Zhenjun Zhao

Care regimen and lens material influence on silicone hydrogel contact lens deposition

Purpose. To quantitatively detect proteins and cholesterol extracted from worn silicone hydrogel contact lenses and determine the effect of various lens care solutions on deposit accumulation. Methods. Contact lenses, made from different polymers and worn on a daily wear schedule with different lens care solutions, were collected. Lipid and protein deposits were extracted by methanol:chloroform (1:1, v/v) and protein extraction solution (containing urea and surfactant), respectively. Lipid extracts were separated and cholesterol quantified using thin layer chromatography. Protein extracts were quantified using standard techniques. Results. Among all lenses tested, Balafilcon A lenses exhibited greatest extracted cholesterol (4.1 to 8.2 μg/lens) and total protein (5.4 to 23.2 μg/lens). AQuify was the most effective solution in reducing extracted deposits, especially extracted protein, from Balafilcon A lenses. AQuify and Opti-Free RepleniSH solutions were most effective in reducing extracted cholesterol from Senofilcon A and Galyfilcon A lenses, respectively. Use of Opti-Free Express solution resulted in more extracted protein from Lotrafilcon B lenses than use of other solutions. Generally, Lotrafilcon B, Senofilcon A, and Galyfilcon A lenses accumulated relatively low amount of proteins. Lotrafilcon B lenses accumulated the least amount of cholesterol deposit among all lenses tested regardless of solution used. Conclusions. Lens polymer (possibly associated with surface characteristics) is a prominent factor affecting lipid and protein accumulation. Within a lens polymer type, lens care solutions exhibit varying effectiveness in reducing protein and lipid accumulation.

Detection and quantification of tear phospholipids and cholesterol in contact lens deposits: the effect of contact lens material and lens care solution.

PURPOSE To examine the deposition of tear phospholipids and cholesterol onto worn contact lenses and the effect of lens material and lens care solution. METHODS Lipids were extracted from tears and worn contact lenses using 2:1 chloroform:methanol and the extract washed with aqueous ammonium acetate, before analysis by electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS Twenty-three molecular lipids from the sphingomyelin (SM) and phosphatidylcholine (PC) classes were detected in tears, with total concentrations of each class determined to be 5 +/- 1 pmol/microL ( approximately 3.8 microg/mL) and 6 +/- 1 pmol/microL ( approximately 4.6 microg/mL), respectively. The profile of individual phospholipids in both of these classes was shown to be similar in contact lens deposits. Deposition of representative polar and nonpolar lipids were shown to be significantly higher on senofilcon A contact lenses, with approximately 59 ng/lens SM, 195 ng/lens PC, and 9.9 microg/lens cholesterol detected, whereas balafilcon A lens extracts contained approximately 19 ng/lens SM, 19 ng/lens PC, and 3.9 microg/lens cholesterol. Extracts from lenses disinfected and cleaned with two lens care solutions showed no significant differences in total PC and SM concentrations; however, a greater proportion of PC than SM was observed, compared with that in tears. CONCLUSIONS Phospholipid deposits extracted from worn contact lenses show a molecular profile similar to that in tears. The concentration of representative polar and nonpolar lipids deposited onto contact lenses is significantly affected by lens composition. There is a differential efficacy in the removal of PC and SM with lens care solutions.

Identification of phospholipids in human meibum by nano-electrospray ionisation tandem mass spectrometry

Meibum is believed to be the major source of tear film lipids, which are vital in the prevention of excess evaporation of the aqueous phase. The complete lipid composition of meibum has yet to be established. While earlier studies reported the presence of phospholipids in human meibum, recent mass spectrometric studies have not detected them. In this study we use electrospray ionisation tandem mass spectrometry to investigate the presence of phospholipids in meibum and provide comparison to the phospholipid profile of tears. Lipids were extracted from human meibum and tear samples using standard biphasic methods and analysed by nano-electrospray ionisation tandem mass spectrometry using targeted ion scans. A total of 35 choline-containing phospholipids were identified in meibum and the profile of these was similar to that observed in tears, suggesting tear lipids are derived from meibum. The results shown here highlight the need for a combination of optimised techniques to enable the identification of the large range of lipid classes in meibum.

β-lactam allergenic determinants: fine structural recognition of a cross-reacting determinant on benzylpenicillin and cephalothin

Background An appreciation of the structural heterogeneity of allergenic determinants on penicillins and cephalosporins reveals the importance of side‐chain groups and their involvement in many allergies to β‐lactam drugs. Although allergenic cross‐reactions between penicillins and cephalosporins are known to occur, the precise molecular bases of such recognitions and cross‐sensitivities have rarely been studied and identified.

Characterization of expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in prostate cancer cell lines.

Stromal expression of some matrix metalloproteinases (MMPs) has been associated with increasing tumour burden in prostate cancer. We investigated the expression of mRNA (by RT-PCR) and protein (by zymography and western blotting) of MMPs and endogenous inhibitors (tissue inhibitors of metalloproteinases, TIMPs) in two parent epithelial prostate cancer cell lines and sublines of increasing invasive/metastatic potential. Expression of membrane type MMPs, MT1-MMP and MT3-MMP mRNA was higher in PC3-derived than in LNCaP-derived lines, whereas MT2-MMP mRNA expression was higher in the LNCaPderived than in PC3-derived cell lines. Active MT1, MT2 and MT3-MMP protein levels were similar in all lines, but processed MT-MMPs, indicative of latent MMP activation, were increased in more aggressive sublines. Expression of MMP-1, MMP-13 and TIMP-1 was higher in the more aggressive sublines and may be implicated in invasive/metastatic ability. Regulation of MMP-1 and MMP-13 expression may offer important therapeutic options for treating patients with prostate cancer.

Influence of Protein Deposition on Bacterial Adhesion to Contact Lenses

Purpose. The aim of the study is to determine the adhesion of Gram positive and Gram negative bacteria onto conventional hydrogel (CH) and silicone hydrogel (SH) contact lens materials with and without lysozyme, lactoferrin, and albumin coating. Methods. Four lens types (three SH—balafilcon A, lotrafilcon B, and senofilcon A; one CH—etafilcon A) were coated with lysozyme, lactoferrin, or albumin (uncoated lenses acted as controls) and then incubated in Staphylococcus aureus (Saur 31) or either of two strains of Pseudomonas aeruginosa (Paer 6294 and 6206) for 24 h at 37°C. The total counts of the adhered bacteria were determined using the 3H-thymidine method and viable counts by counting the number of colony-forming units on agar media. Results. All three strains adhered significantly lower to uncoated etafilcon A lenses compared with uncoated SH lenses (p < 0.05). Lysozyme coating on all four lens types increased binding (total and viable counts) of Saur 31 (p < 0.05). However, lysozyme coating did not influence P. aeruginosa adhesion (p > 0.05). Lactoferrin coating on lenses increased binding (total and viable counts) of Saur 31 (p < 0.05). Lactoferrin-coated lenses showed significantly higher total counts (p < 0.05) but significantly lower viable counts (p < 0.05) of adhered P. aeruginosa strains. There was a significant difference between the total and viable counts (p < 0.05) that were bound to lactoferrin-coated lenses. Albumin coating of lenses increased binding (total and viable counts) of all three strains (p < 0.05). Conclusions. Lysozyme deposited on contact lenses does not possess antibacterial activity against certain bacterial strains, whereas lactoferrin possess an antibacterial effect against strains of P. aeruginosa.

Post-translation modification of proteins in tears

This is the first 2‐DE study using sequential dyes to analyse phospho‐, glyco‐ and total tear protein profiles (Pro‐Q Diamond for phosphoprotein, Pro‐Q Emerald for glycoprotein and Sypro Ruby for total protein). This method minimised the gel–gel variations, allowing better comparisons among the three profiles and generated a whole map of PTM profiles of tear protein. A novel tear protein, dermcidin, was identified for the first time in this study. The identification of this antimicrobial protein suggests a new model of defence in tears. In addition, we are able to present the first experimental evidence of the presence of glycosylated lipocalin 1 and cystatin S. Nucleobindin 2 was only detected using phospho staining, suggesting it is only phosphorylated in tears. This study provides the groundwork for understanding the PTM of tear proteins and consequently these methods could be useful in the search for biomarkers in tears.

Contact lens deposits, adverse responses, and clinical ocular surface parameters.

Purpose. To correlate clinical responses during contact lens wear with the amount of protein or cholesterol extracted from lenses after wear. Methods. Clinical parameters, including adverse response rates and corneal staining, and symptomatology rating during lens wear were collected from a series of clinical tests comprising four different silicone hydrogel lenses with four different multipurpose solutions. To test for correlates, the amount of total protein or cholesterol extracted from lenses after daily wear were compared statistically to clinical parameters. Results. The amount of protein (p = 0.008) or cholesterol (p = 0.01) extracted from lenses was higher for those subjects who showed solution-induced corneal staining. Amount of protein extracted was correlated (p < 0.01) with conjunctival staining (R = −0.23), lens front surface wetting (r = 0.14), and lens fit tightness (R = −0.20). These clinical parameters accounted for 48% of lens protein deposition. The amount of cholesterol extracted from lenses was much more weakly associated with clinical variables. Amount of protein or cholesterol extracted from lenses was not associated with the production of any corneal infiltrative or mechanical adverse event during wear and was only very weakly correlated with insertion comfort of lenses. Conclusions. These results suggest that there may be no physiologically relevant consequence of cholesterol depositing on silicone hydrogel lenses. The amount of protein that deposits onto silicone hydrogel lenses during wear may have more affect on lens performance on-eye. However, the correlations were generally small and may still not indicate any causative relevant physiological response. Further work is required to determine whether there is any direct causative effect to support these correlative findings.

Carboxymethyl Cellulose Stimulates Rabbit Corneal Epithelial Wound Healing

Purpose: Previously, we reported carboxymethyl cellulose (CMC) binding to human corneal epithelial cells and promoting corneal epithelial wound closure in vitro. Using an animal model, the efficacy of CMC in promoting corneal wound healing was examined. Materials and Methods: Following corneal epithelial wounding of NZ white rabbits, CMC (0.2% or 1.0%) or control vehicle (PBS) was administered topically (4 times daily for 3 days) to wounded and unwounded eyes with or without contact lens wear. Wound healing in response to the treatments was measured as percentage reduction of fluorescein-stained wound area 0 to 72 hr post-wounding. Corneas were examined histologically and expression of zonula occludens-1 (ZO-1) tight-junction was detected by immunohistochemistry. Results: Percentage wound reduction in CMC-treated groups was significantly greater than controls (p < 0.05) at 24 and 32 hr. Complete wound closure was observed by 48 hr in 100% of CMC-treated eyes compared to 45% of vehicle-treated eyes. CMC also promoted wound closure dose-dependently. Epithelial cells formed an intact layer following CMC-treatment whereas vehicle-treated cells were less ordered. Strong ZO-1 expression in corneal epithelia of CMC-treated eyes was observed at 72 hr. Contact lens wear appeared to delay wound closure compared to without lens wear during CMC-treatment (p = 0.001). Conclusions: CMC promoted dose-dependent corneal epithelial wound healing. CMC stimulated ZO-1 expression, indicating accelerated corneal epithelial resistance barrier regeneration.

Glycomic Characterization of Basal Tears and changes with Diabetes and Diabetic Retinopathy

As a secreted fluid, the state of tear glycosylation is particularly important in the role of immunity of the ocular surface. Tears are a valuable source of non-invasive biomarkers for disease and there are continued efforts to characterize their components thoroughly. In this study, a small volume of basal tears (5 μL) was collected from healthy controls, patients with diabetes without retinopathy and patients with diabetes and retinopathy. The detailed N- and O-linked tear protein glycome was characterized and the relative abundance of each structure determined. Of the 50 N-linked glycans found, 89% were complex with 50% containing a bisecting N-acetylglucosamine, 65% containing a core fucose whilst 33% were sialylated. Of the 8 O-linked glycans detected, 3 were of cores 1 and 5 of core 2 type, with a majority of them being sialylated (90%). Additionally, these glycan structures were profiled across the three diabetic disease groups. Whilst the higher abundant structures did not alter across the three groups, only five low abundance N-linked glycans and 1 O-linked glycan did alter with the onset of diabetes mellitus and diabetic retinopathy (DR). These results suggest the conservation of glycan types on basal tear proteins between individuals and point to only small changes in glycan expression on the proteins in tears with the development of diabetes and DR.