inoculated were maintained in the laboratory for 2 to 8 weeks and tested for antibody by the Toxoplasma dye test. We found that parasites invaded both epithelial and goblet cells within minutes of inoculation. Replication occurred within 4 hr of inoculation and took place mainly in epithelial cells. Within 48 hr, the organisms were found beneath the basal lamina of the epithelium. The host developed an inflammatory response consisting first largely of polymorphonuclear leukocytes and later largely of macrophages. The parasites also replicated in macrophages, showing their ability to evade host defenses in nonimmune animals. Inoculated guinea pigs kept in the laboratory for 8 weeks survived and developed elevated antibody titers against Toxoplasma. The guinea pig conjunctiva is a suitable tissue for studying the pathogenesis of toxoplasmosis.

To study chlamydial conjunctivitis, conjunctival autografts subcutaneously implanted in pockets on the abdomens of rhesus (Macaca mulatta) and pig-tailed (M. nemestrina) monkeys were inoculated percutaneously (4-10 X 10(4) inclusion-forming units per pocket) with trachoma strains of Chlamydia trachomatis (serovars B & C). These conjunctival pockets were removed on days 2, 3, 4, 5, 6, 7, 8, 10, 14 and 16 post-inoculation (pi). Tissues (at least two samples at each time interval) were prepared either for reisolation of the organisms by cell culture or histologic examination by light and electron microscopy. Control tissue, inoculated with either HeLa cell material or UV-inactivated organisms, were prepared in parallel. Bloods were drawn and tear strips taken at weekly intervals for 8 weeks. A total of 221 bulbar and 99 palpebral conjunctival pockets were established over time with the success rate of 75% and 88%, respectively, in six rhesus and ten pig-tailed monkeys. Histological examination revealed widespread infiltration of mixed polymorphonuclear and mononuclear cells on days 2 and 3 pi. By day 5 pi, lymphocytes had migrated into the thinned epithelial layer. Patches of inflammatory infiltrate similar to trachoma follicles were observed, although distinct germinal centers were absent. Surface morphologies of normal and infected pocket conjunctiva were examined by scanning electron microscopy. Normal tissue was characterized by a regular mosaic pattern of closely packed epithelial cells containing numerous microvilli. Infected tissue was edematous, and the continuity of the mucosal surface had been altered. Chlamydiae were reisolated from the pockets on days 2, 3, 6, 8 and 10 pi.(ABSTRACT TRUNCATED AT 250 WORDS)