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The conventional microelectrodes for recording neuronal activities do not have innate selectivity to cell type, which is one of the critical limitations for the detailed analysis of neuronal circuits. In this study, we engineered a downsized variant of the artificial synapse organizer based on neurexin1β and a peptide-tag, fabricated gold microelectrodes functionalized with the receptor for the organizer, and performed validation experiments in primary cultured neurons. Successful inductions of synapse-like junctions were detected at the sites of contact between neurons expressing the engineered synapse organizer and functionalized microelectrodes, but not in the negative control experiment in which the electrode functionalization was omitted. Such a molecularly inducible neuron-microelectrode junction could be the basis for the next-generation electrophysiological technique enabling cell type-selective recording., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

It has been proposed that cell-type-specific bioelectronic interfaces for neuronal circuits could be established by utilizing the function of synapse organizers. For this purpose, using neurexin-1β and a peptide tag, we engineered compact synapse organizers that do not interact with the naturally occurring receptors but induce presynaptic differentiation upon contact with nanobody-decorated objects in cultured mammalian and chick forebrain neurons. In chick neurons, the engineered organizer exerted synaptogenesis typically in ∼4 h after the contact, even under an air atmosphere at room temperature, thereby providing a useful cellular model for establishing the molecularly inducible neuron-microelectrode interface., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Reverse pH-dependent fluorescent protein, including dKeima, is a type of fluorescent protein in which the chromophore protonation state depends inversely on external pH. The dependence is maintained even when immobilized at the metal-solution interface. But, interestingly, its responses to the hydrogen evolution reaction (HER) at the interface are not reversed: HER rises the pH of the solution around the cathode, but, highly active HER induces chromophore deprotonation regardless of the reverse pH dependence, reflecting an interface-specific deprotonation effect by HER. Here, we exploit this phenomenon to perform scanning-less, real-time visualization of interfacial proton dynamics during HER at a wide field of view. By using dKeima, the HER-driven deprotonation effect was well discriminated from the solution pH effect. In the electrodes of composite structures with a catalyst, dKeima visualized keen dependence of the proton depletion pattern on the electrode configuration. In addition, propagations of optical signals were observed, which seemingly reflect long-range proton hopping confined to the metal-solution interface. Thus, reverse pH-dependent fluorescent proteins provide a unique tool for spatiotemporal analysis of interfacial proton dynamics, which is expected to contribute to a better understanding of the HER process and ultimately to the safe and efficient production of molecular hydrogen., (© 2023. Springer Nature Limited.)

Clustering of neurexin-1β occurs through the formation of a trans-cellular complex with neuroligin-1, which promotes the generation of presynapse. While the extracellular region of neurexin-1β functions to constitute the heterophilic binding interface with neuroligin-1, it has remained unclear whether the region could also play any key role in exerting the intracellular signaling for presynaptic differentiation. In this study, we generated neurexin-1β lacking the binding site to neuroligin-1 and with a FLAG epitope at the N-terminus, and examined its activity in cultured neurons. The engineered protein still exhibited robust synaptogenic activities upon the epitope-mediated clustering, indicating that the region for complex formation and that for transmitting presynapse differentiation signals are structurally independent of each other. Using a fluorescence protein as an epitope, synaptogenesis was also induced by a gene-codable nanobody. The finding opens possibilities of neurexin-1β as a platform for developing various molecular tools which may allow, for example, precise modifications of neural wirings under genetic control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Lactoferrin (LF) is an iron-binding basic glycoprotein that has an antimicrobial effect against certain microbes. The purpose of this study is to evaluate the amoebicidal effect of bovine milk LF (bLF) against Acanthamoeba clinical-isolate trophozoites, which cause severe keratitis. Most of the risk factor for Acanthamoeba keratitis is from wearing soft contact lenses (SCLs). Acanthamoeba trophozoites were incubated in bovine LF (bLF) solution, and the ratios of viability and encystment were determined with microscopic analysis of cyst formation. The amoebicidal effect of bLF was assessed by Trypan blue assay. The ratios of viable cells in the presence of iron-free bLF (apo-bLF), native-bLF, and iron-saturated bLF (Fe-bLF) at the concentration of 10 μmol/L for 60 min were 7.7% ± 4.6%, 80.7% ± 10.1%, and 97.3% ± 1.5%, respectively. Apo-bLF showed potent amoebicidal effect against Acanthamoeba trophozoites, but Fe-bLF did not have this effect. After treating with apo-bLF, most dead cells were nonglobular forms of trophozoites but not cystic forms. Encystment of Acanthamoeba was assessed by the sarkosyl-calcofluor white assay. The encystment ratios treated with 0.5% propylene glycol (positive control) and 10 μmol/L apo-bLF for 24 h were 96.12% ± 10.6% and 0.47% ± 0.5%, respectively. These results suggest that the amoebicidal effect of apo-bLF without encystment might lead to the prevention of contamination of Acanthamoeba in SCL stock cases.

Human papillomavirus type 16 (HPV16) is a major cause of cervical cancer. We previously demonstrated that C-to-T and G-to-A hypermutations accumulated in the HPV16 genome by APOBEC3 expression in vitro. To investigate in vivo characteristics of hypermutation, differential DNA denaturation-PCR (3D-PCR) was performed using three clinical specimens obtained from HPV16-positive cervical dysplasia, and detected hypermutation from two out of three specimens. One sample accumulating hypermutations in both E2 and the long control region (LCR) was further subjected to Next-Generation Sequencing, revealing that hypermutations spread across the LCR and all early genes. Notably, hypermutation was more frequently observed in the LCR, which contains a viral replication origin and the early promoter. APOBEC3 expressed abundantly in an HPV16-positive cervix, suggesting that single-stranded DNA exposed during viral replication and transcription may be efficient targets for deamination. The results further strengthen a role of APOBEC3 in introducing HPV16 hypermutation in vivo., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) proteins are cellular DNA/RNA-editing enzymes that play pivotal roles in the innate immune response to viral infection. APOBEC3 (A3) proteins were reported to hypermutate the genome of human papillomavirus 16 (HPV16), the causative agent of cervical cancer. However, hypermutation did not affect viral DNA maintenance, leaving the exact role of A3 against HPV infection elusive. Here we examine whether A3 proteins affect the virion assembly using an HPV16 pseudovirion (PsV) production system, in which PsVs are assembled from its capsid proteins L1/L2 encapsidating a reporter plasmid in 293FT cells. We found that co-expression of A3A or A3C in 293FT cells greatly reduced the infectivity of PsV. The reduced infectivity of PsV assembled in the presence of A3A, but not A3C, was attributed to the decreased copy number of the encapsidated reporter plasmid. On the other hand, A3C, but not A3A, efficiently bound to L1 in co-immunoprecipitation assays, which suggests that this physical interaction may lead to reduced infectivity of PsV assembled in the presence of A3C. These results provide mechanistic insights into A3s' inhibitory effects on the assembly phase of the HPV16 virion., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: The purpose of this study was first to evaluate the effect of multipurpose contact lens care solutions (MPSs) on the expression of membrane-associated mucins (MUC1 and MUC16) in SV40-transformed human corneal epithelial (HCE-T) cells and in vivo rat cornea. The second aim of this study was to determine the role of the common MPS additive boric acid in reducing mucin expression and release., Methods: The HCE-T cells were exposed to different concentrations of MPS-F, MPS-G, MPS-H, MPS-I, and MPS-J with 100% treatment for 30 minutes and 10% treatment for 24 hours. MUC1 and MUC16 expressions were subsequently analyzed by Western blotting. Wister rats were also subjected to MPS-A, MPS-B, MPS-C, MPS-D, and MPS-E and received phosphate-buffered saline exposure (1 drop in the right eye every 10 minutes for 1 hour). The left eye was used as control. Cornea sections and lysates were used for the immunohistochemical assay of MUC1 and MUC16 expressions. Conditioned media from treated HCE-T cells were also analyzed using Western blotting., Results: The MPSs containing boric acid downregulated MUC1 and MUC16 in the rat cornea, whereas MPSs without boric acid had no effect as demonstrated by the Western blotting and immunohistochemical analysis. Conditioned media from MPS-containing boric acid revealed some trace of MUC16., Conclusions: The clinical use of MPSs containing boric acid that reduce MUC1 and MUC16 availability should be avoided. Additionally, the presence of MUC16 in the conditioned media suggests that boric acid may have enhanced cleavage of MUC16 at the cell membrane surface.

Glaucoma is a degenerative optic neuropathy that is associated with elevated intraocular pressure. Primary open angle glaucoma is the most common type of glaucoma in canines, and its highest incidence among dog breeds has been reported in Shiba-Inus, followed by Shih-Tzus. These breeds are known to have an abnormal iridocorneal angle and dysplastic prectinate ligament. However, the hereditary and genetic backgrounds of these dogs have not yet been clarified. In this study, we investigated the association between polymorphisms of the glaucoma candidate genes, SRBD1, ELOVL5, and ADAMTS10, and glaucoma in Shiba-Inus and Shih-Tzus. We analyzed 11 polymorphisms in these three genes using direct DNA sequencing. Three SRBD1 SNPs, rs8655283, rs22018514 and rs22018513 were significantly associated with glaucoma in Shiba-Inus, while rs22018513, a synonymous SNP in exon 4, showed the strongest association (P = 0.00039, OR = 3.03). Conditional analysis revealed that rs22018513 could account for most of the association of these SNPs with glaucoma in Shiba-Inus. In Shih-Tzus, only rs9172407 in the SRBD1 intron 1 was significantly associated with glaucoma (P = 0.0014, OR = 5.25). There were no significant associations between the ELOVL5 or ADAMTS10 polymorphisms and glaucoma in Shiba-Inus and Shih-Tzus. The results showed that SRBD1 polymorphisms play an important role in glaucoma pathology in both Shiba-Inus and Shih-Tzus. SRBD1 polymorphisms have also been associated with normal- and high-tension glaucomas in humans. Therefore, SRBD1 may be a common susceptibility gene for glaucoma in humans and dogs. We anticipate that the nucleotide sequencing data from this study can be used in genetic testing to determine for the first time, the genetic status and susceptibility of glaucoma in dogs, with high precision. Moreover, canine glaucoma resulting from SRBD1 polymorphisms could be a useful animal model to study human glaucoma.

Purpose: To evaluate simultaneously the effects of multipurpose contact lens care solution (MPS) on the viability and encystment of Acanthamoeba using flow cytometry., Methods: Viability and encystment rate were evaluated using Acanthamoeba castellanii (ATCC 50514 and ATCC 50370) and three clinical strains of Acanthamoeba spp. isolated from patients with Acanthamoeba keratitis. Acanthamoeba trophozoites (1.0 × 10(5) cells/mL) were exposed to four kinds of commercially available MPSs for 24 hours. After dispensing the cell suspension into two portions, one portion was stained with 0.004% Congo Red (CR), a fluorescence dye to stain the inner cell wall of cysts, and the other portion was stained with a mixture of Congo Red and 3% sarkosyl (CRS), a detergent to lyse the trophozoites and pseudocysts. Flow cytometric analysis of the treated portions was then carried out on an EPICS ALTRA flow cytometer. The encystment rate and disinfecting efficacies (percentage of rounded trophozoites, "pseudocyst") were calculated by the rates of CR-stained, CR-nonstained, and CRS-stained populations, respectively. Ultrastructural features of resistant (mature or immature) cysts and pseudocysts were observed by transmission electron microscopy., Results: Resistant cysts and rounded trophozoites (pseudocysts) were stained with CR, whereas native (unrounded) trophozoites were not. Resistant cysts were also stained with CRS unlike pseudocysts. Three clinical isolates showed higher resistance and higher encystment rates than two ATCC strains when treated with encystment-positive control solution. Disinfecting efficacy of each MPS was not directly related to each encystment rate. Transmission electron microscopy observations showed basic differences in the ultrastructure of pseudocysts produced by MPSs and resistant cysts., Conclusions: These results suggest that viability and encystment of Acanthamoeba are independent phenomena, and therefore disinfecting efficacy of MPS and encystment rates of Acanthamoeba should be evaluated, respectively. Thus, it is important to evaluate simultaneously the disinfecting efficacies and encystment rates of newly developed premarket MPS using the authors' novel flow cytometric methods.

Purpose: The aim of this study was to simulate the biofilm formation in contact lens (CL) case under poor hygiene behaviors; antimicrobial efficacies of multipurpose solutions (MPSs) against biofilm on the lens case were evaluated., Methods: Five MPSs (Epica Cold, Complete 10 min, ReNu MultiPlus, SoftOne Mois, and OPTI-FREE Plus) were tested. Lens cases containing ACUVUE2 were inoculated with 1×10, 10, or 10 colony-forming units (CFUs) of Staphylococcus epidermidis (SE). Each lens case was treated with 1 MPS for 4 hrs followed by the estimation of the number of SE by the CFU method. Disinfection efficacies of MPSs against SE biofilm were evaluated by biomicroscopy with safranin staining and scanning electron microscopy., Results: Lens cases, inoculated with 1×10 CFU, were disinfected by all MPSs. Epica Cold, Complete 10 min, ReNu MultiPlus, and OPTI-FREE showed almost a 2-log reduction of the CFU, whereas SoftOne Mois effect was almost a 1 log reduction, significantly lower than other MPSs (P<0.05). No biofilm formations were observed in Epica Cold, Complete 10 min, ReNu MultiPlus, and OPTI-FREE Plus-treated groups unlike significant biofilm formation in the SoftOne Mois-treated group (P<0.01)., Conclusions: Greater efforts to educate patients regarding compliant lens care behavior are needed to reduce the incidence of CL-associated microbial keratitis.

Purpose: To evaluate the effect of 7 multipurpose contact lens care solutions (MPSs) on the adhesion of Acanthamoeba (AC) to 5 silicone hydrogel contact lenses (SHCLs)., Methods: Acanthamoeba castellanii (ATCC50370) trophozoites were inoculated onto disks trimmed from SHCLs, Asmofilcon A, Galyfilcon A, Senofilcon A, Lotrafilcon B, and Balafilcon A. After 4-hour incubation, the number of adherent AC trophozoites on SHCL was counted under phase contrast microscopy. AC trophozoites mixed with 7 MPSs were inoculated onto Balafilcon A and incubated for 24 hours followed by direct counting, phase contrast microscopy, and scanning electron microscopy. AC cysts were also inoculated onto Balafilcon A followed by counting using phase contrast microscopy., Results: Adhesion of AC trophozoites to Lotrafilcon B and Balafilcon A was 10 times higher in comparison with the other 3 SHCLs. Twenty four-hour treatment of AC trophozoites with Epica Cold, Epica Cold Aquamore, ReNu MultiPlus, OptiFree Plus, and Complete DoubleMoist reduced the numbers of adherent AC to less than 25% of control, whereas the numbers of AC treated with Complete AminoMoist and C3 SoftOne Moist was about 50% and 75% of control, respectively. Normal AC trophozoites without any treatments showed 25 times higher adhesion rates compared with normal AC cysts., Conclusions: The adhesion rates of AC trophozoites to SHCL varied depending on the type of MPSs used. Appropriate uses of MPS could reduce adhesion rates of AC to SHCL and potentially decrease clinical rates of Acanthamoeba keratitis.

Purpose: Membrane-associated mucins play an important role for protecting epithelial cells at the ocular surface from microbial invasion. The purpose of this study was to determine whether multipurpose solutions (MPSs) for contact lens care and their ingredients alter the expression of membrane-associated mucins in human corneal epithelial (HCE-T) cells., Methods: SV40-immortalized HCE-T cells were cultured in Dulbecco's modified Eagle medium/F12 medium with 5% fetal bovine serum to confluence and were then exposed to 10% dilutions of five different MPSs, or to their representative ingredients, 0.1% macrogolglycerol hydroxystearate, 0.1% poloxamer, 0.1% poloxamine, 1 and 5 ppm polyhexamethylene biguanide, or 0.05% and 0.1% boric acid for 24 hr. Quantitative real-time polymerase chain reaction was used to investigate the gene expression of MUC1, MUC4, and MUC16. Immunofluorescence staining of MUC16 protein on the surface of the HCE-T cells exposed to 10% diluted MPSs for 24 hr or undiluted MPSs for 30 min was observed by laser confocal scanning microscopy followed by quantitative image analysis., Results: Three MPSs containing boric acid significantly reduced gene expressions of MUC1 from 20.2% to 56.7% (P<0.01). Gene expressions of MUC4 and MUC16 were also reduced by these MPSs; however, there were no significant differences. Among ingredients, 0.1% boric acid significantly reduced gene expressions of MUC1 and MUC16 by 7.4% and 18.9%, respectively (P<0.01). Immunofluorescence microscopy also demonstrated that in undiluted form, three MPSs containing boric acid significantly reduced the expression of MUC16 protein., Conclusions: The MPSs containing boric acid downregulate membrane-associated mucins as compared with MPSs that do not contain boric acid. There may be some subtle membrane or other interactions between ingredients in lens-care solutions that adversely alter corneal cell mucins.

Transient receptor potential vanilloid 3 (TRPV3), a member of the calcium-permeable thermosensitive TRP (thermoTRP) subfamily of receptors, is an important cutaneous sensor that detects thermal and chemical stimuli. TRPV3 is activated by innocuous warm temperature stimuli (>33 degrees C) and a variety of physiologically active substances. While the corneal epithelium is known to respond to such stimuli, it is unknown whether TRPV3 is involved in this phenomenon. We show here that TRPV3 mRNA and protein are abundantly expressed in the epithelial cells of human and mouse cornea. Carvacrol, an agonist of TRPV3, elevated cytosolic Ca2+ concentration in both primary mouse corneal epithelial cells and cultured human corneal epithelial cells (HCE-T cells). The response to carvacrol was inhibited by ruthenium red, a TRPV channel antagonist. Moreover, repetitive agonist stimulation sensitized the response with gradually increasing amplitude, suggesting that the TRPV3 in the cornea has similar physiological and pharmacological characteristics to that in skin keratinocytes. Finally, a wound healing assay revealed that appropriate calcium ion influx via activated TRPV3 in corneal epithelial cells accelerated their proliferation. Thus, functional TRPV3 is present in corneal epithelial cells and may play a role not only in thermosensation, but also in the regulation of cell proliferation.

Purpose: To study the adhesion of Acanthamoeba castellanii treated with multipurpose contact lens care solutions (MPSs) to human corneal epithelial cells., Methods: Cell suspensions of A. castellanii (ATCC50514) trophozoites were mixed with six MPSs: MPS-A (polyhexamethylene biguanide [PHMB], macrogolglycerol hydroxystearate, propylene glycol), MPS-B (PHMB, poloxamine, boric acid), MPS-C (polyquad, poloxamine, boric acid), MPS-D (PHMB, poloxamer, propylene glycol), MPS-E (PHMB, poloxamer), or MPS-F (PHMB, poloxamer) for 4 hr. Morphologic changes of A. castellanii after exposure with MPSs were observed with scanning electron microscopy. A. castellanii cells treated with MPS for 4 hr were inoculated onto cultured SV40-immortalized human corneal epithelial cells. After 2-hr incubation, the number of adherent A. castellanii was assessed by 18S-rDNA quantification using real-time polymer chain reaction., Results: After 4-hr incubation, MPS-A- and MPS-B-treated A. castellanii have changed from trophozoite morphology into cyst form; however, MPS-E- and MPS-F-treated A. castellanii maintained trophozoite morphology. In contrast, both cyst and trophozoite forms were observed in MPS-C- and MPS-D-treated A. castellanii. The adherence rate of A. castellanii was approximately two times higher in MPS-E (not significant), and more than three times higher in MPS-F (P<0.05) compared with MPS-A, which produced the lowest adhesiveness as estimated by real-time polymer chain reaction., Conclusions: Taken together, these results support the possibility that chronic use of MPS with the lowest efficacies on promoting encystment of A. castellanii (MPS-E and MPS-F) by hydrogel contact lens wearers may increase adhesiveness of A. castellanii to corneal epithelial cells.

Purpose: To study the adhesion of Pseudomonas aeruginosa (PA) to human corneal epithelial cells treated with multipurpose contact-lens care solutions (MPSs)., Methods: SV40-immortalized human corneal epithelial cells (svHCET cells) were cultured on collagen-coated culture slides for 7 days. The svHCET cells were exposed to three MPSs: MPS-A (polyhexamethylene biguanide, macrogolglycerol hydroxystearate), MPS-B (polyhexamethylene biguanide, Poloxamer, and boric acid) or MPS-C (Polyquad, Poloxamine, and boric acid) for 60 min. PA cells (ATCC27853) were inoculated onto cultured svHCET cells and adhesion was observed with a PKH67 fluorescent dye-labeling method using a confocal laser scanning microscope. The number of adherent PA was assessed by 16S-rDNA quantification using real-time polymerase chain reaction and confocal laser scanning microscope imaging. PA adhesion and inter- and intracellular invasion into svHCET cells were also observed with scanning electron microscopy and transmission electron microscopy., Results: PA adhesion was more than three times higher in MPS-B-treated cells and six times higher in MPS-C-treated cells compared with control estimated by real-time polymerase chain reaction (P < 0.05). MPS-A-treated cells showed no significant increase in PA adhesion. With scanning electron microscopy and transmission electron microscopy, PA were observed to enter opened cell-cell borders between adjacent svHCET cells treated with MPS-B and C, but not with MPS-A., Conclusions: Taken together, these results support the possibility that chronic use of MPS containing boric acid (MPS-B and MPS-C) by hydrogel contact lens wearers may lead to increased risk for associated microbial corneal infection with PA.

Purpose: To compare the effects of four commercially available multipurpose solutions (MPSs) on the structure and barrier function of corneal epithelial tight junctions., Methods: Human corneal epithelial cells were cultured on collagen-coated slides and then exposed to MPS A (polyhexamethylene biguanide, macrogolglycerol hydroxystearate), MPS B (polyhexamethylene biguanide, poloxamine), MPS C (Alexidine, poloxamine), and MPS D (POLYQUAD, poloxamine) for 60 minutes. Tight junction integrity of the corneal epithelial cells was evaluated with ZO-1 (tight junction-related protein) labeling under laser confocal microscopy. To investigate the changes of ultrastructure in tight junctions of human corneal epithelial cells, an ultrathin cross-section of the cell on collagen membrane was also observed by transmission electron microscopy. For quantitative evaluation of barrier functions, transepithelial electrical resistance of the epithelial cell was measured 30, 60, and 120 minutes after MPS exposure by using a volt ohmmeter., Results: The control (i.e., without MPS treatment) and MPS A-treated epithelial cells showed a normal, continuous linear pattern in ZO-1 staining along with cell-cell junctions. However, epithelial cells treated with MPS B, MPS C, and MPS D showed discontinuous, disrupted line structures at cell-cell borders. This may correspond to a partial breakdown of epithelial tight junctions. Treatment of epithelial monolayers with MPS B, MPS C, and MPS D caused a time-dependent decrease in transepithelial electrical resistance, whereas there was no significant difference between the MPS A-treated group and the control group., Conclusions: These results suggest the possibility that frequent use of a MPS with high cytotoxicity may lead to the breakdown of epithelial barrier functions and increase the risk of associated microbial infections in hydrogel lens wearers.

Purpose: To investigate the role of mitogen-activated protein kinase (MAPK), such as p44/42 MAPK, p38 MAPK and stress-activated protein kinase (SAPK), in corneal epithelial cells during the wound healing process., Methods: A single non-penetrating incision was produced on rat cornea. Then the corneal wound healing process was observed with an immunocytochemical technique using specific antibodies reacting only with phosphorylated p44/42 MAPK, p38 MAPK or SAPK. Cell lysates of corneal epithelial cells in rabbits stimulated with keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were processed for Western blot using antibodies to phosphorylated p44/42 MAPK., Results: Maximum activation of p44/42 MAPK was observed in wing and basal cells at wounded regions in rat cornea at 1 hour after the incision. Activation of p44/42 MAPK was still detected in all basal and wing cells at wounded regions at up to 24 hours when the incisions were completely closed, and then receded to normal intensity after 7 days. Neither p38 MAPK nor SAPK were activated during the wound healing process. Western blot analysis of cultured corneal epithelial cells in rabbits showed phosphorylation of p44/42 MAPK after 30 minutes in response to KGF and HGF, whereas non-activated p44/42 MAPK was ordinarily detected even at the absence of KGF or HGF., Conclusions: These results demonstrate that p44/42 MAPK is activated during the corneal wound healing process and suggest that KGF and HGF play an important role in initiation of cell migration and proliferation in the initial wound healing process by activating p44/42 MAPK.

With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.

Purpose: To assess the chronic effects of rigid gas-permeable (RGP) contact lenses on corneal swelling and glucose-lactate metabolism in the rabbit cornea during 1 month of continuous extended wear and to establish the relationship between these effects and the oxygen transmissibility (Dk/L) of the test lens polymer., Methods: Four RGP lenses of varying Dk/L were tested in 8 rabbits per test group (left eyes served as controls). After 7 days and 1 month extended wear, the concentrations of lactate and glucose in the corneal epithelium, stroma and aqueous humor were determined by enzyme assay; and epithelial and stromal ATP concentrations were separately measured by bioluminescence techniques. Corneal thickness was measured at a standard morning time by ultrasonic pachymetry before and after 1, 7, 15 days and 1 month extended wear., Results: After 7 days and 1 month extended wear, generalized decreases were found in aqueous humor lactate levels for all test lenses, while concomitant increased aqueous glucose concentrations were observed. Total epithelial lactate levels correlated inversely with decreasing Dk/L levels for lower oxygen transmissible lenses (R = 0.951, P = 0.0051); and remained unchanged after extended wear of the hyper-oxygen transmissible Dk/L 125 test lens. By contrast, stromal lactate levels consistently decreased at all time points measured forextended wear of all test lenses. As expected, both epithelial and stromal ATP concentrations simultaneously decreased in extended wear. Overnight corneal swelling values after 24 hours wear of Dk/L = 27, 43, 70 and 125 test lenses were increased by 9.8, 7.1, 5.5, and 5.2% while persistent (residual) stromal swelling after one month extended wear was 16.8, 10.1, 8.6, and 5.6% respectively, in excess of baseline values., Conclusions: Chronic RGP contact-lens induced hypoxia is associated with altered glucose-lactate metabolism in the cornea and aqueous humor with excess production of increased levels of lactate in the epithelium for lower Dk/L test lenses, but decreased lactate concentration in the stroma and aqueous humor. Extended wear of the hyper-oxygen transmissible test lens (Dk/L = 125) however, produced no increase in epithelial lactate levels. Expected lens-induced decreases in epithelial and stromal ATP were not dependent on lens-oxygen transmissibility. Despite the persistence of lower than normal stromal levels of lactate during 1 month of extended wear for all test lenses, residual corneal swelling values remained consistently elevated above baseline values. Taken together, these data establish that increased stromal lactate accumulation cannot account for persistent stromal edema in chronic extended wear of RGP lenses; and that this effect appears to be independent of lens-oxygen transmissibility and may thus represent the prolonged mechanical effect of lens wear itself.

We evaluated the effects on the rabbit cornea of daily wear of rigid gas permeable (RGP) contact lenses treated with preserved care solutions by measuring concomitant tear lactate dehydrogenase (LDH) activity followed by in vivo tandem scanning confocal microscopy (TSCM). In vivo morphologic changes were confirmed by in vitro scanning electron microscopy (SEM). Two standard commercial RGP lens wetting and soaking solutions from the same manufacturer were tested: solution A with 0.004% benzalkonium chloride (BAK) and solution B with 0.003% chlorhexidine digluconate (CHX) and 0.002% thimerosal. Two experimental PBS-based wetting and soaking solutions were also tested: solution C with 0.005% BAK and 2% hydroxypropylmethylcellulose (HPMC) and solution D with 0.005% BAK without HPMC. Instillation of solution A without contact lens wear caused significant (P < 0.01) increases in desquamation of the superficial corneal epithelium and tear LDH activity compared with control eyes. After 3 weeks of RGP contact lens daily wear (8 hours/day), modified Draize scores of ocular surface lesions on the eyes wearing RGP lenses treated with solution A increased according to the duration of lens wear. Solution B did not produce significant change. With daily wear for 4 days (8 hours/day), RGP lenses treated with solution C and solution D produced increased corneal epithelium desquamation and an increase of LDH activity in tears. These effects were greater with HPMC (solution C) than without HPMC (solution D).(ABSTRACT TRUNCATED AT 250 WORDS)

Purpose: To assess adverse effects of contact lens-induced hypoxia on the rabbit cornea in vivo and determine the relation between binding of Pseudomonas aeruginosa and oxygen transmissibility for rigid and hydrogel lenses., Methods: Six rigid lenses with Dk/Ltotal values between 0 and 97 x 10(-9) (cm/second) (ml O2/ml mmHg) and four hydrogel lenses (Dk/Ltotal 9, 20, 39, 51) were tested. All lenses had 14.0-mm diameters and a thickness (parallel) of 0.12 or 0.15 mm. Tear lactate dehydrogenase activity and tandem scanning confocal microscopy determinations were performed after the lens was worn for 24 hours. Binding of P. aeruginosa then was separately determined by the colony-forming unit method. Scanning electron microscopy was used to confirm in vivo tandem scanning confocal microscopy findings., Results: Lens oxygen transmissibility determines binding of P. aeruginosa to the cornea after the lens is worn for 24 hours; epithelial damage produced by lenses of lower Dk/Ltotal appears to be the dominant biologic factor for P. aeruginosa binding and not lens rigidity., Conclusions: These results suggest that the risk of P. aeruginosa keratitis developing with overnight wear will be enhanced significantly for contact lenses with Dk/Ltotal values less than 50 x 10(-9) (cm/second) (ml O2/ml mmHg) (human equivalent oxygen percentage < or = 15%), and this risk will increase with further decreases in oxygen transmissibility. Because no hydrogel lenses approved by the Food and Drug Administration are available with oxygen transmission at this level, patients should be made aware of the increased risk of infectious keratitis associated with the overnight wear of current extended wear hydrogel lenses. Results of this study also demonstrate that quantitative clinical tandem scanning confocal microscopy imaging and tear lactate dehydrogenase activity measurements can provide prospective, noninvasive methods for assessing the ongoing interaction between contact lens and cornea in vivo.

We used noninvasive biochemical techniques to study the effects on rabbit corneas of 7-day extended wear of rigid gas permeable (RGP) contact lenses of varying oxygen transmissibilities. Corneal effects were assessed through measurement of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities and albumin levels in tears. The RGP contact lenses used had Dk/Ltotal values ranging from 33 to 64 x 10(-9) (cm/sec) (mL O2/mL mmHg) and were of uniform 0.15 mm center thickness. Extended wear of high Dk (Dk/Ltotal = 34) and super high Dk (Dk/Ltotal = 56) lenses caused an increase in tear LDH activity from 1,190 U/L (before lens wear) to over 18,000 U/L during 7 days of continuous wear. These contact lenses also caused gradual increases in tear MDH activity from 431 U/L (before lens wear) to over 750 U/L after 7 days of continuous wear. Extended wear of the ultra high Dk lens (Dk/Ltotal = 64), however, caused no significant increase in LDH or MDH activity in tears. Tear albumin levels in all contact lens wearing eyes increased after 1 day of lens wear, then gradually recovered to normal values after 2 days of continuous wear. The changes in albumin levels did not correlate with Dk/Ltotal values of lenses worn.(ABSTRACT TRUNCATED AT 250 WORDS)

We have established a quantitative method for evaluation of ocular surface lesions on the basis of lactate dehydrogenase (LDH) activity, malate dehydrogenase (MDH) activity, and albumin levels in rabbit tears. Lesions were produced with solutions of 0.005-0.02% benzalkonium chloride (BAK), 0.01-0.03% chlorhexidine digluconate (CHX), and 0.01-0.03% polyhexamethylene biguanide hydrochloride (PHMB), all of which are widely used in topical ophthalmic preparations. Two drops of test solution were instilled 15 times into rabbit eyes at 5 minute intervals. Sixty minutes after 0.02% BAK instillation, tear LDH activity increased from 1,840 U/L (without instillation) to 26,100 U/L, and concomitantly tear albumin levels rose from 0.11 mg/mL (without instillation) to 9.48 mg/mL. Instillation of 0.03% CHX and 0.03% PHMB caused smaller increases in LDH and MDH activity and albumin tear levels. LDH activity and albumin levels in tears were significantly correlated with the degree of total ocular surface lesions in both cornea and conjunctiva as observed by slit lamp biomicroscopy quantified using a modified Draize score. Based on the results of this study, we believe that LDH activity and albumin level in tears can be used as objective indicators for the quantitative evaluation of ocular surface lesions on both cornea and conjunctiva following application of topical ophthalmic preparations containing cytotoxic preservatives in animals and man.

Lactate dehydrogenase (LDH) activity in tears was measured in 202 myopic rigid gas permeable (RGP) and 79 hydrogel human contact lens wearers and 48 normal controls by noninvasive microcapillary sampling. The oxygen permeabilities (Dk) of five selected RGP contact lenses ranged between 0 and 230 x 10(-11) (cm2/s) ml O2/ml mm Hg), and the water content (WC) of the hydrogel lenses was 38, 72, and 80%. When normal diurnal variation of tear LDH activity and tear sample volume (0.3-0.6 microliters) were carefully controlled, the tear LDH activity of RGP lens wearers in daily and extended wear correlated as an inverse function of Dk/L, with the highest enzyme activity observed in wearers of polymethylmethacrylate daily wear lenses (347.4 U/L). The tear LDH activity in Menicon EX (Dk 108) lens wearers was higher in the extended wear (192.7 U/L) than in the daily wear group (161.9 U/L). There was no significant difference in tear LDH activity between the Menicon SF-P (Dk 230) extended wear group (132.0 U/L) and controls (133.5 U/L). In the hydrogel lens daily wear group, tear LDH activity of Experimental 72 (WC 72%) and Experimental 80 (WC 80%) was higher than that of hydroxyethylmethacrylate (HEMA) despite having high Dk value. Experimental 80 extended wearers showed lower LDH activity in tears sampled between 0.3 and 0.6 microliters than that of HEMA wearers. These results suggest that sequential measurement of LDH levels in tears may offer a new and unique method for the assessment of the physiologic effects of contact lens wear on the ocular surface, and provide a new clinical paradigm for the interaction of the contact lens with the cornea.