Masaki Imayasu

The Relation between Contact Lens Oxygen Transmissibility and Binding of Pseudomonas aeruginosa to the Cornea after Overnight Wear

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.

Effects of multipurpose contact-lens care solutions on adhesion of pseudomonas aeruginosa 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.

Effects of multipurpose contact lens care solutions on the adhesiveness of Acanthamoeba to corneal epithelial cells.

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.

Effects of multipurpose contact lens care solutions and their ingredients on membrane-associated mucins of human corneal epithelial cells.

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 Dulbeccos 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.

Dogs and Humans Share a Common Susceptibility Gene SRBD1 for Glaucoma Risk

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.

Effects of multipurpose contact lens care solutions on the adhesion of Acanthamoeba to silicone hydrogel contact lenses.

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.

Assessment of effects of multipurpose contact lens care solutions on human corneal epithelial cells.

To the Editor: Contact lens wearers use multipurpose contact lens solutions (MPSs) to clean, rewet, and disinfect their lenses. However, the effects of MPSs and their constituents on the ocular surface integrity and the tear film mucin layer are not understood. Here, we confirm by immunoblotting that polyhexamethylene biguanide (PHMB)based and polyquaternium-1 (Polyquad)-based multipurpose solutions including boric acid reduce membrane-associated mucins (MUC1 and MUC16) in human corneal epithelial (HCE-T) cells. These results confirm an urgent need to look for similar effects in an in vivo model, especially if found to be associated with solutionenhanced binding of microbial pathogens to the corneal surface. Membrane-associated mucins play an important role in the stability of the tear film and the maintenance of the integrity of the ocular surface. To protect the ocular surface from microbial invasion, contact lens wearers use MPSs to disinfect their contact lenses. Currently, there are many different types of MPSs with different formulations in the contact lens business market. However, in the past years, several MPS-related microbial outbreaks have been reported. These microbial outbreaks have led to investigations on both contact lens wearers’ habits and the effect of MPS constituents on the ocular surface. Quantitative real time polymerase chain reaction (qPCR) and immunostaining studies have shown that PHMB-based and Polyquad-based multipurpose solutions including boric acid reduce the mRNA and the expression of membraneassociated mucins (MUC1, MUC4, and MUC16). In this study, we showed that MPSs quantitatively reduce the protein levels of MUC1 and MUC16 in vitro using SV40-immortalized HCE-T cells. Commercially available membrane-associated mucin antibodies (MUC1 antibody: cat# sc7313, VU4H5, Santa Cruz Biotechnology, CA; and MUC16 [OV185:1]: cat# GTX20697, GeneTex, Inc., Irvine, CA) were used in this study for the Western blot analysis of mucin (MUC1 and MUC16) expression. We compared the effect of six commercially available multipurpose solutions (MPS-A, MPS-B, MPS-C, MPS-D, and MPS-E) (Table 1) on MUC1 and MUC16 expression by immunostaining in HCE-T cells. In this follow-on study, after 24 hrs of 10% MPS treatments, membrane-associated mucin (MUC1 and MUC16) expressions were analyzed by Western blotting. Quantitative analysis of the Western blot bands confirmed that MPSs containing PHMB, Polyquad, and boric acid seem to reduce MUC1 and MUC16 expression in HCE-T cells (Figs. 1 and 2). We also tested the ‘‘stand-alone’’ effects of six different MPS common ingredients (0.1% macrogolglycerol hydroxystearate [hydrogenated caster oil], 0.1% poloxamer, 0.1% poloxamine, 5 pm PHMB, 0.05% boric acid, and 0.1% boric acid) on MUC1 and MUC16 protein expression levels after 24-hr treatments using HCE-T cells. The HCE-T cells were cultured to 100% confluence and treated with the above six common MPS ingredients. The HCE-T cells

Effects of multipurpose solutions on the viability and encystment of acanthamoeba determined by flow cytometry.

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 × 105 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.

Effects of lactoferrin on the viability and the encystment of Acanthamoeba trophozoites1

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.

Contact lens care solutions downregulate membrane-associated mucins 1 and 16 in cultured human corneal epithelial cells and at the rat corneal surface in Vivo

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.