A novel role for CRIM1 in the corneal response to UV and pterygium development.

Pterygium is a pathological proliferative condition of the ocular surface, characterised by formation of a highly vascularised, fibrous tissue arising from the limbus that invades the central cornea leading to visual disturbance and, if untreated, blindness. Whilst chronic ultraviolet (UV) light exposure plays a major role in its pathogenesis, higher susceptibility to pterygium is observed in some families, suggesting a genetic component. In this study, a Northern Irish family affected by pterygium but reporting little direct exposure to UV was identified carrying a missense variant in CRIM1 NM_016441.2: c.1235 A > C (H412P) through whole-exome sequencing and subsequent analysis. CRIM1 is expressed in the developing eye, adult cornea and conjunctiva, having a role in cell differentiation and migration but also in angiogenesis, all processes involved in pterygium formation. We demonstrate elevated CRIM1 expression in pterygium tissue from additional individual Northern Irish patients compared to unaffected conjunctival controls. UV irradiation of HCE-S cells resulted in an increase in ERK phosphorylation and CRIM1 expression, the latter further elevated by the addition of the MEK1/2 inhibitor, U0126. Conversely, siRNA knockdown of CRIM1 led to decreased UV-induced ERK phosphorylation and increased BCL2 expression. Transient expression of the mutant H412P CRIM1 in corneal epithelial HCE-S cells showed that, unlike wild-type CRIM1, it was unable to reduce the cell proliferation, increased ERK phosphorylation and apoptosis induced through a decrease of BCL2 expression levels. We propose here a series of intracellular events where CRIM1 regulation of the ERK pathway prevents UV-induced cell proliferation and may play an important role in the in the pathogenesis of pterygium.

Staphylococcus aureus activates the NLRP3 inflammasome in human and rat conjunctival goblet cells.

The conjunctiva is a moist mucosal membrane that is constantly exposed to an array of potential pathogens and triggers of inflammation. The NACHT, leucine rich repeat (LRR), and pyrin domain-containing protein 3 (NLRP3) is a Nod-like receptor that can sense pathogens or other triggers, and is highly expressed in wet mucosal membranes. NLRP3 is a member of the multi-protein complex termed the NLRP3 inflammasome that activates the caspase 1 pathway, inducing the secretion of biologically active IL-1ß, a major initiator and promoter of inflammation. The purpose of this study was to: (1) determine whether NLRP3 is expressed in the conjunctiva and (2) determine whether goblet cells specifically contribute to innate mediated inflammation via secretion of IL-1ß. We report that the receptors known to be involved in the priming and activation of the NLRP3 inflammasome, the purinergic receptors P2X4 and P2X7 and the bacterial Toll-like receptor 2 are present and functional in conjunctival goblet cells. Toxin-containing Staphylococcus aureus (S. aureus), which activates the NLRP3 inflammasome, increased the expression of the inflammasome proteins NLRP3, ASC and pro- and mature caspase 1 in conjunctival goblet cells. The biologically active form of IL-1ß was detected in goblet cell culture supernatants in response to S. aureus, which was reduced when the cells were treated with the caspase 1 inhibitor Z-YVAD. We conclude that the NLRP3 inflammasome components are present in conjunctival goblet cells. The NRLP3 inflammasome appears to be activated in conjunctival goblet cells by toxin-containing S. aureus via the caspase 1 pathway to secrete mature IL1-ß. Thus goblet cells contribute to the innate immune response in the conjunctiva by activation of the NLRP3 inflammasome.

Development of allele-specific therapeutic siRNA for keratin 5 mutations in epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is an incurable, inherited skin-blistering disorder predominantly caused by dominant-negative mutations in the genes encoding keratins K5 or K14. RNA interference, particularly in the form of small interfering RNA (siRNA), offers a potential therapy route for EBS and related keratin disorders by selectively silencing the mutant allele. Here, using a systemic screening system based on a luciferase reporter gene assay, we have developed mutant-specific siRNAs for two independent EBS-causing missense mutations in the K5 gene (p.Ser181Pro and p.Asn193Lys). The specificity of the allele-specific inhibitors identified in the screen was subsequently confirmed at the protein level, where the lead inhibitors were shown to strongly knock down the expression of mutant proteins with negligible effect on wild-type K5 expression. In a cell-based model system, the lead inhibitors were able to significantly reverse the cytoskeletal aggregation phenotype. Overall, this approach shows promise for the treatment of EBS and paves the way for future clinical trials.

Effect of tear hyperosmolarity and signs of clinical ocular surface pathology upon conjunctival goblet cell function in the human ocular surface.

PURPOSE: To investigate the effect of tear hyperosmolarity and signs of clinical ocular surface pathology on conjunctival goblet cell population. METHODS: 111 participants were assessed using tear osmolarity (TO) measurements and a comprehensive selection of clinical ophthalmic tests. The resultant clinical database was assessed for evidence of patterns of composite increasing pathology. The total, filled, and empty goblet cell numbers were measured: total number of goblet cells as per cytokeratin 7 (CK7) immunofluorescence and number of filled goblet cells as per periodic acid Schiff's reagent (PAS) or lectin helix pomatia agglutinin (HPA). Goblet cell profile was correlated with composite clinical pathologic grades. RESULTS: No significant correlation was found between TO and goblet cell number or function (as indicated by number of filled or unfilled goblet cells). Distinct composite clinical pathologic groups 0-IV with increasing pathology were created based on the frequency of positive pathologic signs, which adhered to the Dry Eye Workshop purported mechanism. Only in group IV was there significantly increased mean tear osmolarity of 344 mOsm/L (P < 0.000) along with significantly decreased empty goblet cell number (CK7+ and HPA-) compared to filled (CK7+ and HPA+, P = 0.000). When the number of filled goblet cells (PAS+) was analyzed there was significant increase in tear osmolarity for the two most severe grades; 3 and 4. CONCLUSIONS: The goblet cell population does not appear to be affected by isolated tear hyperosmolarity. Hyperosmolarity when combined with other ocular surface pathology or inflammation alters the goblet cell population.

Spherical aberration and higher order aberrations with Balafilcon A (PureVision) and Comfilcon A (Biofinity).

BACKGROUND: Advances in the optical design of soft contact lenses have seen certain manufacturers incorporate aspheric optics into soft lenses in an attempt to reduce spherical aberration, to provide superior visual performance. The aim of this study is to determine the on-eye differences in spherical aberration and higher order aberrations (HOA) between the Bausch and Lomb PureVision (Balafilcon A) and the CooperVision Biofinity (Comfilcon A). METHODS: Twenty subjects were recruited in a prospective, randomized, unilateral study. The right eye was dilated and HOA measured with the NIDEK OPD-Scan. Each eye was fitted randomly with a -3.00D PureVision and a -3.00D Biofinity, and HOA were measured with lenses in situ across a 6 mm pupil. Paired t-tests were performed to determine HOA differences with the lenses in situ compared to baseline. RESULTS: Aberrometry was successfully performed on all subjects. Statistical analysis indicated no changes in spherical aberration, but changes in other HOA. With the PureVision, there were increases in Zernike terms Z (3) (1) (from 0.01 µm to -0.11 µm), Z (4) (-2) (from 0.01 µm to 0.13 µm) and Z (5) (-1) (from -0.01 µm to 0.03 µm). With the Biofinity there was an increase in Zernike term Z (3) (3) (from 0.00 µm to 0.09 µm). CONCLUSIONS: No statistically significant changes occurred in spherical aberration. The PureVision caused statistically significant increases in Z (3) (1) , Z (4) (-2) and Z (5) (-1) , and the Biofinity caused an increase in Z (3) (3) . Clinically significant changes (>0.1 µm) occurred with terms Z (3) (1) and Z (4) (-2) with the PureVision only.

Femtosecond laser in situ keratomileusis versus conductive keratoplasty to obtain monovision in patients with emmetropic presbyopia.

PURPOSE: To compare visual outcomes, complications, and patient satisfaction after femtosecond laser in situ keratomileusis (LASIK) and conductive keratoplasty (CK). SETTING: Private laser clinics, Reading and Southampton, United Kingdom. METHODS: In this retrospective consecutive single-surgeon comparative study, presbyopic emmetropia was treated with femtosecond LASIK or CK to achieve monovision by targeting -1.50 diopters (D) of myopia in the nondominant eye after a successful monovision contact lens trial. The CK treatments were performed with a ViewPoint CK system using the light-touch technique. The femtosecond LASIK was performed using an IntraLase FS/FS30 and EC-5000 platform with OPDCAT wavefront treatment. RESULTS: The mean spherical equivalent 12 months postoperatively was -1.63 D +/- 0.68 (SD) in the femtosecond LASIK group and -0.97 +/- 0.82 D in the CK group (P<.001). The mean vector value of astigmatism at 12 months was 0.32 +/- 0.32 D and 1.00 +/- 0.75 D, respectively (P<.0001). The mean induced higher-order aberration (HOA) was 0.45 +/- 0.28 microm in the femtosecond LASIK group and 1.13 +/- 0.25 microm in the CK group (P<.0001). The retreatment rate was 3% after femtosecond LASIK and 50% after CK (P<.0001). On a questionnaire administered at 12 months, 20 patients (62.5%) in the femtosecond LASIK group and 11 patients (34.4%) in the CK group reported being satisfied (P = .02). CONCLUSIONS: In emmetropic presbyopic cases, femtosecond LASIK monovision provided stable correction with less induced astigmatism and HOA. Eyes with CK monovision had regression and induced astigmatism.

Phacoemulsification after deep anterior lamellar keratoplasty.

PURPOSE: To evaluate the results of phacoemulsification and intraocular lens implantation after deep anterior lamellar keratoplasty (DALK). METHODS: Retrospective, consecutive, noncomparative, single-surgeon series. RESULTS: Sixteen eyes of 16 patients were included (mean age: 51 years). Five eyes had phacoemulsification because of cataract, and 11 eyes for myopic refractive lens exchange. No intraoperative or postoperative complications were noted. Mean spherical equivalent (SE) improved from -8.69 D (SD 3.74) to -0.97 D (SD 1.13). Mean preoperative defocus equivalent (DE) improved from 10.32 D (SD 4.04) to 2.57 D (SD 0.92). Mean preoperative best spectacle-corrected visual acuity improved from 0.48 logMAR (SD 0.60) to 0.13 D (SD 0.005). Mean postoperative uncorrected visual acuity was 0.675 logMAR (SD 0.252). Safety index was 2.33, efficacy index was 0.70, and endothelial cell loss was not significant. CONCLUSIONS: Phacoemulsification can provide safe and predictable visual rehabilitation for cataract and refractive errors resulting after DALK.

Assessment of toric intraocular lens alignment by a refractive power/corneal analyzer system and slitlamp observation.

PURPOSE: To assess the validity of an internal optical path difference map of a refractive power/corneal analyzer system in determining the alignment of toric intraocular lenses (IOLs). SETTINGS: Private practices, Spring Hill, Brisbane, and Chermside, Australia. METHODS: This retrospective study comprised patients with more than 1.5 diopters of preexisting corneal astigmatism who had phacoemulsification and AcrySof toric IOL implantation. Preoperatively, the surgical eye was marked at the slitlamp microscope using a 4-point technique. The desired IOL orientation was marked with a Mendez marker based on the steep corneal axis. The toric IOL axis was measured 3 weeks postoperatively by rotating the slitlamp beam to align with the IOL axis indicator marks and using the Internal OPD Map on the Nidek OPD-Scan system. Uncorrected (UDVA) and corrected (CDVA) distance visual acuities, residual refractive sphere, and residual keratometric and refractive cylinders were also measured at 3 weeks. RESULTS: Postoperatively, the mean UDVA was 0.17 logMAR +/- 0.18 (SD) and the mean CDVA, -0.01 +/- 0.12 logMAR; 88.2% of eyes had a UDVA of 0.3 or better, and no eye lost lines of visual acuity. There was an 82.33% reduction in defocus equivalent and a 64.62% reduction in refractive cylinder. The mean IOL misalignment measured by slitlamp was 2.55 +/- 2.76 degrees and by the internal map, 2.65 +/- 1.98 degrees. The correlation between the 2 methods was highly significant (r = 0.99, P<.001). CONCLUSIONS: Both refractive power/corneal analyzer system and slitlamp observation were reliable and predictable methods of assessing IOL alignment. The 4-point preoperative marking technique yielded clinically acceptable, accurate toric IOL alignment.

Attitudes towards diagnostic tests and therapies for dry eye disease.

AIM: The purpose of this study was to survey the attitudes of optometrists and ophthalmologists, located in a number of different countries, towards diagnostic tests and therapies for dry eye disease. METHODS: A web-based questionnaire was used to survey attitudes using forced-choice questions and Likert scales. RESULTS: Sixty-one respondents (23 ophthalmologists and 38 optometrists) reported a wide range of patient dry eye symptoms. A large variation in use of diagnostic tests was noted. Patient symptoms and fluorescein staining were reported to be significantly more valuable and more frequently performed than any other test. Artificial tear supplements and improved lid hygiene were the preferred therapeutic options selected by the entire group. The results demonstrated a wide variation in attitudes in relation to satisfaction with the range of available diagnostic and therapeutic options. CONCLUSIONS: This study indicates that the interest for the issue of dry eye is relatively limited amongst eye professionals, as demonstrated by the poor participation in the questionnaire.

Difficulties imaging herpes simplex keratitis with fluorescein isothiocynate-labeled anti-HSV-1 antibodies in an ex vivo model.

AIM: The aim of this study was to attempt to visualize herpes simplex keratitis in an ex vivo model using currently available ophthalmological equipment and anti-herpes simplex virus 1 (HSV-1) fluorescein isothiocynate-labeled antibody. METHODS: Sixteen donor human corneas were included in this study. Eight corneas were infected with HSV-1, whereas 8 remained uninfected. Abrasions were made on 2 infected and 2 uninfected corneas to assess a possible nonspecific absorption of antibodies in the sites of corneal epithelial defects. Corneas were examined before and after antibody application using a slit lamp, the fluorescein enhancing filter settings of fundus camera, and Confoscan 3. All corneas were further imaged using multiphoton laser confocal microscopy. RESULTS: Before anti-HSV-1 antibody application, no fluorescence was detected in donor corneas with the blue light of the slit lamp and fundus camera at fluorescein enhancing filter settings. Examination with the fundus camera after antibody application detected increased background fluorescence in all the corneas with more highlighted areas of epithelial defects in abraded infected and uninfected corneas. Confoscan 3 did not show a significant difference between the appearances of HSV-1-infected and control corneas with and without application of the antibody. However, specific staining was confirmed by multiphoton laser confocal microscopy in all infected corneas. CONCLUSION: Further refinement of currently available ophthalmological tools is required to aid in vivo visualization of herpes simplex keratitis using fluorescein isothiocynate-labeled antibodies.

Hypothesis about mechanisms through which nicotine might exert its effect on the interdependence of inflammation and gut barrier function in ulcerative colitis.

Ulcerative colitis (UC) is characterized by impairment of the epithelial barrier and the formation of ulcer-type lesions, which result in local leaks and generalized alterations of mucosal tight junctions. Ultimately, this results in increased basal permeability. Although disruption of the epithelial barrier in the gut is a hallmark of inflammatory bowel disease and intestinal infections, it remains unclear whether barrier breakdown is an initiating event of UC or rather a consequence of an underlying inflammation, evidenced by increased production of proinflammatory cytokines. UC is less common in smokers, suggesting that the nicotine in cigarettes may ameliorate disease severity. The mechanism behind this therapeutic effect is still not fully understood, and indeed it remains unclear if nicotine is the true protective agent in cigarettes. Nicotine is metabolized in the body into a variety of metabolites and can also be degraded to form various breakdown products. It is possible these metabolites or degradation products may be the true protective or curative agents. A greater understanding of the pharmacodynamics and kinetics of nicotine in relation to the immune system and enhanced knowledge of gut permeability defects in UC are required to establish the exact protective nature of nicotine and its metabolites in UC. This review suggests possible hypotheses for the protective mechanism of nicotine in UC, highlighting the relationship between gut permeability and inflammation, and indicates where in the pathogenesis of the disease nicotine may mediate its effect.