The Marine Neurotoxin Brevetoxin (PbTx-2) Inhibits Karenia brevis and Mammalian Thioredoxin Reductases by Targeting Different Residues.

The brevetoxins, neurotoxins produced by Karenia brevis, the Florida red tide dinoflagellate, effect fish and wildlife mortalities and adverse public health and economic impacts during recurrent blooms. Knowledge of the biochemical consequences of toxin production for K. brevis could provide insights into an endogenous role of the toxins, yet this aspect has not been thoroughly explored. In addition to neurotoxicity, the most abundant of the brevetoxins, PbTx-2, inhibits mammalian thioredoxin reductase (TrxR). The thioredoxin system, composed of the enzymes TrxR and thioredoxin (Trx), is present in all living organisms and is responsible in part for maintaining cellular redox homeostasis. Herein, we describe the cloning, expression, and semisynthesis of the selenoprotein TrxR from K. brevis (KbTrxR) and reductase activity toward a variety of substrates. Unlike mammalian TrxR, KbTrxR reduces oxidized glutathione (GSSG). We further demonstrate that PbTx-2 is an inhibitor of KbTrxR. Covalent adducts between KbTrxR and rat TrxR were detected by mass spectrometry. While both enzymes are adducted at or near the catalytic centers, the specific residues are distinct. Biochemical differences reported for high and low toxin producing strains of K. brevis are consistent with the inhibition of KbTrxR and suggest that PbTx-2 is an endogenous regulator of this critical enzyme.

Cytotoxicity and Genotoxicity of a New Intracanal Medicament, 2-hydroxyisocaproic Acid-An In Vitro Study.

INTRODUCTION: A successful outcome of root canal therapy relies on effective disinfection of the root canal system, including the use of intracanal medicaments, which vary in their bactericidal and cytotoxic properties. Assessing the benefits and risks associated with the use of these medicaments is of extreme importance, especially in regenerative endodontic procedures, because residual stem cells may be harmed. In this study, we tested the cytotoxicity and genotoxicity of a novel agent, 2-hydroxyisocaproic acid (HICA), and compared its properties with those of a well-established medicament, calcium hydroxide. METHODS: Human periodontal ligament fibroblasts were exposed to varying concentrations of HICA (1, 2.5, 5, 10, 20, and 40 mg/mL) for 24 hours, and a dose-response curve was generated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Immunofluorescence for 2 markers of DNA double-strand breaks, phosphorylated γH2AX and 53BP1, was used to establish the genotoxicity of HICA at various half maximal effective concentration (EC50) fractions. Cytotoxicity and genotoxicity of HICA and calcium hydroxide at 1 mg/mL were compared at 24 and 48 hours using the same methods. RESULTS: At 10 mg/mL and higher, HICA was significantly more cytotoxic and genotoxic than the control (P < .05 and P < .0001, respectively). Calcium hydroxide at 1 mg/mL was more cytotoxic than HICA at 1 mg/mL at 24 and 48 hours (P < .05 for both), whereas no difference in the accumulated DNA damage was observed. CONCLUSIONS: HICA is not cytotoxic and genotoxic at concentrations <10 mg/mL. At the concentration of 1 mg/mL, HICA is significantly less cytotoxic than calcium hydroxide.

Interleukins in gingival crevicular fluid in patients with definitive full-coverage restorations.

The objective of this study was to determine interleukin (IL)-1α and IL-8 levels in the gingival crevicular fluid (GCF) of patients with different levels of crown margin placements. Samples of GCF were obtained from 12 study participants with definitive full-coverage restorations with supragingival or equigingival crown margin placements. The periodontal status of the volunteers ranged from healthy to generalized severe periodontitis. Pocket depth and bleeding on probing were assessed at the clinical examination, and interleukin concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Analysis of variance (ANOVA) was used to statistically compare interleukin concentrations between the control, supragingival, and equigingival GCF samples. Compared to controls (60.4 ± 8.9 pg/mL), the average IL-1α concentration in the GCF samples surrounding the supragingival margins was 53.8 ± 9.7 pg/mL and was 110.5 ± 23.3 pg/mL in the equigingival margins. Compared to controls (59.0 ± 14.1 pg/mL), the average IL-8 concentration in the supragingival margins was 46.9 ± 9.7 pg/mL and was 131.4 ± 27.5 pg/mL in the equigingival margins. The trend of higher levels of interleukins in GCF corresponding to equigingival margins was consistent, as was the trend of lower concentrations in supragingival margins compared to the controls; however, statistical significance was not achieved because of the wide biological variation within and between patients. In conclusion, differences in GCF IL-1α and IL-8 concentrations were observed when comparing fixed crown restorations with equigingival and supragingival margins. Gingival inflammation may be dependent on the periodontal condition in addition to restoration or margin placement.

Comparison of cone-beam computed tomography and periapical radiography in predicting treatment decision for periapical lesions: a clinical study.

Objectives. To compare the ability of endodontists to determine the size of apical pathological lesions and select the most appropriate choice of treatment based on lesions' projected image characteristics using 2 D and 3 D images. Study Design. Twenty-four subjects were selected. Radiographic examination of symptomatic study teeth with an intraoral periapical radiograph revealed periapical lesions equal to or greater than 3 mm in the greatest diameter. Cone-beam Computed tomography (CBCT) images were made of the involved teeth after the intraoral periapical radiograph confirmed the size of lesion to be equal to greater than 3 mm. Six observers (endodontists) viewed both the periapical and CBCT images. Upon viewing each of the images from the two imaging modalities, observers (1) measured lesion size and (2) made decisions on treatment based on each radiograph. Chi-square test was used to look for differences in the choice of treatment among observers. Results. No significant difference was noted in the treatment plan selected by observers using the two modalities (χ(2)(3) = .036, P > 0.05). Conclusion. Lesion size and choice of treatment of periapical lesions based on CBCT radiographs do not change significantly from those made on the basis of 2 D radiographs.

Cytotoxicity comparison of three current direct pulp-capping agents with a new bioceramic root repair putty.

INTRODUCTION: The purpose of this in vitro study was to compare the cytotoxicity of white mineral trioxide aggregate cement (AMTA, MTA-Angelus), Brasseler Endosequence Root Repair Putty (ERRM), Dycal, and Ultra-blend Plus (UBP) by using human dermal fibroblasts and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. METHODS: Cultured adult human dermal fibroblasts were exposed to multiple concentrations of material elutes. The test material samples were immersed and incubated in the culture medium for 2, 5, or 8 days at 37°C. The cytotoxic effects were recorded by using an MTT-based colorimetric assay. Positive and negative controls were used. The results were statistically examined by one-way analysis of variance and Tukey post tests. RESULTS: The cell viability of cultures exposed to all dilutions of AMTA, ERRM, and UBP was statistically similar to the negative control at 2 and 5 days. Only the Dycal-exposed specimens exhibited a statistically significant increase in cytotoxicity at the 2 initial evaluation periods. After exposure to the 8-day elutes, the respective percentage of cell survivability was 91% (Brasseler), 88% (MTA-Angelus), 76% (Ultra-blend Plus), and 37% (Dycal). CONCLUSIONS: From the data in this in vitro study, AMTA, ERRM, and UBP had statistically similar adult human dermal fibroblast cytotoxicity levels. Relative to the negative control, only Dycal was shown to have a statistically significant cytotoxic effect to adult human dermal fibroblasts at all tested intervals.

Topical flagellin-mediated innate defense against Candida albicans keratitis.

PURPOSE: This study was conducted to investigate whether flagellin, the sole ligand of Toll-like receptor-5 (TLR5), induces an innate defense that is sufficient to protect injured corneas from Candida albicans. METHODS: Scarified corneas of adult B6, TLR5(-/-), Camp(-/-) (cathelicidin-related antimicrobial peptide), or PMN-depleted mice were pretreated with Pseudomonas aeruginosa flagellin or a mutant and then were inoculated with C. albicans. The corneas were compared for disease progression, cytokine and Camp expression, and PMN infiltration before and after C. albicans infection. Disease progress was recorded by digital photography and clinical scoring, cytokine levels were determined by ELISA, the levels of Camp gene product were assessed by Western blot, and PMN infiltration was measured by MPO determination and immunohistochemistry. RESULTS: Topical application of flagellin induced profound protection against Candida keratitis in a TLR5-dependent manner. The improved disease outcome including reduced tissue inflammation and rapid functional recovery can be attributed to a marked decrease in fungal burden at the early stage of C. albicans infection in flagellin-exposed B6 mouse corneas. Although both PMN infiltration and Camp upregulation contributed to corneal innate defense against fungal infection, Camp ablation totally, and PMN depletion partially, abrogated flagellin-induced fungal clearance in B6 mouse corneas. CONCLUSIONS: Flagellin induces a strong innate defense and promotes robust resistance to C. albicans infection in the cornea. Topical flagellin or its mimetic may become a new prophylactic agent for preventing contact lens or trauma/injury-associated microbial keratitis.

Thymosin beta4 inhibits TNF-alpha-induced NF-kappaB activation, IL-8 expression, and the sensitizing effects by its partners PINCH-1 and ILK.

The mechanisms by which thymosin ß 4 (Tß(4)) regulates the inflammatory response to injury are poorly understood. Previously, we demonstrated that ectopic Tß(4) treatment inhibits injury-induced proinflammatory cytokine and chemokine production. We have also shown that Tß(4) suppresses TNF-α-mediated NF-κB activation. Herein, we present novel evidence that Tß(4) directly targets the NF-κB RelA/p65 subunit. We find that enforced expression of Tß(4) interferes with TNF-α-mediated NF-κB activation, as well as downstream IL-8 gene transcription. These activities are independent of the G-actin-binding properties of Tß(4). Tß(4) blocks RelA/p65 nuclear translocation and targeting to the cognate κB site in the proximal region of the IL-8 gene promoter. Tß(4) also inhibits the sensitizing effects of its intracellular binding partners, PINCH-1 and ILK, on NF-κB activity after TNF-α stimulation. The identification of a functional regulatory role by Tß(4) and the focal adhesion proteins PINCH-1 and ILK on NF-κB activity in this study opens a new window for scientific exploration of how Tß(4) modulates inflammation. In addition, the results of this study serve as a foundation for developing Tß(4) as a new anti-inflammatory therapy.

Cytotoxicity comparison of mineral trioxide aggregates and EndoSequence bioceramic root repair materials.

INTRODUCTION: The purpose of this bench top evidence level 5 in vitro study was to compare the cytotoxic effect of 2 brands of white mineral trioxide aggregate cement (ProRoot MTA and MTA-Angelus), Brasseler EndoSequence Root Repair Material, and Brasseler EndoSequence Root Repair Putty by using human dermal fibroblasts. METHODS: The cells were cultured in recommended culture conditions and exposed to the tested materials. The cytotoxic effects were recorded at an observation period of 24 hours by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay. Results were analyzed by using one-way analysis of variance with significance of p < .05. RESULTS: All materials tested demonstrated cell viability ≥ 91.8%. Overall, there was no statistically significant difference in cell viability of ProRoot MTA, MTA-Angelus, and Brasseler EndoSequence Root Repair Material. However, there was a statistically significant difference negatively associated with the cell viability of human dermal fibroblasts in association with the Brasseler EndoSequence Root Repair Putty. CONCLUSIONS: The Brasseler EndoSequence Root Repair Materials were shown to have similar cytotoxicity levels to those of ProRoot MTA and MTA-Angelus.

Recruitment, development, and retention of dental faculty in a changing environment.

The scarcity of full-time clinical faculty members in dental schools across the country is a major crisis confronting dentistry. Dental schools are experiencing critical faculty shortages and are struggling to maintain appropriate faculty to student ratios. The adage of "doing more with less" applies, in many ways, to dental schools and their mission of educating future dentists. Solutions to the problem have included plans to recruit, develop, and mentor faculty members. However, progress has been slow. Dental schools have created incentive programs to help with faculty shortages and develop future educators in dentistry. This requires commitment and resources and will take years before these efforts and their impact can be assessed. Creative solutions need to be developed both nationally and locally to reverse the trend more rapidly.

Thymosin beta4 and corneal wound healing: visions of the future.

Persistent corneal epithelial defects and inflammation within the central cornea can directly distort visual acuity and may lead to permanent visual loss. Therefore, treatments with agents that enhance corneal reepithelialization and regulate the inflammatory response without the deleterious side effects of currently used agents such as corticosteroids would result in improved clinical outcome and would represent a major advance in the field. Despite much progress in the areas of corneal wound healing research, clinically available pharmacological therapies that can promote repair and limit the visual complications from persistent corneal wounds are severely limited and remains a major deficiency in the field. Prior studies from our laboratory have demonstrated the potent wound healing and anti-inflammatory effects of thymosin beta4 (Tbeta(4); Tbeta4) in numerous models of corneal injury. We are studying the mechanisms by which Tbeta(4) suppresses inflammation and promotes repair. Herein, we discuss some of our new basic scientific directions that may lead to the use of Tbeta(4) as a novel corneal wound healing and anti-inflammatory therapy.

Biological activities of thymosin beta4 defined by active sites in short peptide sequences.

Thymosin beta(4), a small ubiquitous protein containing 43 aa, has structure/function activity via its actin-binding domain and numerous biological affects on cells. Since it is the major actin-sequestering molecule in eukaryotic cells and is found essentially in all cells and body fluids, thymosin beta(4) has the potential for significant roles in tissue development, maintenance, repair, and pathology. Several active sites with unique functions have been identified, including the amino-terminal site containing 4 aa (Ac-SDKP) that generally blocks inflammation and reduces fibrosis. Another active site at the amino terminus contains 15 aa, including Ac-SDKP, and promotes cell survival and blocks apoptosis, while a short sequence containing LKKTETQ, the central actin-binding domain (aa 17-23) plus 1 additional amino acid (Q), promotes angiogenesis, wound healing, and cell migration. Several additional biological activities have been identified but not yet localized in the molecule, including its antimicrobial activity, the induction of various genes (including laminin-5, MMPs, TGF beta, zyxin, terminal deoxynucleotidyl transferase, and angiogenesis-related proteins), and the ability to activate ILK/PINCH/Akt, and other signaling molecules important in both apoptosis and inflammatory pathways. This review details these important physiologically and pathologically active sites and their potential therapeutic uses.

Lack of MD-2 expression in human corneal epithelial cells is an underlying mechanism of lipopolysaccharide (LPS) unresponsiveness.

In the present study we tested the responsiveness of human corneal epithelial cells (HCECs) and corneal fibroblasts to lipopolysaccharide (LPS), a Toll-like receptor (TLR) 4 ligand. Purified Pseudomonas aeruginosa LPS was used to stimulate telomerase-immortalized HCECs (HUCL) and stromal fibroblast (THK) cell lines. Exposure of cells to LPS induced a time-dependent activation of NF-kappaB in THK but not in HUCL cells, as assessed by an increase in IkappaB-alpha phosphorylation and degradation. Concomitant with NF-kappaB activation, LPS-treated THK cells, but not HUCL cells, produced a significantly larger number of cytokines than control untreated cells. A cell surface biotinylation assay revealed that HUCL cells express TLR4 intracellularly, whereas TLR5 is expressed on the cell surface. Furthermore, reverse transcriptase-PCR analysis revealed that HUCL and primary HCECs, in contrast to THK cells, do not express myeloid differentiation (MD)-2. Thus, our results demonstrate that the LPS unresponsiveness of HCECs might be due to deficient expression of MD-2, an essential component for LPS-TLR4 signaling.

Thymosin beta4 is cytoprotective in human gingival fibroblasts.

Thymosin beta4 (Tbeta(4)) is a naturally occurring, ubiquitous, non-toxic protein with documented wound-healing, anti-inflammatory, anti-apoptotic, and tissue-repair properties in skin, the ocular surface, and the heart. The ability of Tbeta(4) to demonstrate similar protective properties in cells of the oral cavity was analyzed using an in vitro model of cultured human gingival fibroblasts. Thymosin beta 4 significantly suppressed the secretion of interleukin-8 (IL-8) following stimulation with tumor necrosis factoralpha (TNF-alpha), suggesting that it may suppress the inflammatory response initiated by pro-inflammatory cytokines. By contrast, Tbeta(4) was not effective in protecting fibroblasts from challenge with lipopolysaccharide purified from Porphyromonas gingivalis or Escherichia coli. Thymosin beta 4 was able to protect gingival fibroblasts against the known cytotoxic effects of chlorhexidine digluconate, a mouthrinse containing chlorhexidine digluconate, and carbamide peroxide. Additionally, Tbeta(4) was able to protect gingival fibroblasts from the apoptosis that is induced by stimulation with TNF-alpha or by exposure to chlorhexidine. Because of its multifunctional roles in protecting cells against damage, Tbeta(4) may have significant potential for use as an oral heathcare aid with combined antimicrobial, anti-inflammatory, anti-apoptotic, and cytoprotective properties.

Thymosin beta-4 and the eye: I can see clearly now the pain is gone.

The cornea epithelium responds to injury by synthesizing several cytokines, growth factors, and tissue remodeling molecules. Proinflammatory cytokines have been implicated in the inflammation that follows corneal epithelial injury and cytokine-mediated processes play a significant role in corneal epithelial wound healing. Poorly regulated corneal inflammatory reactions that occur after injury can retard healing. In turn, persistent corneal epithelial defects and inflammation may lead to ocular morbidity and permanent visual loss. Therefore, treatments with agents that enhance corneal reepithelialization and regulate the inflammatory response without the deleterious side effects of currently used agents, such as corticosteroids, would result in improved clinical outcome and would represent a major advance in the field. Evidence is mounting to support the idea that thymosin beta-4 (Tbeta-4) has multiple, seemingly diverse, cellular functions. In the cornea, as in other tissues, Tbeta-4 promotes cell migration and wound healing, has anti-inflammatory properties, and suppresses apoptosis. Prior studies from our laboratory have demonstrated the potent wound healing and anti-inflammatory effects of Tbeta-4 in numerous models of corneal injury. Recently, we demonstrated that Tbeta-4 suppresses the activation of the transcription factor, nuclear factor-kappa b (NF-kappaB) in TNF-alpha-stimulated cells. TNF-alpha initiates cell signaling pathways that converge on the activation of NF-kappaB, thus both are known mediators of the inflammatory process. These results have important clinical implications for the potential role of Tbeta-4 as a corneal anti-inflammatory and wound-healing agent.

Matrix metalloproteinase activity is necessary for thymosin beta 4 promotion of epithelial cell migration.

Studies from our laboratory provide substantial evidence that thymosin beta 4, (Tbeta(4)), an actin-sequestering protein, promotes corneal wound healing through its ability to stimulate epithelial cell migration. Matrix metalloproteinases (MMPs), which are expressed in a wide variety of tissues including the cornea, also play a key role in epithelial cell migration and wound healing. In this study we investigated the role of MMPs in Tbeta(4)-stimulated corneal epithelial cell migration. In Boyden chamber assays, XG076, an inhibitor of the conversion of pro- to active MMPs, had no effect on epithelial cell migration stimulated by exogenous activated MMP-1. However, in in vitro migration assays where the activation of pro-MMPs was blocked, XG076 significantly inhibited cell migration and wound healing in the presence or absence of Tbeta(4). GM6001, a broad-spectrum inhibitor of active MMPs and selective MMP inhibitors, also suppressed Tbeta(4)-stimulated cell migration. Tbeta(4) upregulated MMP-1 gene and protein expression in primary human corneal epithelial cells and in transformed human corneal epithelial cells following scrape wounding. From these results we conclude that MMP catalytic activity is necessary for Tbeta(4) promotion of epithelial cell migration. These novel findings are the first to demonstrate a functional link between the two.

Thymosin beta 4 suppression of corneal NFkappaB: a potential anti-inflammatory pathway.

The purpose of this study was to determine the effect of thymosin beta 4 (Tbeta4) on NFkappaB protein levels, activation, phosphorylation, and nuclear translocation in a model of tumor necrosis factor (TNF)-alpha-mediated corneal inflammation. Transformed and primary (HCET and HCEC) human corneal epithelial cells were stimulated with the pro-inflammatory cytokine TNF-alpha and treated or not with Tbeta4. Nuclear NFkappaB p65 subunit protein levels were assayed using ELISA, and activity was measured by determining NFkappaB binding to consensus oligonucleotides. NFkappaB p65 protein phosphorylation was also measured by ELISA. Nuclear translocation of NFkappaB p65 subunit was assayed by immunofluorescence microscopy. Compared to non-treated controls, Tbeta4 treatment significantly decreased nuclear NFkappaB protein levels, NFkappaB activity and p65 subunit phosphorylation in corneal epithelial cells after TNF-alpha stimulation. In TNF-alpha-stimulated corneal epithelial cells, NFkappaB p65 subunit translocation to the nucleus was observed using immunofluorescence microscopy. In contrast, Tbeta4 blocked nuclear translocation of the NFkappaB p65 subunit in TNF-alpha-stimulated corneal epithelial cells. TNF-alpha initiates cell signaling pathways that converge on the activation of NFkappaB, thus both are known mediators of the inflammatory process. Tbeta4, a protein with diverse cellular functions including wound healing and suppression of inflammation, inhibits the activation of NFkappaB in TNF-alpha-stimulated cells. These results have important clinical implications for the potential role of Tbeta4 as a corneal anti-inflammatory agent.

Thymosin beta 4: A novel corneal wound healing and anti-inflammatory agent.

Thymosin beta 4 (Tbeta(4)) is a low molecular weight protein present in all cells except erythrocytes. Although Tbeta(4) is the major monomeric actin-sequestering peptide in cells and can depolymerize F-actin, evidence is mounting to support the idea that it has multiple, seemingly diverse, cellular functions. In cornea, as in other tissues, Tbeta(4) promotes cell migration and wound healing, has anti-inflammatory properties, and suppresses apoptosis. In this review we discuss the current state of knowledge regarding the effects of Tbeta(4) in maintaining the healthy, functional cornea. The clinical implications of the use of Tbeta(4) as a wound healing and anti-inflammatory agent are discussed.

Thymosin beta4 inhibits benzalkonium chloride-mediated apoptosis in corneal and conjunctival epithelial cells in vitro.

Thymosin beta-4 (Tbeta(4)) is known to promote ocular wound healing, to decrease ocular inflammation, and to have anti-apoptotic effects on corneal epithelium. In this study, the effect of Tbeta(4) on the survival of human ocular surface epithelial cells exposed to benzalkonium chloride (BAK) was measured. Human conjunctival epithelial cells (HC0597) or human corneal epithelial cells (HCET) were treated with 0%, 0.001%, 0.01%, or 0.1% BAK for 15 min. After 3 or 24h of recovery in culture medium containing 1 microg/ml Tbeta(4), a dosage that has been demonstrated effective in several published studies, DNA synthesis was measured using a colorimetric BrdU incorporation assay. Both conjunctival and corneal epithelial DNA synthesis was inhibited by BAK in a dose-dependent manner. Tbeta(4) did not protect the epithelial cells from BAK-induced inhibition of proliferation. To assess the ability of Tbeta(4) to prevent apoptosis, epithelial cells were treated with 0.01% BAK+Tbeta(4) and cell death was measured using a colorimetric assay. BAK-induced apoptosis increased throughout the duration of the assay, which was carried out to 5 days in culture. Treatment of HC0597 cells with Tbeta(4) significantly inhibited the apoptosis shown to be initiated by BAK. Treatment of non-transformed human corneal epithelial cells (HCEC) with Tbeta(4) also significantly inhibited the apoptosis shown to be initiated by BAK at later times in culture. Ocular solutions containing BAK as a preservative are typically used for extended periods of time. This study suggests that Tbeta(4) may be able to overcome the apoptotic side effect of BAK, and may be a useful additive to solutions containing this preservative.

Plasminogen activator inhibitor-1 (PAI-1) stimulates human corneal epithelial cell adhesion and migration in vitro.

In addition to its role as an inhibitor of urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) is hypothesized to regulate epithelial cell adhesion and migration. We have previously reported that PAI-1 may be an important regulatory factor of the uPA system in cornea. The purpose of this study was to extend those observations by determining the effect of exogenous PAI-1 on the migration and adhesion of human corneal epithelial cells (HCEC) in vitro. The expression of PAI-1 in non-transformed early passage HCEC was confirmed by immunofluorescence microscopy and Western blot analysis. Colorimetric assays coupled with function-inhibiting antibody studies using the matrix assembled in situ by cultured cells demonstrate that immobilized PAI-1 serves as an efficient substrate for HCEC adhesion. HCEC attachment to PAI-1 is comparable to that of laminin-10, a known strong adhesion protein for epithelial cells. In addition to serving as an adhesion substrate, PAI-1 also functions as a chemotactic agent for corneal epithelium. Additionally it promotes the random migration of HCEC, from an initial cell cluster, along a culture substrate. Our results in corneal epithelium are consistent with reports from other investigators showing that PAI-1 facilitates both epithelial adhesion and migration. From our studies we conclude that PAI-1 may play a dual role in corneal wound healing. Initially PAI-1 may function to stimulate migration and facilitate the reepithelialization of the wound bed. Post-reepithelization, PAI-1 may ensure corneal epithelial cell adhesion to matrix to promote successful wound healing.

Thymosin-beta4 inhibits corneal epithelial cell apoptosis after ethanol exposure in vitro.

PURPOSE: The purpose of this study was to determine the effect of thymosin beta 4 (Tbeta(4)) treatment on human corneal epithelial cells exposed to ethanol in vitro. The efficacy of Tbeta(4) in preventing mitochondrial disruption and in inhibiting caspase-mediated apoptosis was examined. METHODS: Nontransformed human corneal epithelial cells (HCECs) at passage 4 were untreated or treated with ethanol (20% for 20 seconds) or a combination of ethanol and Tbeta(4). The cells were allowed to recover from ethanol treatment for 24 hours. Mitochondrial membrane integrity and the release of cytochrome c to the cytoplasm were assessed using microscopy, Western blot, and ELISA. Bcl-2 expression and cell proliferation were measured using ELISA. Colorimetric activity assays were completed for caspase-2, -3, -8, and -9. RESULTS: Tbeta(4) treatment decreased deleterious mitochondrial alterations, significantly decreased cytochrome c release from mitochondria, and increased Bcl-2 expression in ethanol-exposed human corneal epithelial cells. In ethanol-exposed corneal epithelium Tbeta(4) treatment inhibited caspase-2, -3, -8, and -9 activity, with caspase-8 showing the most significant inhibition. Tbeta(4) treatment resulted in no significant effect on the proliferation of human corneal epithelial cells after ethanol exposure. CONCLUSIONS: Tbeta(4) plays an antiapoptotic role under conditions of epithelial cell challenge with an external stress such as exposure to ethanol. Tbeta(4) may function as an antiapoptotic agent by inhibiting the release of cytochrome c from mitochondria and by suppressing the activation of caspases.