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We previously reported the use of an ex vivo model of bovine corneal organ culture for predicting ocular irritation and evaluating acute chemical toxicity. Since the ability to recover after chemical exposure is a key parameter for assessing ocular toxicity of test chemicals, we sought to determine if cultured porcine cornea is able to recover after surfactant exposure and if functional recovery might be used as an ex vivo parameter for an ocular toxicity assay. Corneal chemical injury was created by placing a 7‐mm circular filter paper soaked in different concentrations of sodium dodecyl sulfate (SDS) on the center of porcine corneas for 2 minutes. The corneas exposed to surfactant with mild irritation (1% and 3% SDS, Draize score < 16.9) were able to recover from the initial damage, but no evidence of recovery was observed after exposure to surfactant with severe irritation (15% SDS, Draize score 59.2) as assessed by reestablishment of epithelial barriers. There was a concentration‐dependent increase in...

OBJECTIVES: To evaluate the efficacy of systemic or topical administration of cyclosporin A (CsA) after limbal transplantation of stem cell allografts in rabbits. METHODS: Thirty-six rabbits underwent corneal epithelial debridement and limbal ablation to induce ocular surface disease and were then treated by limbal allograft transplantation. Animals received either systemic CsA (10 mg/kg per day, intramuscularly), 1% CsA eyedrops, or vehicle eyedrops immediately after transplantation and 28 days thereafter. Concentration of CsA in plasma and aqueous humor was determined by fluorescence polarization immunoassay after 4 weeks of therapy. Graft survival was inspected clinically. RESULTS: Both systemic and topical administration of CsA resulted in a significant prolongation of graft survival. In addition, one of seven of the limbal allografts in either group of systemic and topical CsA survived >60 days on cessation of CsA. There was no significant difference in mean survival time between systemic and topical application, although plasma levels of CsA were significantly higher after systemic administration. However, a significant higher aqueous concentration was found in topical treatment. CONCLUSIONS: Limbal allografts were stable in maintaining the reconstructed ocular surface under attentive postoperative immunosuppression. Topically administered CsA was as effective as systemic use.

BACKGROUND--The study sought to investigate the histogenesis of retinoblastoma. METHODS--One hundred specimens of retinoblastomas were examined along with those of 18 astrocytic gliomas and 15 medulloblastomas to compare similarities of glial differentiation in retinoblastoma and the two types of brain tumour. Employing avidin-biotin immunoperoxidase technique, antibodies were applied against neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S-100 protein (S-100). RESULTS--Most rosettes and fleurettes, and some undifferentiated cells in retinoblastomas were NSE positive, but GFAP and S-100 negative. GFAP and S-100 positive cells in retinoblastomas were detected mostly in well differentiated glial cells which were interpreted as reactive or non-neoplastic cells. Some of the GFAP and S-100 positive cells in retinoblastomas were defined as tumour cells that resembled neoplastic astrocytes in astrocytic gliomas and medulloblastomas. CONCLUSION--Retinoblastoma may arise from primitive bipotential or multipotential cells capable of neuronal and glial differentiation.

PURPOSE. The purpose of the study was to investigate the effects of hyperglycemia on EGFR (epidermal growth factor receptor)-mediated wound response and signal transduction in the corneal epithelium of rats with type I diabetes mellitus (DM). METHODS. Corneal epithelia were removed from streptozotocin (STZ)- and weight-matched normal rats. Wound healing was monitored by fluorescein staining at 24 or 48 hours after epithelial debridement. Phosphorylation of EGFR, AKT, ERK, and BAD was determined by Western blot analysis. The distribution of phospho-AKT and proliferating cell nuclear antigen (PCNA) in rat corneas was examined by immunohistochemistry. Cell death was evaluated by TUNEL staining. RESULTS. A significant delay in corneal epithelial wound healing was observed 48 hours after wounding in the diabetic rats compared with the weight-matched control rats. In the DM rat corneas, epithelial cells demonstrated diminished responses to wounding, as assessed by the phosphorylation of EGFR and its downstream signaling molecules, AKT and ERK. Furthermore, although the distribution pattern of phospho-AKT suggested a role for AKT in epithelial migration and proliferation in the normoglycemic rat corneas, it was abrogated in the healing epithelia of the DM rats. Consistent with impaired AKT activity, the number of PCNA-stained cells was also greatly reduced in the healing corneas of the diabetic rats. Finally, decreases in pBAD (Ser(136) and Ser(112)) and increases in TUNEL-positive cells were observed in both the uninjured and healing corneal epithelia of the DM rats, but not of the control rats. CONCLUSIONS. In the corneas of SZT rats, EGFR-PI3K-AKT and ERK, as well as their downstream BAD signaling pathways in migratory epithelium, were altered, resulting in increased apoptosis, decreased cell proliferation, and delayed wound closure.

BackgroundMutations in a novel gene, UBIAD1, were recently found to cause the autosomal dominant eye disease Schnyder corneal dystrophy (SCD). SCD is characterized by an abnormal deposition of cholesterol and phospholipids in the cornea resulting in progressive corneal opacification and visual loss. We characterized lesions in the UBIAD1 gene in new SCD families and examined protein homology, localization, and structure.Methodology/principal findingsWe characterized five novel mutations in the UBIAD1 gene in ten SCD families, including a first SCD family of Native American ethnicity. Examination of protein homology revealed that SCD altered amino acids which were highly conserved across species. Cell lines were established from patients including keratocytes obtained after corneal transplant surgery and lymphoblastoid cell lines from Epstein-Barr virus immortalized peripheral blood mononuclear cells. These were used to determine the subcellular localization of mutant and wild type protein, and to examine cholesterol metabolite ratios. Immunohistochemistry using antibodies specific for UBIAD1 protein in keratocytes revealed that both wild type and N102S protein were localized sub-cellularly to mitochondria. Analysis of cholesterol metabolites in patient cell line extracts showed no significant alteration in the presence of mutant protein indicating a potentially novel function of the UBIAD1 protein in cholesterol biochemistry. Molecular modeling was used to develop a model of human UBIAD1 protein in a membrane and revealed potentially critical roles for amino acids mutated in SCD. Potential primary and secondary substrate binding sites were identified and docking simulations indicated likely substrates including prenyl and phenolic molecules.Conclusions/significanceAccumulating evidence from the SCD familial mutation spectrum, protein homology across species, and molecular modeling suggest that protein function is likely down-regulated by SCD mutations. Mitochondrial UBIAD1 protein appears to have a highly conserved function that, at least in humans, is involved in cholesterol metabolism in a novel manner.

In this article, we briefly review recent findings in the effects of growth factors including the EGF family, KGF, HGF, IGF, insulin, and TGF-beta on corneal epithelial wound healing. We discuss the essential role of EGFR in inter-receptor cross-talk in response to wounding in corneal epithelium and bring forward a concept of "alarmins" to the field of wound healing research.

OBJECTIVEPatients with diabetes are at an increased risk for developing corneal complications and delayed wound healing. This study investigated the effects of high glucose on epidermal growth factor receptor (EGFR) signaling and on epithelial wound healing in the cornea.RESEARCH DESIGN AND METHODSEffects of high glucose on wound healing and on EGFR signaling were investigated in cultured porcine corneas, human corneal epithelial cells, and human corneas using Western blotting and immunofluorescence. Effects of high glucose on reactive oxygen species (ROS) and glutathione levels and on EGFR pathways were assessed in porcine and primary human corneal epithelial cells, respectively. The effects of EGFR ligands and antioxidants on high glucose–delayed epithelial wound healing were assessed in cultured porcine corneas.RESULTSHigh glucose impaired ex vivo epithelial wound healing and disturbed cell responses and EGFR signaling to wounding. High glucose suppressed Akt phosphorylation in an ROS-sensitive manner and decreased intracellular glutathione in cultured porcine corneas. Exposure to high glucose for 24 h resulted in an increase in ROS-positive cells in primary human corneal epithelial cells. Whereas heparin-binding EGF-like growth factor and antioxidant N-acetylcysteine had beneficial effects on epithelial wound closure, their combination significantly accelerated high glucose–delayed wound healing to a level similar to that seen in control subjects. Finally, Akt signaling pathway was perturbed in the epithelia of human diabetic corneas, but not in the corneas of nondiabetic, age-matched donors.CONCLUSIONSHigh glucose, likely through ROS, impairs the EGFR–phosphatidylinositol 3-kinase/Akt pathway, resulting in delayed corneal epithelial wound healing. Antioxidants in combination with EGFR ligands may be promising potential therapeutics for diabetic keratopathy.

In response to pathologic conditions, retinal pigment epithelial (RPE) cells initiate a wound-healing process and become transformed from a stationary epithelial state to a migratory and proliferative mesenchymal state, leading to the epiretinal membrane formation associated with the development of proliferative vitreoretinopathy (PVR).1 It is thought that activation of several autocrine or paracrine loops by growth factors and their receptors is critical for RPE transformation and PVR progression.2 Prominent among these factors are hepatocyte growth factor (HGF)/scatter factor (SF) and the epidermal growth factor (EGF) family. HGF is involved in cell scattering and migration and from epithelial to mesenchymal transition (EMT).3,4 The EGF receptor tyrosine kinase (RTK) family has been characterized in many cell systems, including RPE,5-7 and is known to participate in a wide variety of biological responses, including cell migration, proliferation, and differentiation. HGF is a multipotential cytokine that has been implicated in diverse events in organ development, tissue maintenance and homeostasis, and wound healing. At the cellular level, HGF can promote other bioactivities, such as junctional breakdown, cell scattering, migration, cell survival, and invasive behavior.8,9 HGF is thought to be synthesized by mesenchymally derived cells, typically fibroblasts, which primarily target epithelial cells in a paracrine manner through c-Met, the only known receptor for HGF that mediates all HGF-induced biological activities.8,10,11 c-Met consists of an α/β heterodimer at the cell surface, with α as an extracellular subunit and β as a subunit containing an extracellular domain, a membrane-spanning domain, and a cytoplasmic tyrosine kinase domain.12 On HGF stimulation, the c-Met receptor is tyrosine phosphorylated; this is followed by the recruitment of a group of signaling molecules, adaptor proteins, or both to its cytoplasmic domain and to its multiple docking sites. This action leads to the activation of several different signaling cascades, including extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3′-kinase (PI3K), that form a signaling network of intracellular and extracellular responses. Unlike HGF, the EGFR ligand family of growth factors consists of more than 10 members, including EGF13 and HB-EGF.14 These factors act through the stimulation of specific cell-surface receptors of the erbB or EGFR family. There are four related RTKs: EGFR/erbB1, erbB2, erbB3, and erbB4.15-18 Activation of erbBs, similar to c-Met, elicits myriad signaling events, including ERK and PI3K.19-21 EGFR ligand stimulation promotes RPE cell proliferation and survival, signaling through both ERK/MAPK and PI3K pathways.5,6 Recently, HB-EGF has been implicated in driving the uncontrolled wound-healing process of the retina during proliferative retinopathy.7 Although various reactions have been described, wounding or breakdown of the tight junction barrier in vivo results in the availability of circular or otherwise segregated22 growth factors, such as HGF and EGFR ligands to their receptors, leading to the initiation of a wound healing response. Hence, the multiplicity of cell surface receptors activated by endogenous signals is contrasted by the relative uniformity of intracellular signaling pathways triggered by these receptors. In particular, the activation of EGFR and c-Met may elicit similar signal transduction pathways in cells. Thus, cross talk of these growth factor receptors may affect the strength, duration, or both of shared downstream signaling pathways. Whether c-Met and EGFR influence each other’s activity and how the cross talk between these RTKs determines cell signaling remains to be fully explored. Hence, we investigated the role of HGF and HB-EGF in mediating RPE wound healing and the cross talk between these two growth factors using cultured human ARPE-19 cells.

We have shown previously that wounding of human corneal epithelial (HCE) cells resulted in epidermal growth factor receptor (EGFR) transactivation through ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF). However, the initial signal to trigger these signaling events in response to cell injury remains elusive. In the present study, we investigated the role of ATP released from the injured cells in EGFR transactivation in HCE cells as well as in BEAS 2B cells, a bronchial epithelial cell line. Wounding of epithelial monolayer resulted in the release of ATP into the culture medium. The wound-induced rapid activation of phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) pathways in HCE cells was attenuated by eliminating extracellular ATP, ADP and adenosine. The nonhydrolyzable ATP analog ATP-γ-S induced rapid and sustained EGFR activation that depended on HB-EGF shedding and ADAM (a disintegrin and metalloproteinase). Targeting pathways leading to HB-EGF shedding and EGFR activation attenuated ATP-γ-S-enhanced closure of small scratch wounds. The purinoceptor antagonist reactive blue 2 decreased wound closure and attenuated ATP-γ-S induced HB-EGF shedding. Taken together, our data suggest that ATP, released upon epithelial injury, acts as an early signal to trigger cell responses including an increase in HB-EGF shedding, subsequent EGFR transactivation and its downstream signaling, resulting in wound healing.

Purpose To identify the underlying mechanisms by which lipid mediator lysophosphatidic acid (LPA) acts as a growth factor in stimulating extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3K) during corneal epithelial wound healing. Methods Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial (THCE) cells were allowed to heal in the presence or absence of an epidermal growth factor receptor (EGFR) inhibitor (tyrphostin AG1478), a matrix metalloproteinase inhibitor (GM6001), or a heparin-binding EGF-like growth factor (HB-EGF) antagonist (CRM197) with or without LPA. EGFR activation was analyzed by immunoprecipitation using EGFR antibodies and Western blotting with phosphotyrosine antibodies. Phosphorylation of ERK and AKT (a major substrate of PI3K) was analyzed by Western blotting with antibodies specific to the phosphorylated proteins. Wound- and LPA-induced shedding of HB-EGF was assessed by measuring the release of alkaline phosphatase (AP) in a stable THCE cell line that expressed HB-EGF with AP inserted in the heparin-binding site. Results In organ and cell culture models, LPA enhanced corneal epithelial wound healing. LPA-stimulated and spontaneous wound closure was attenuated by AG1478, GM6001, or CRM197. Consistent with the effects on epithelial migration, these inhibitors, as well as the Src kinase inhibitor (PP2), retarded LPA-induced activation of EGFR and its downstream effectors ERK and AKT in THCE cells. Unlike exogenously added HB-EGF, LPA stimulated moderate EGFR phosphorylation; the level of phosphorylated EGFR was similar to that induced by wounding. However, LPA appeared to prolong wound-induced EGFR signaling. The release of HB-EGF assessed by AP activity increased significantly in response to wounding, LPA, or both, and the release of HB-EGF-AP induced by LPA was inhibited by PP2 and GM6001. Conclusions LPA accelerates corneal epithelial wound healing through its ability to induce autocrine HB-EGF signaling. Transactivation of EGFR by LPA represents a convergent signaling pathway accessible to stimuli such as growth factors and ligands of G-protein-coupled receptors in response to pathophysiological challenge in human corneal epithelial cells.

Purpose Epithelial wound healing is, at least in part, mediated in an autocrine fashion by epidermal growth factor (EGF) receptor (EGFR)-ligand interactions. This study sought to identify the endogenous EGFR ligand and the mechanism by which it is generated in response to wounding in cultured porcine corneas and human corneal epithelial cells. Methods Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial (THCE) cells were allowed to heal in the presence of tyrphostin AG1478 (an EGFR inhibitor), GM6001 (a matrix metalloproteinase [MMP] inhibitor), or CRM197 (a diphtheria toxin mutant), with or without HB-EGF. The activation of EGFR and extracellular signal-regulated kinase (ERK) was analyzed by immunoprecipitation using EGFR antibodies and Western blot analysis with phosphotyrosine antibody. Wound induced HB-EGF shedding was assessed by isolation of secreted HB-EGF from wounded THCE cells and by measuring the release of alkaline phosphatase (AP) in THCE stable cell lines expressing HB-EGF-AP. Results In THCE cells, wound-induced EGFR phosphorylation and ERK activation. In both organ and cell culture models, epithelial wounds were healed in basal media and inhibition of EGFR activation by AG1478 blocked wound closure with or without exogenously added HB-EGF. GM6001 delayed wound closure. Its effects diminished in the presence of exogenous EGF or HB-EGF, suggesting that the MMP inhibitor primarily blocks the release of EGFR ligands. CRM197, a highly specific antagonist of HB-EGF, impaired epithelial wound closure, suggesting that HB-EGF is an endogenous ligand released on epithelial wounding. Consistent with the effects on epithelial migration, all inhibitors as well as HB-EGF function-blocking antibodies retarded wound-induced EGFR phosphorylation in cultured THCE cells. The release of HB-EGF in response to wounding was demonstrated by the fact that heparin-binding proteins isolated from wounded, but not control, THCE-conditioned medium stimulated EGFR and ERK phosphorylation and by the expression of HB-EGF-AP in THCE cells, in which wounding induced the release of AP activity in an MMP-inhibitor-sensitive manner. Conclusions HB-EGF released on wounding acts as an autocrine-paracrine EGFR ligand. HB-EGF shedding and EGFR activation represent a critical event during corneal epithelial wound healing, suggesting a possible manipulation of wound healing during the early phases.

To investigate the role of protein kinase C (PKC) isozymes in epithelial growth factor (EGF)-induced activation of extracellular signal-regulated kinase (ERK) and cell proliferation in cultured human corneal epithelial cells.Simian virus (SV)40 stably transfected human corneal epithelial (THCE) cells were cultured in keratinocyte growth medium. PKC isozymes and phosphorylation of ERK in THCE cells were assessed by Western blot analysis. Translocation of the PKC isozyme was determined by subcellular fractionation followed by Western blot analysis. Cell proliferation was measured by incorporation of [(3)H]-thymidine into DNA.Six PKC isozymes-PKC-alpha, -betaI, -betaII, -delta, - epsilon, and - micro -were found in THCE cells. Phorbol 12-myristate 13-acetate (PMA) caused PKC-alpha, -betaI, and - epsilon, initially present in the cytoplasm, to be translocated to the membrane and nuclear subcellular fractions and PKC-delta to be depleted from the cytoskeleton. The PKC inhibitor GF109203X inhibited PMA-induced, but not basal or EGF-induced, phosphorylation of ERK, whereas the EGF receptor inhibitor tyrphostin AG1478 blocked basal and EGF-, but not PMA-, induced phosphorylation of ERK. Depletion of PMA-sensitive PKC isozymes including PKC-alpha, -betaI, -betaII, -delta, and - epsilon, inhibited PMA-, but not EGF-, induced phosphorylation of ERK. Depletion of these PKC isozymes blocked PMA-, but not EGF-, induced cell proliferation.Although activation of PKC by PMA results in activation of ERK, EGF-induced phosphorylation of ERK and/or cell proliferation is independent of the conventional and novel isozymes PKC-alpha, -betaI, -betaII, -delta, and - epsilon in human corneal epithelial cells.

We previously reported an increased secretion of amyloid precursor-like protein 2 (APLP2) in the healing corneal epithelium. The present study sought to investigate signal transduction pathways involved in APLP2 shedding in vitro. APLP2 was constitutively shed and released into culture medium in SV40-immortalized human corneal epithelial cells as assessed by Western blotting, flow cytometry, and indirect immunofluorescence. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) caused significant increases in APLP2 shedding. This was inhibited by staurosporine and a PKC-ε-specific, N-myristoylated peptide inhibitor. Epidermal growth factor (EGF) also induced APLP2 accumulation in culture medium. Basal APLP2 shedding as well as that induced by PMA and EGF was blocked by a mitogen-activated protein kinase (MAPK) kinase inhibitor, U-0126. Our results suggest that MAPK activity accounts for basal as well as PKC- and EGF-induced APLP2 shedding. In addition, PKC-ε may be involved in the induction of APLP2 shedding in corneal epithelial cells.

The main goal of the present study was to investigate the response of cultured bovine corneas to the application of irritant substances and its potential use for predicting ocular irritancy in humans. We hypothesized that chemicals causing eye irritation may induce disruption of epithelial tight junctions and trigger cell stress responses modulated via transcription factors such as AP-1 and NF-kappaB. A simple air-lifted corneal organ culture system was used as an ex vivo model for ocular irritancy test. The effects of two surfactants, sodium dodecyl sulfate (SDS) and benzalkonium chloride (BAK), on corneal epithelial permeability and DNA-binding activity of AP-1 and NF-kappaB were studied in cultured bovine corneas. Both SDS and BAK induced tight junction disruption and increased permeability of corneal epithelium assessed using surface biotinylation in a concentration- and time-dependent manner. An increase in DNA-binding activity measured using electrophoretic mobility shift assay was observed when cultured corneas were treated with surfactants at concentrations causing minimal to mild ocular irritation, indicating epithelial cell stress response. Furthermore, exposure of cultured corneas to SDS or BAK at concentrations causing severe ocular irritancy resulted in a decrease in DNA-binding activity of these transcription factors in epithelial cells. These results indicate that the combination of corneal organ culture and measurements of corneal epithelial permeability and DNA-binding activity of stress-response transcription factors following chemical exposure has the potential to be used as a mechanistically based alternative to in vivo animal testing.

Our purpose was to investigate the relationship between insufficient tear secretion and decreased corneal sensitivity. We studied 59 patients with dry eye, 15 of whom had Sjogren's syndrome (SS), and 26 healthy subjects. Corneal sensitivity was measured by the esthesiometer of Cochet and Bonnet. Schirmer test with or without anesthesia, tear clearance rate, tear function index, and rose bengal and fluorescein staining were also evaluated. The mean corneal sensitivity of either dry-eye group (4.6 +/- 1.2 and 4.5 +/- 1.2 cm for non-SS and SS dry eye, respectively) was significantly lower than that of the control (5.8 +/- 0.4 cm, p < 0.001). Corneal sensitivity correlated significantly with the Schirmer values with anesthesia and the tear function index in the two dry-eye groups and the control (p < 0.05). There were significant relationships between corneal sensitivity and the rose bengal and fluorescein scores in the three groups (p < 0.05). Hyposecretion of tears in dry eye may lead to pathologic changes in corneal epithelium and a decline in corneal sensitivity. Prompt treatment of dry eye is essential to maintain a normal corneal protective mechanism.

BACKGROUND--The study sought to determine dynamic changes and theoretical bases of a clinical diagnostic test, the tear clearance rate. METHODS--Thirty four healthy subjects ranging in age from 22 to 84 years underwent examination of tear clearance rate, the Schirmer test with anaesthesia, as well as fluorophotometric measurement of tear turnover, tear volume, and tear flow. By applying 0.5% fluorescein into the conjunctival sac and subsequently measuring colour fades on a Schirmer strip, the tear clearance rate for assessing tear drainage was divided into nine grades. The results of the tear clearance rate were compared with those of the basal tear turnover and tear flow obtained from fluorophotometry. RESULTS--Significant relations were found between the tear clearance rate and the basal tear turnover or tear flow (r = 0.91 and 0.79, respectively, p = 0.0001). Considering the grades of progression from low to high, each grade of tear clearance rate showed a 12.5% increase in basal tear turnover (3.59%/min) and tear flow (0.38 microliter/min). There was no significant correlation between age and the basal tear turnover, tear volume, tear flow, or the tear clearance rate. CONCLUSION--The tear clearance rate is proposed as a simple and useful way to estimate basal tear turnover and tear flow, and measure tear drainage indirectly.

Objective: To develop an efficient way to evaluate tear dynamics clinically. Participants: Three hundred fifty-two patients with dry eye, 64 of whom had Sjogren syndrome, and 55 normal subjects. Design: Because various forces that affect tear drainage are reflected in the values of the Schirmer test with anesthesia and the tear clearance rate, we introduced a new measure of tear dynamics, the tear function index, which is the value obtained from dividing the value of the Schirmer test with anesthesia by the tear clearance rate. Results: The tear function index was more specific (91.8%) and sensitive (78.9%) in diagnosing dry eye associated with Sjogren syndrome than was the Schirmer or tear clearance rate test alone. Tear function indexes below 96 were consistent with dry eye and those below 34 were seen primarily in patients with Sjogren syndrome. Conclusions: The tear function index offers a new method to evaluate tear production with the Schirmer test, introduces an extended way to measure tear flow combining with tear drainage, and gives a practical measure to diagnose dry eye.

Human corneal epithelial cells (HCECs) were functionally depleted of erbB2 to elucidate its role in epidermal growth factor (EGF) receptor (EGFR) activation-dependent cell migration.The retrovirus pBabe-5R, which encodes an erbB2 single-chain antibody with an endoplasmic reticulum (ER)-targeting sequence, and control pBabe-puro were used to infect THCE cells (an SV40-immortalized HCEC line). Several cell lines expressing 5R were selected along with a pBabe-puro control line. The depletion of erbB2 was verified by cell surface biotinylation of proteins, followed by streptavidin precipitation and subsequent detection of erbB2 by immunoblot analysis. Activation of erbBs was analyzed by immunoprecipitation using the phosphotyrosine antibody pY20, followed by Western blot analysis with erbB1 or erbB2 antibodies. Phosphorylation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3K) was analyzed by Western blot with antibodies specific to phosphorylated proteins. Effects of erbB2 depletion on heparin-binding EGF-like growth factor (HB-EGF)-induced cell migration were determined by Boyden chamber migration assay and by scratch wound assay.Wounding induced erbB2 tyrosine phosphorylation. Expression of 5R encoding an erbB2 single-chain antibody with an endoplasmic reticulum-targeting sequence depleted the cell surface expression of erbB2 in HCECs. Wounding resulted in a rapid increase in the phosphorylation of erbB1 in both 5R-expressing and control cells, whereas wound-induced erbB2 phosphorylation in 5R-expressing cells was not detectable. Depletion of functional erbB2 attenuated the healing of scratch wounds in the presence of HB-EGF and impaired both chemotactic migration stimulated by HB-EGF and haptotactic migration toward a fibronectin-collagen I (3:1; FNC) coating mix. Expression of 5R affected both the intensity and the duration of wound-induced, EGFR-elicited ERK and PI3K activation. Inhibition of ERK and PI3K pathways in cultured porcine corneas impaired ex vivo epithelial wound healing.ErbB2 serves as a critical component that couples erbB receptor tyrosine kinase to the migration machinery of corneal epithelial cells.

Purpose Flagellin is the major structural protein of the flagella of Gram-negative bacteria and is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms. In this study, we sought to determine whether flagellin induces an inflammation response in cultured human corneal epithelial (HCE) cells and to determine the underlying mechanisms. Methods Flagellin was purified from Pseudomonas aeruginosa (PA) strain PAO1 with ammonium sulfate gradient precipitation and lipopolysaccharide in flagellin preparation was removed by ion exchange chromatography. Purified flagellin was used to challenge HUCL, a telomerase-immortalized HCE cell line, and primarily cultured HCE cells. Inhibitory (I)kappaB-alpha phosphorylation and degradation were detected by Western blot. Interleukin (IL)-6 and -8 expression in mRNA levels and secretion were assessed using RT-PCR and enzyme-linked immunosorbent assay, respectively. TLR5 localization in human cornea was analyzed by immunohistochemistry using anti-TLR5 antibody. Anti-flagellum antiserum and anti-TLR5 antibody were used for functional blocking of flagellin stimulation and TLR5 activation. Results Exposure of both HUCL and primary HCE cells to purified PA flagellin (250 ng/mL) resulted in IkappaB-alpha phosphorylation and degradation in a time-dependent manner. Concomitant with NF-kappaB activation, transcriptional expression and subsequent secretion of IL-6 and -8 in these cells were also induced by flagellin. Toll-like receptor (TLR)-5, an innate immunity receptor for flagellin, was expressed in HUCL cells and located at the cell surface of the basal and wing, but not in superficial, cells of human corneal epithelium. Presence of flagellum- or TLR5-antisera in culture medium attenuated flagellin-induced IkappaB-alpha phosphorylation and degradation as well as IL-6 and -8 production. Conclusions Flagellin of Gram-negative pathogens such as PA contributes to the inflammatory responses of corneal epithelium in a TLR5-NF-kappaB signaling pathway-dependent manner.