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Role of Contact Lens Wear, Bacterial Flora, and a Mannose-Induced Protease in the Pathogenesis of Acanthamoeba Keratitis
TEAR FILM & OCULAR SURFACE CONTACT LENS AND TEAR FILM THICKNESS MEASURES ASSOCIATED WITH DIFFERENT MULTIPURPOSE CARE SOLUTIONS. Jason J. Nichols, P. Ewen King-Smith. The Ohio State University College of Optometry, Columbus, OH USA. Purpose. To determine differences in tear film thickness parameters during contact lens wear that might be associated with compositionally different multipurpose contact lens care solutions. Methods. A randomized, crossover, investigator-masked clinical trial was conducted to compare AMO’s Complete MoisturePlus (CMP) to Alcon’s Opti-Free Express (OFX). The primary outcome was a comparison of pre-lens tear film thickness between solutions during the first two hours after lens insertion. Secondary outcomes of the study included a comparison of post-lens tear film thicknesses, pre-lens tear film thinning rates, symptom scores, and patient-reported overall preferences. Tear film thickness measures were made using a previously described ‘wavelength-dependent’ interferometric system. Results. The average overall PLTF thickness over the 120-minute time period after using CMP was 3.02 ± 1.07μm, while it was 2.72 ± 0.86μm after using OFX (p = 0.003). For both solutions, the PLTF was fitted best by the sum of an exponential decay plus a constant. The PLTF decay amplitudes did not statistically differ when comparing solutions (p = 0.11). There was no difference in overall PoLTF thicknesses when comparing the average values after using each solution (p = 0.57), although there was a difference immediately after the lens was applied to the eye (CMP average = 4.10 vs. OFX average = 3.48, p = 0.02). The PoLTF also demonstrated an exponential decay for both solutions. The PoLTF decay amplitudes did not statistically differ (p = 0.14). Twenty subjects (64.5%) preferred CMP to OFX (35.5%) (p = 0.11), and nearly every subject (90.3%) suggested ‘comfort’ as their reason for preference. Conclusions. CMP is associated with a thicker PLTF when compared to OFX in contact lens wearers without dry eye symptoms. Future studies will address the impact of these and other contact lens solutions on the thickness of the tear film in contact lens-related dry eye individuals. Support: Advanced Medical Optics, Inc DIAGNOSIS AND OUTCOME MEASURES FOR OCULAR SURFACE DISEASE: REFLECTIONS ON THE PAST AND THE FUTURE. Kelly K. Nichols. The Ohio State University College of Optometry, Columbus, OH, USA. Purpose. Numerous advances have been made in the field of ocular surface disease over the past 15 years. While our understanding of the underlying mechanisms of dry eye disease has significantly increased, particularly in the areas of immunology, endocrinology, and surface chemistry, the translation of this knowledge to clinical research and patient care has lagged behind. However, several large multi-centered clinical trials have been completed or are underway, which has led to progress in the selection of appropriate outcome measures for dry eye studies. While global acceptance of a “dry eye definition” has not been achieved, some consensus of symptom assessment and clinical testing has been reached. On a smaller scale, numerous investigators have taken existing diagnostic techniques or new technology and have adapted these techniques for improved diagnostic ability. Functional aspects of visual acuity, tear film stability, and patient symptomatology have been evaluated. Improved grading scales and digital photo documentation and grading of corneal and conjunctival staining and infrared meibography have been considered as techniques for improving consistency between examiners. Current clinical techniques for diagnosis reported in recent large-scale studies will be discussed. While diagnostic tests based on the evaluation of immune markers in the tears and tear film osmolarity have not been marketed, new tests and instruments for research and clinical care can be expected in the upcoming years.
ANALYSIS OF MEIBOMIAN LIPID USING ELECTROSPRAY IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY. Kelly K. Nichols,1 Corrie Ziegler,1 Kari B. Green-Church,2 Jason J. Nichols.1 The Ohio State University College of Optometry, Columbus, OH, USA;1 The Ohio State University Mass Spectrometry and Proteomics Facility, Columbus, OH, USA.2 Purpose. To use electrospray ionization time-of-flight mass spectrometry (ESI-TOF) to evaluate meibomian gland secretions. In addition, the stability of meibomian gland secretions stored over time by freezing was evaluated by ESI to determine if this is associated with lipid degradation. Methods. Meibomian gland secretions were collected using a sterile 1.5mm curette probe from the lower eyelids of a male and female subject (mean age = 48.5) over a period of five consecutive days. Each sample was stored in a 2:1 chloroform/methanol solution. Secretions collected from the left eye were immediately processed with ESI, while secretions from the right eye were frozen at -70ºC. Ten days after the initial sample was taken (five days after the last sample), the frozen meibomian gland secretions from the right eye were processed by ESI. All samples were directly infused at 20uL/min to a Micromass LCT equipped with an orthogonal electrospray source (Zspray) operated in positive ion mode. ESI conditions that produced the best results included capillary voltage 2500V, source temperature 100ºC, and a cone voltage of 35V. Data was attained in continuum mode until suitable averaged data was collected (approximately 1 minute). Results. Inter- and intra-subject agreement for samples analyzed immediately was good. Peaks were consistently observed within- and between-subjects at the following masses (m/z): 261.1, 489.2, 585.5, 727.5, 955.7. Also, observed masses were replicated upon the analysis of frozen samples both within- and between-subjects. Conclusions. Observed peaks were within the expected ranges of both lipid and phospholipid masses. No significant changes occurred when frozen samples were analyzed, suggesting that freezing periods of one week or less is not associated with lipid degradation. Further study is necessary to determine if longer freeze periods is associated with any degradation. Thin layer chromatography will be used to aid in the identification of the mass peaks and will be presented. ROLE OF CONTACT LENS WEAR, BACTERIAL FLORA, AND A MANNOSE-INDUCED PROTEASE IN THE PATHOGENESIS OF ACANTHAMOEBA KERATITIS. Jerry Y. Niederkorn, Hassan Alizadeh, Sudha Neelam, Michael Hurt. University of Texas Southwestern Medical Center, Dallas, Texas, USA. Purpose. This study examined the role of a 133 kDa mannose-induced protease (MIP133) in the pathogenesis of Acanthamoeba keratitis and the efficacy of mucosal immunization with MIP133 in mitigating Acanthamoeba keratitis. Methods. Acanthamoeba trophozoites were exposed to either 100 mM mannose or mannose-coated latex beads and the secretion of MIP133 was assessed. The capacity of bacterial mannose and mannosylated proteins on contact lenses and on the corneal epithelium to induce Acanthamoeba trophozoites to secrete MIP133 was also examined. In vivo studies examined the effect of a mannose-containing bacterium, Corynebacterium xerosis, which has been associated with Acanthamoeba keratitis, on the pathogenicity of Acanthamoeba trophozoites. Organcultured pig eyes were used to ascertain the effect of contact lens wear on binding of Acanthamoeba trophozoites to the corneal surface and the elaboration of MIP133. Chinese hamsters were mucosally immunized with MIP133 as a means of mitigating the pathogenesis of Acanthamoeba keratitis. Results. Bacteria with high mannose contents (C. xerosis) induced MIP133 secretion and increased the pathogenic behavior of Acanthamoeba trophozoites. Mannosylated proteins, either on contact lenses or on corneas preconditioned by contact lens wear, induced steep increases in MIP133 secretion by trophozoites and exacerbated Acanthamoeba keratitis. Mucosal immunization with MIP133 induced the production of anti-MIP133 IgA antibodies in the tears and mitigated Acanthamoeba keratitis. Conclusions. The results show the importance of a mannose-induced protease (MIP133) in the pathogenesis of Acanthamoeba keratitis and demonstrate the feasibility of an “anti-disease” vaccine for mitigating the pathogenic processes invoked by microorganisms that are either poorly immunogenic or that have highly evolved immune escape mechanisms. Commercial Relationship(s): None; Support: NIH grant EY 09756 and an unrestricted grant from Research to Prevent Blindness, Inc
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ANALYSIS OF MEIBOMIAN LIPID USING ELECTROSPRAY IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY. Kelly K. Nichols,1 Corrie Ziegler,1 Kari B. Green-Church,2 Jason J. Nichols.1 The Ohio State University College of Optometry, Columbus, OH, USA;1 The Ohio State University Mass Spectrometry and Proteomics Facility, Columbus, OH, USA.2 Purpose. To use electrospray ionization time-of-flight mass spectrometry (ESI-TOF) to evaluate meibomian gland secretions. In addition, the stability of meibomian gland secretions stored over time by freezing was evaluated by ESI to determine if this is associated with lipid degradation. Methods. Meibomian gland secretions were collected using a sterile 1.5mm curette probe from the lower eyelids of a male and female subject (mean age = 48.5) over a period of five consecutive days. Each sample was stored in a 2:1 chloroform/methanol solution. Secretions collected from the left eye were immediately processed with ESI, while secretions from the right eye were frozen at -70ºC. Ten days after the initial sample was taken (five days after the last sample), the frozen meibomian gland secretions from the right eye were processed by ESI. All samples were directly infused at 20uL/min to a Micromass LCT equipped with an orthogonal electrospray source (Zspray) operated in positive ion mode. ESI conditions that produced the best results included capillary voltage 2500V, source temperature 100ºC, and a cone voltage of 35V. Data was attained in continuum mode until suitable averaged data was collected (approximately 1 minute). Results. Inter- and intra-subject agreement for samples analyzed immediately was good. Peaks were consistently observed within- and between-subjects at the following masses (m/z): 261.1, 489.2, 585.5, 727.5, 955.7. Also, observed masses were replicated upon the analysis of frozen samples both within- and between-subjects. Conclusions. Observed peaks were within the expected ranges of both lipid and phospholipid masses. No significant changes occurred when frozen samples were analyzed, suggesting that freezing periods of one week or less is not associated with lipid degradation. Further study is necessary to determine if longer freeze periods is associated with any degradation. Thin layer chromatography will be used to aid in the identification of the mass peaks and will be presented. ROLE OF CONTACT LENS WEAR, BACTERIAL FLORA, AND A MANNOSE-INDUCED PROTEASE IN THE PATHOGENESIS OF ACANTHAMOEBA KERATITIS. Jerry Y. Niederkorn, Hassan Alizadeh, Sudha Neelam, Michael Hurt. University of Texas Southwestern Medical Center, Dallas, Texas, USA. Purpose. This study examined the role of a 133 kDa mannose-induced protease (MIP133) in the pathogenesis of Acanthamoeba keratitis and the efficacy of mucosal immunization with MIP133 in mitigating Acanthamoeba keratitis. Methods. Acanthamoeba trophozoites were exposed to either 100 mM mannose or mannose-coated latex beads and the secretion of MIP133 was assessed. The capacity of bacterial mannose and mannosylated proteins on contact lenses and on the corneal epithelium to induce Acanthamoeba trophozoites to secrete MIP133 was also examined. In vivo studies examined the effect of a mannose-containing bacterium, Corynebacterium xerosis, which has been associated with Acanthamoeba keratitis, on the pathogenicity of Acanthamoeba trophozoites. Organcultured pig eyes were used to ascertain the effect of contact lens wear on binding of Acanthamoeba trophozoites to the corneal surface and the elaboration of MIP133. Chinese hamsters were mucosally immunized with MIP133 as a means of mitigating the pathogenesis of Acanthamoeba keratitis. Results. Bacteria with high mannose contents (C. xerosis) induced MIP133 secretion and increased the pathogenic behavior of Acanthamoeba trophozoites. Mannosylated proteins, either on contact lenses or on corneas preconditioned by contact lens wear, induced steep increases in MIP133 secretion by trophozoites and exacerbated Acanthamoeba keratitis. Mucosal immunization with MIP133 induced the production of anti-MIP133 IgA antibodies in the tears and mitigated Acanthamoeba keratitis. Conclusions. The results show the importance of a mannose-induced protease (MIP133) in the pathogenesis of Acanthamoeba keratitis and demonstrate the feasibility of an “anti-disease” vaccine for mitigating the pathogenic processes invoked by microorganisms that are either poorly immunogenic or that have highly evolved immune escape mechanisms. Commercial Relationship(s): None; Support: NIH grant EY 09756 and an unrestricted grant from Research to Prevent Blindness, Inc
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