Search

The article discusses a study that examines atopic dermatitis (AD) as risk factor for intrastromal corneal ring segment (ICRS) extrusion. The study included patients treated with ICRS at the Helsinki University Eye Hospital that developed corneal melts necessitating ICRS removal. It suggests that patients with a history of AD/AKC treated with ICRS implantation should be performed by femtosecond laser-assisted system.

Purpose: To describe epidemiology, causes, treatments and outcomes of all ocular injuries in southern Finland among people aged 61 and older. Methods: All new ocular trauma patients, admitted to the Helsinki University Eye Hospital, during 1 year in 2011–2012. The data were from hospital records and prospectively from patient questionnaires. The follow‐up time was 3 months. Results: The incidence for ocular injuries among the elderly was 38/100 000/year. From 118 patients 69% were men. The mean age was 70.9 years old (median 67). The hospitalization rate was 14%. Injury types were minor traumas (48%), contusions (22%), chemical injuries (10%), eyelid wounds (8%), open globe injuries (OGI; 7%) and orbital fractures (5%). The injuries occurred at home (58%), institutions (12%) and in other public places (12%). The main causes of ocular injury were falls (22%), sticks (19%), superficial foreign bodies (18%) and chemicals (12%). All OGI and 88% of contusions needed a lifelong follow‐up. A permanent visual or functional impairment occurred in 15 (13%) patients. Of these 53% were OGI, 40% contusions and 7% chemical injuries. The causes of permanent injuries were falls (seven cases, 47%), work tools, sports equipment, sticks, chemicals and eyeglasses. The incidence for legal blindness was 2.3/100 000. Conclusion: Minor trauma was the most frequent type, and home was the location of the most occurred eye injuries. Falls were the most frequent and serious cause, but behavioural causes were not significant. Preventive measures should be directed towards the main identified causes and risk factors of the eye injuries in the elderly. [ABSTRACT FROM AUTHOR]

The article discusses a study on the combined use of corneal cross-linking (CXL) and human amniotic membrane transplantation for the treatment of pseudomonas aeruginosa (PA) keratitis. Its aim is to achieve faster recovery and add the beneficial antimicrobial and fibrosis inhibiting properties of amniotic membrane to the antimicrobial and cornea strengthening effects of CXL. It concludes that the combined treatment can be used in the primary treatment of serious vision-threatening PA keratitis.

Highlights: [•] We simulate interactions of pyrene-linked lipids in a lipid membrane. [•] We study the effects of where pyrenes are attached to the chains of their host lipids. [•] Pyrene-induced perturbations are largely similar in all cases in a DOPC environment. [•] Transition to membrane-spanning pyrene dimers is very abrupt. [ABSTRACT FROM AUTHOR]

. Purpose: Angiogenesis in diabetic retinopathy (DR) is a multifactorial process regulated by hypoxia-induced growth factors and inflammatory cytokines. In addition to the angiogenic switch, the proteolytic processing and altered synthesis of the extracellular matrix are critical steps in this disease. This study was performed to evaluate the levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2 and MMP-9), angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2), vascular endothelial growth factor (VEGF), erythropoietin (EPO) and transforming growth factor-β1 (totalTGFβ1) in the vitreous of diabetic eyes undergoing vitrectomy compared with control eyes operated because of macular hole or pucker. Methods: Prospective consecutive controlled observational study performed in the unit of vitreoretinal surgery in Finland during the years 2006-2008. Vitreous samples were collected before the start of the conventional 3-ppp vitrectomy. Vitreous MMP-2 and MMP-9, Ang-1 and Ang-2, VEGF, EPO and TGFβ1 concentrations were measured from 69 patients with Type 1 or 2 diabetes and 40 controls. Results: Comparison of eyes with DR with controls revealed that the mean vitreous concentrations of proMMP-2 (p = 0.0015), totalMMP-2 (p = 0.0011), proMMP-9 (p = 0.00001), totalMMP-9 (p < 0.00001), Ang-2 (p < 0.00001), VEGF (p < 0.00001), EPO (p < 0.00001) and totalTGFβ1 (p = 0.000026) were significantly higher in the former group. A multivariate logistic regression analysis suggested intravitreal Ang-2 concentration being the key marker of PDR (p = 0.00025) (OR = 1507.9). Conclusion: The main new finding is that the intravitreal concentrations of Ang-2 correlated significantly with MMP-9, VEGF, EPO and TGFβ1 levels in diabetic eyes undergoing vitrectomy. Thus, these factors could promote retinal angiogenesis synergistically. [ABSTRACT FROM AUTHOR]

. Purpose: To retrospectively evaluate the visual and refractive outcome of visually impaired adults treated with refractive surgery (photorefractive keratectomy or laser-assisted in situ keratomileusis). Methods: We searched a refractive surgery database comprising 1716 mildly visually impaired patients [best spectacle-corrected visual acuity (BSCVA) on a logMAR scale ≤ −0.1 (Snellen ≤ 0.8)] who had undergone either PRK or LASIK ( n = 96). PRK patients who had visits at 5-7, 8-13 and 14-24 months postoperatively were selected. Eleven patients and nine PRK control myopic patients were found (cohort 1). From the same database, 41 visually impaired patients and 54 controls who had a postoperative control at 14-24 months postoperatively were chosen. These patients formed cohort 2. Results: Preoperatively, in cohort 1, the mean BSCVA on a logMAR scale was −0.15 ± 0.13 (Snellen 0.73 ± 0.16) in visually impaired patients and 0.04 ± 0.02 (Snellen 1.11 ± 0.17) in myopic controls. At 14-24 months postoperatively, the mean BSCVA improved to 0.05 ± 0.04 (Snellen 1.13 ± 0.10) in visually impaired patients and 0.05 ± 0.08 (Snellen 1.13 ± 0.21) in control patients. In cohort 2, preoperatively the mean BSCVA on a logMAR scale was −0.15 ± 0.12 (Snellen 0.74 ± 0.14) in visually impaired patients and 0.01 ± 0.03 (Snellen 1.04 ± 0.10) in myopic controls. At 14-24 months postoperatively, the mean BSCVA improved to 0.02 ± 0.07 (Snellen 1.06 ± 0.16) in visually impaired patients and 0.06 ± 0.06 (Snellen 1.15 ± 0.16) in control patients. Conclusion: Refractive surgery improves BSCVA in visually impaired patients, possibly through plastic changes in the visual cortex. Consequently, refractive surgery may be used successfully for the treatment of visually impaired adults to enhance their visual acuity. [ABSTRACT FROM AUTHOR]

Purpose: To compare the long-term results of photorefractive keratectomy (PRK) for low to moderate myopia performed using a broad-beam laser system or a scanning-slit laser system. Setting: Department of Ophthalmology, University of Helsinki, Helsinki, Finland. Design: Case-control study. Methods: This follow-up study comprised eyes with myopia (−1.25 to −7.00 diopters [D]) or myopic astigmatism (astigmatism lower than −2.50 D) corrected by PRK using a broad-beam (Visx) or scanning-slit (Nidek) laser. Follow-up included a visit at 3 months and at more than 8 years. The uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), and manifest refraction were recorded. Results: The broad-beam group comprised 27 eyes and the scanning-slit group, 34 eyes. At the last postoperative follow-up, the UDVA was 0.0 or better in 55% of eyes in the broad-beam group and 65% of eyes in the scanning-slit group. The CDVA was 0.0 or better in all eyes in the broad-beam group and 96% of eyes in the scanning-slit group. Regarding predictability, 48% and 73% of the eyes, respectively, were within ±0.50 D of the intended spherical equivalent refraction. There were no statistically significant differences between the 2 laser groups in any preoperative or postoperative parameter. Conclusion: There were no significant differences in UDVA, CDVA, or SE after PRK for low to moderate myopia between the broad-beam laser system and the scanning-slit laser system. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned. [Copyright &y& Elsevier]

Abstract: Fatal drug overdoses are a cause for concern all over the world. We present here a lipid-based formulation which has a strong affinity for some common illicit street drugs and can be used in vivo as a lipid ‘sink’. In this study, the in vitro interactions of nine lipophilic drugs and three lipid dispersions were determined by electrokinetic capillary chromatography and fluorescence polarization. Two lipid dispersions, zwitterionic 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and an anionic mixture of POPC and 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) were tested and compared with a commercial lipid dispersion Intralipid®, which has been successfully used for resuscitation of patients in cases of anesthetic overdoses. The interactions between dispersions and the drugs were quantified by means of retention factors and distribution constants, which makes the results highly comparable to those obtained from any other formulation of lipids. The results demonstrate a stronger interaction between the drugs and an artificial liposome dispersion than with the commercial Intralipid dispersion. The liposome dispersion composed of POPC and POPG functions as a lipid ‘sink’ for efficient entrapment of various lipophilic drugs. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to describe the morphology, corneal topography and sensitivity in individuals with Dystrophia Helsinglandica. This autosomal dominant corneal disease is characterized by recurrent corneal erosive episodes and progressive subepithelial fibrosis not significantly affecting visual acuity. Methods: The corneas of nine affected and nine unaffected individuals were examined using slit-lamp biomicroscopy, in vivo confocal microscopy (IVCM) and videokeratography. Corneal mechanical sensitivity was also measured using a non-contact esthesiometer. Results: Slit-lamp biomicroscopy revealed that the affected individuals represented different stages of corneal changes, from a nearly normal cornea to subepithelial fibrosis of the central cornea. Corneal changes in affected individuals did not significantly decrease the best spectacle-corrected visual acuity. In vivo confocal microscopy detected morphological changes in the epithelium and stroma. Subepithelial opacity formation including altered keratocytes could be found in the anterior stroma in all affected eyes. With the exception of two eyes (one affected and one unaffected), all videokeratographies showed irregular astigmatism. Corneal sensitivity was significantly lower in affected individuals (p = 0.01). Age and corneal sensitivity showed no correlation. Conclusion: The main morphological findings in affected individuals were discrete and progressive subepithelial fibrosis, in the in vivo confocal microscope corresponding to optically dense extracellular matrix and activated keratocytes. Subbasal nerve morphology was changed in the affected family members who also showed a decreased corneal sensitivity. The findings are per se not specific to the disease. The changes probably reflect a healing response to erosive events on the corneal surface influenced by the genotype. [ABSTRACT FROM AUTHOR]

Components of the ocular surface synergistically contribute to maintaining and protecting a smooth refractive layer to facilitate the optimal transmission of light. At the air-water interface, the tear film lipid layer (TFLL), a mixture of lipids and proteins, plays a key role in tear surface tension and is important for the physiological hydration of the ocular surface and for ocular homeostasis. Alterations in tear fluid rheology, differences in lipid composition, or downregulation of specific tear proteins are found in most types of ocular surface disease, including dry eye disease (DED). Artificial tears have long been a first line of treatment in DED and aim to replace or supplement tears. More recently, lipid-containing eye drops have been developed to more closely mimic the combination of aqueous and lipid layers of the TFLL. Over the last 2 decades, our understanding of the nature and importance of lipids in the tear film in health and disease has increased substantially. The aim of this article is to provide a brief overview of our current understanding of tear film properties and review the effectiveness of lipid-based products in the treatment of DED. Liposome lid sprays, emulsion eye drops, and other lipid-containing formulations are discussed. [ABSTRACT FROM AUTHOR]

Liposomes were used as biomimetic models in capillary electrokinetic chromatography (EKC) for the determination of distribution constants (K D ) of certain local anesthetics and a commonly used preservative. Synthetic liposomes comprised phosphatidylcholine and phosphatidylglycerol phospholipids with and without cholesterol. In addition, ghost liposomes made from red blood cell (RBC) lipid extracts were used as pseudostationary phase to acquire information on how the liposome composition affects the interactions between anesthetics and liposomes. These results were compared with theoretical distribution coefficients at pH 7.4. In addition to 25 °C, the distribution constants were determined at 37 and 42 °C to simulate physiological conditions. Moreover, the usability of five electroosmotic flow markers in liposome (LEKC) and micellar EKC (MEKC) was studied. LEKC was proven to be a convenient and fast technique for obtaining data about the distribution constants of local anesthetics between liposome and aqueous phase. RBC liposomes can be utilized for more representative model of cellular membranes, and the results indicate that the distribution constants of the anesthetics are greatly dependent on the used liposome composition and the amount of cholesterol, while the effect of temperature on the distribution constants is less significant. [ABSTRACT FROM AUTHOR]

Purpose: To refine the diagnostic criteria for peripheral hypertrophic subepithelial corneal degeneration (PHSD) and characterize its clinical phenotype, histopathology and immunohistochemical features. Methods: Diagnostic criteria were refined on the basis of literature data. Fourteen patients (13 women and one man; median age 52 years, range 33-66) were identified based on these criteria. Keratectomy specimens were evaluated via routine and immunohistochemical stainings. The main outcome measures were symptoms, clinical phenotype, immunological status and histopathologic results. Results: We defined the diagnostic criteria of typical PHSD as elevated circumferential and perilimbal subepithelial fibrosis with focal superficial corneal neovascularization, which were supported by female sex (93%), bilaterality (86%), the centre being in the upper quadrants (81%) and irregular astigmatism of two dioptres or more. The typical symptoms were reduced vision (86%) and the symptoms of ocular surface disease (64%). Light microscopy showed fibrosis with abundant collagen deposition but no inflammation in all patients. An immunohistochemical analysis of nine patients showed uniform staining for vimentin in three distinct types of fibroblasts in variable proportions: keratocytelike cells that were positive for CD34, myofibroblasts that were positive for smooth muscle actin (SMA) and fibroblasts that were negative for CD34 and SMA. Small numbers of CD68-positive macrophages were also found. Conclusions: Peripheral hypertrophic subepithelial degeneration is characteristic of middle-aged women, in whomit is typically a bilateral idiopathic degeneration of the cornea associated with ocular surface disease and reduced vision. The fibrotic lesions probably undergo remodelling, inducing changes in corneal contour. A smouldering low-grade inflammation favouring low TGF-β1 concentrations is postulated as the primary pathological process leading to PHSD. [ABSTRACT FROM AUTHOR]

. Purpose: To prospectively study the effect of refractive surgery in the primary visual cortex of adult anisometropic and isometropic myopic patients. Methods: Two anisometropic and two isometropic myopic patients were examined with multifocal functional magnetic resonance imaging technique (mffMRI) before refractive surgery and at 3, 6, 9 and 12 months postoperatively. Two controls without refractive surgery were also examined with mffMRI in the beginning and in the end of the study. Anisometropic patients had only their more myopic eye operated to correct the anisometropia. The myopic isometropic patients had their both eyes operated. Results: Operated anisometropic eyes showed 65% reduced amount of active voxels in foveal data at 12 months postoperatively compared with the preoperative situation. In unoperated anisometropic eyes, the corresponding value was 86% and in myopic patients and controls 31% and 1%, respectively. To confirm this finding, the number of activated voxels representing the innermost ring of the stimulus was also calculated, and an exactly similar phenomenon was encountered in the anisometropic patients. Both anisometropic patients improved the best-spectacle-corrected visual acuity in the operated eye after refractive surgery. Conclusion: Our results suggest that plastic changes may take place in the primary visual cortex of anisometropic adult patients after refractive surgery. [ABSTRACT FROM AUTHOR]

Abstract: Intravenous lipid emulsion is recommended as treatment for local anesthetic intoxication based on the hypothesis that the lipophilic drug is entrapped by the lipid phase created in plasma. We compared a 15.6mM 80/20mol% phosphatidyl choline (PC)/phosphatidyl glycerol (PG)-based liposome dispersion with the commercially available Intralipid® emulsion in a pig model of local anesthetic intoxication. Bupivacaine–lipid interactions were studied by electrokinetic capillary chromatography. Multilamellar vesicles were used in the first in vivo experiment series. This series was interrupted when the liposome dispersion was discovered to cause cardiovascular collapse. The toxicity was decreased by an optimized sonication of the 50% diluted liposome dispersion (7.8mM). Twenty anesthetized pigs were then infused with either sonicated PC/PG liposome dispersion or Intralipid®, following infusion of a toxic dose of bupivacaine which decreased the mean arterial pressure by 50% from baseline. Bupivacaine concentrations were quantified in blood samples using liquid chromatography/mass spectrometry. No significant difference in the context-sensitive plasma half-life of bupivacaine was detected (p =0.932). After 30min of lipid infusion, the bupivacaine concentration was 8.2±1.5mg/L in the PC/PG group and 7.8±1.8mg/L in the Intralipid® group, with no difference between groups (p =0.591). No difference in hemodynamic recovery was detected between groups (p >0.05). [Copyright &y& Elsevier]

Abstract: The purpose of this review is to provide insight into the biophysical properties and functions of tear fluid and lung surfactant – two similar fluids covering the epithelium of two distinctive organs. Both fluids form a layer-like structure that essentially comprise of an aqueous layer next to the epithelium and an anterior lipid layer at the air-water interface. The aqueous layers contain soluble proteins and metabolites, and they are responsible for the host defence system and nutrition of the organ. However, many proteins also interact with the lipid layer and are important for the surface-active function of the fluid film. The lipid layer of lung surfactant comprises mainly of phospholipids, especially phosphatidylcholines, and only small amounts of non-polar lipids, mainly cholesterol. In contrast, tear fluid lipid layer comprises of a mixture of polar and non-polar lipids. However, the relative proportion and the spectrum of different polar and non-polar lipids seem to be more extensive in tear fluid than in lung surfactant. The differing lipid compositions generate distinctive lipid layer structures. Despite the structural differences, these lipid layers decrease the surface tension of the air-water interface. The structure of the tear film lipid layer also minimises the evaporation of the tear fluid. In lung surfactant surface activity is crucial for the function of the organ, as the lipid layer prevents the collapse of the lung alveoli during the compression-expansion cycle of breathing. Similarly the tear film experiences a compression-expansion cycle during blinking. The dynamics of this cycle have been studied to a lesser extent and are not as clear as those of lung surfactant. The common structure and properties suggest a similar behaviour under rapid compression–expansion for both fluids. [Copyright &y& Elsevier]

Interactions between Intralipid dispersion and local anesthetics (bupivacaine, prilocaine, and lidocaine) were investigated. The amount of bupivacaine (the most cardiotoxic analyte of the local anesthetics studied) entrapped in Intralipid in the presence of plasma was studied using an off-line filtration and solid phase extraction method combined with capillary zone electrophoresis for quantification of free unbound bupivacaine. To confirm interactions between the analytes and Intralipid at lower concentrations, direct injection mass spectrometry was used. The use of immobilized Intralipid chromatography-atmospheric pressure ionization–ion trap mass spectrometry in the study of interactions between drugs and Intralipid dispersion is demonstrated. Finally, interactions between Intralipid dispersion and local anesthetics were investigated by electrokinetic capillary chromatography. The electrophoretic mobility of the Intralipid dispersed phase was calculated using the iterative procedure and a homologous series of alkyl phenyl benzoates (C1–C6), and the retention factors for the analytes were determined. [ABSTRACT FROM AUTHOR]

Abstract: Fusidic acid (FA) is a potent antibiotic and blocks the protein synthesis by binding to elongation factor G (EF-G) directly. Here we hypothesized that the antibiotic activity of FA would be potentiated by several orders of magnitude if both FA and EF-G would be residing in the lipid membranes and, hence, the probability of interaction would transform from three-dimensional to two-dimensional. Such detailed information could lead to more effective therapeutic interventions if they are understood on a molecular level. Interactions between FA and various lipid membranes composed of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (Chol) were studied by capillary electrochromatography (CEC). The influence of the lipid vesicle size—sonicated liposomes and liposomes extruded through 30-, 50-, and 100-nm filters—on the packing of vesicles on the silica capillary surface was investigated by CEC and dissipative quartz crystal microbalance. The CEC results evidenced that FA interacts with and resides in phospholipid membranes. Likewise, monolayer, asymmetrical flow field flow fractionation, and CEC studies confirmed that EF-G is hydrophobic and incorporated into POPC and POPC/Chol membranes. Including EF-G in phospholipid vesicles did not improve the binding of FA to the membranes. [Copyright &y& Elsevier]

The human tear fluid film consists of a superficial lipid layer, an aqueous middle layer, and a hydrated mucin layer located next to the corneal epithelium. The superficial lipid layer protects the eye from drying and is composed of polar and neutral lipids provided by the meibomian glands. Excess accumulation of lipids in the tear film may lead to drying of the corneal epithelium. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. To gain insight into the formation of tear film, we investigated whether PLTP and CETP are present in human tear fluid. Tear fluid samples were collected with microcapillaries. The presence of PLTP and CETP was studied in tear fluid by Western blotting, and the PLTP concentration was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. Size-exclusion and heparin-affinity chromatography assessed the molecular form of PLTP. PLTP is present in tear fluid, whereas CETP is not. Quantitative assessment of PLTP by ELISA indicated that the PLTP concentration in tear fluid, 10.9 ± 2.4 μg/mL, is about 2-fold higher than that in human plasma. PLTP-facilitated phospholipid transfer activity in tears, 15.1 ± 1.8 μmol mL-1 h-1 was also significantly higher than that measured in plasma. Inactivation of PLTP by heat treatment (+58 °C, 60 min) or immunoinhibition abolished the phospholipid transfer activity in tear fluid. Size-exclusion chromatography of tear fluid indicated that PLTP eluted in a position corresponding to a size of 160-170 kDa. Tear fluid PLTP was quantitatively bound to Heparin-Sepharose and could be eluted as a single peak by 0.5 M NaCl. These data indicate that human tear fluid contains catalytically active PLTP protein, which resembles the active form of PLTP present in plasma. The results suggest that PLTP may play a role in the formation of the tear film by supporting phospholipid transfer. [ABSTRACT FROM AUTHOR]

Dry eye syndrome (DES), one of the most common ophthalmological diseases, is typically caused by excessive evaporation of tear fluid from the ocular surface. Excessive evaporation is linked to impaired function of the tear film lipid layer (TFLL) that covers the aqueous tear film. The principles of the evaporation resistance of the TFLL have remained unknown, however. We combined atomistic simulations with Brewster angle microscopy and surface potential experiments to explore the organization and evaporation resistance of films composed of wax esters, one of the main components of the TFLL. The results provide evidence that the evaporation resistance of the TFLL is based on crystalline-state layers of wax esters and that the evaporation rate is determined by defects in the TFLL and its coverage on the ocular surface. On the basis of the results, uncovering the nonequilibrium spreading and crystallization of TFLL films has potential to reveal new means of treating DES.

Purpose: Most pure glaucoma drugs (pGDs) are hydrophobic substances intended to reduce elevated intraocular pressure. The aims of our study were to determine the toxicity of pGDs (brimonidine tartrate, brinzolamide, latanoprost, timolol maleate, and pilocarpine hydrochloride) on ocular surface cells and to establish whether their toxicity is subsequent to cellular membrane destabilization., Methods: The toxicity of clinically efficient doses of pGDs was measured at different time points in a cell culture of human corneal epithelial cells using a redox indicator. pGD interaction with the plasma membrane was analyzed using a hemolysis assay and liposome electrokinetic chromatography. The capacity of pGDs to induce endoplasmic reticulum stress was investigated by immunoblotting., Results: The toxicity assay showed that all pGDs decrease the viability of the epithelial cells to variable degrees. Early toxicity was measured for 4% pilocarpine and 0.15% brimonidine with 60% cell death at 4 hours, whereas 2% pilocarpine and 0.005% latanoprost showed almost 100% toxicity but only after 16 hours. The hemolysis assay and liposome electrokinetic chromatography experiments suggested that interaction between pGDs and lipid membranes is weak and cannot explain cell death through lysis. Immunoblotting revealed that the drugs activate endoplasmic reticulum stress and, with the exception of pilocarpine, have the capacity to induce apoptosis through upregulation of C/EBP homologous protein., Conclusions: Our study indicates that all studied pGDs decrease the viability of the corneal epithelial cells, but none of the tested compounds were able to destabilize cellular membranes. The pGDs seem to be internalized and can induce apoptosis through C/EBP homologous protein recruitment.

The members of the Tear Film Subcommittee reviewed the role of the tear film in dry eye disease (DED). The Subcommittee reviewed biophysical and biochemical aspects of tears and how these change in DED. Clinically, DED is characterized by loss of tear volume, more rapid breakup of the tear film and increased evaporation of tears from the ocular surface. The tear film is composed of many substances including lipids, proteins, mucins and electrolytes. All of these contribute to the integrity of the tear film but exactly how they interact is still an area of active research. Tear film osmolarity increases in DED. Changes to other components such as proteins and mucins can be used as biomarkers for DED. The Subcommittee recommended areas for future research to advance our understanding of the tear film and how this changes with DED. The final report was written after review by all Subcommittee members and the entire TFOS DEWS II membership., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Climatic droplet keratopathy (CDK) is an acquired and potentially handicapping cornea degenerative disease that is highly prevalent in certain rural communities around the world. It predominantly affects males over their forties. It has many other names such as Bietti's band-shaped nodular dystrophy, Labrador keratopathy, spheroidal degeneration, chronic actinic keratopathy, oil droplet degeneration, elastoid degeneration and keratinoid corneal degeneration. CDK is characterized by the haziness and opalescence of the cornea's most anterior layers which go through three stages with increasing severity. Globular deposits of different sizes may be histopathologically observed under the corneal epithelium by means of light and electron microscopy. The coalescence and increased volume of these spherules may cause the disruption of Bowman's membrane and the elevation and thinning of the corneal epithelium. The exact aetiology and pathogenesis of CDK are unknown, but they are possibly multifactorial. The only treatment in CDK advanced cases is a corneal transplantation, which in different impoverished regions of the world is not an available option. Many years ago, the clinical and histological aspects of this disease were described in several articles. This review highlights new scientific evidence of the expanding knowledge on CDK's pathogenesis which will open the prospect for new therapeutic interventions., (© 2015 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Purpose: To explore factors related to pathogenesis of rhegmatogenous retinal detachment (RRD) and development of proliferative vitreoretinopathy (PVR), vitreous levels of angiopoietin-1 and -2 (Ang-1 and -2), previously undefined in RRD, transforming growth factor-(TGF) β1, vascular endothelial growth factor (VEGF), erythropoietin (EPO) and proteolytic mediators of extracellular matrix remodelling (MMP-2 and -9) were compared in eyes with RRD and eyes with idiopathic macular hole or pucker., Methods: Vitreous samples were collected from 117 eyes with RRD (study group) and 40 eyes with macular hole or pucker (control group). Growth factors were measured by ELISA and matrix metalloproteinases (MMPs) by gelatin zymography., Results: The mean vitreous concentrations of Ang-2, MMP-2, and MMP-9 were higher (all p < 0.01), whereas concentration of VEGF was lower (p = 0.01) in eyes with RRD relative to controls. Logistic regression analysis identified Ang-2 concentration as a novel marker of RRD (p = 0.0001, OR 48.7). Ang-1, EPO, and total TGF-β1 levels were not significantly different between the groups. However, TGF-β1 and MMP-2 were increased in eyes with total RRD compared to those with local RRD (p ≤ 0.05). In eyes with PVR, no differences were observed in any studied marker as compared with non-PVR eyes., Conclusions: Current results reveal Ang-2 as a key factor upregulated in RRD. It may co-operate with fibrosis-associated factors and contribute to vascular complications such as breakdown of blood-eye barrier and PVR development.

Purpose: To evaluate the efficacy of keratectomy in treating irregular astigmatism caused by peripheral hypertrophic subepithelial corneal degeneration (PHSD) and to study the possible underlying immunological risk factors., Materials and Methods: Patients (14 eyes) with diagnosed PHSD were treated with superficial keratectomy with or without the assistance of phototherapeutic keratectomy (VisX S4; VisX Inc., Santa Ana, CA, USA). Thirteen patients were subjected to analysis of human leucocyte antigen (HLA) genes, complement C4 gene numbers and total plasma immunoglobulin levels. Immunological risk factors between patients and a control group comprising 150 individuals were compared., Results: The mean preoperative best spectacle corrected visual acuity (BCVA) improved from 0.16 ± 0.22 (LogMAR scale range 0-0.7) to 0.06 ± 0.13 (-0.1-0.4) (p < 0.01). The mean preoperative astigmatism decreased significantly from 3.8 ± 2.1 D (range 1.2-8.2) to 2.1 ± 1.4 (range 0.6-5.0, p = 0.02) based on corneal topography. The HLA-B*44 allele and the ancestral haplotype (AH) 8.1 were found significantly more often in PHSD patients than in controls (both p = 0.03). No differences in the C4 genes were found., Conclusions: Astigmatism secondary to PHSD can be effectively treated with keratectomy. Peeling of the fibrotic tissue reduced astigmatism and improved visual performance. We suggest that HLA-B*44 allele and AH 8.1 haplotype are immunological factors predisposing to the development of PHSD. The consequent disruption/alteration of the limbal barrier may lead to corneal peripheral fibrous formation inducing astigmatism., (© 2013 The Authors. Acta Ophthalmologica © 2013 Acta Ophthalmologica Scandinavica Foundation.)

Purpose: To report the corneal thinning during and after corneal cross-linking (CXL)., Design: Prospective, nonrandomized, single-center observational study., Methods: Thirty patients (30 eyes; 9 female, 21 male; age, 38 ± 12 years) were consecutively scheduled for CXL between January 23 and July 6, 2009. Twenty-four eyes had progressive keratoconus, 2 had pellucid marginal degeneration, 3 eyes had progressive keratectasia after a LASIK operation, and 1 eye had pseudophakic bullous keratopathy. Riboflavin-ultraviolet A (UVA)-induced CXL included the instillation of 0.1% riboflavin drops for 30 minutes followed by riboflavin instillation combined with UVA irradiation for another 30 minutes. Corneal thickness was measured preoperatively, during CXL, and after 1 and 6 months using an ultrasound pachymeter. Changes in the endothelial cell count, corneal steepness, refraction, and visual performance are also given., Results: On average, the corneas thinned 87 ± 40 μm (range, 37-206 μm; 19% ± 7%) during a 60-minute CXL treatment. In 1 patient, the cornea did not swell, even with hypotonic solution such that CXL would be safe. After 1 month, the corneal thickness was lower than the preoperative thickness, but after 6 months, the corneas had regained their original thicknesses. The endothelial cell count and corneal steepness were unchanged after CXL. The UCVA (uncorrected visual acuity) and BSCVA (best spectacle-corrected visual acuity) were improved 6 months after CXL., Conclusions: Corneal thickness decreases significantly during CXL, even to a level where the health of the endothelium and cornea is jeopardized. Visual performance is improved 6 months after CXL., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: Climatic droplet keratopathy (CDK) is an acquired corneal disease characterized by progressive scarring of the cornea. In several corneal diseases, matrix metalloproteinases (MMPs) are upregulated during the degradation of epithelial and stromal tissues. We investigated the levels, degree of activation and molecular forms of MMP-2, MMP-9, MMP-8 and MMP-13 and their tissue inhibitors TIMP-1 and TIMP-2 in tear fluid of patients with CDK., Methods: Seventeen CDK patients and 10 controls living in Argentine Patagonia received a complete eye examination, and MMPs and TIMP-1/2 were determined by immunofluorometric assay (IFMA), gelatin zymography and quantitative Western immunoblot analysis in tear samples., Results: The MMPs were detected mostly in their latent forms. The levels of MMP-9 and MMP-2 were found to be significantly elevated in CDK patients, whereas latent and active MMP-8 levels were significantly enhanced in controls. There was no significant difference in the level of MMP-13. TIMPs were found as part of complexes, and the TIMP-1 levels were significantly lower in patients than controls., Conclusion: Elevated MMP-2 and MMP-9 levels have been implicated in the failure of corneal re-epithelialization, and enhanced MMP-2 and MMP-9 levels in CDK patients suggest that these MMPs may play a role in corneal scarring in CDK. Elevated levels of MMP-8 suggest a defensive role for this MMP in inflammatory reactions associated with recurring corneal traumas. Decreased expression of TIMP-1 in CDK patients suggest deficient antiproteolytic shield likely to render the corneas of CDK patients vulnerable to enhanced MMPs. Overall, these data suggest a mechanistic link between MMPs and TIMP-1 level in cornea and tears with corneal scarring in CDK., (© 2009 The Authors. Journal compilation © 2009 Acta Ophthalmol.)

In the tear fluid the outermost part facing the tear-air interface is composed of lipids preventing evaporation of the tears. Phospholipid transfer protein (PLTP) mediates phospholipid transfer processes between serum lipoproteins and is also a normal component of human tears. To study whether PLTP plays any functional role in tear fluid we investigated PLTP-deficient mice, applying functional and morphologic analyses under normal housing and experimentally induced dry eye conditions. Aqueous tear fluid production, corneal epithelial morphology, barrier function, and occludin expression were assessed. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression were shown. These pathologic signs were worsened by experimentally induced dry eye both in wild-type and PLTP knock-out mice. Deficiency in the production of tear PLTP in mice is accompanied by corneal epithelial damage, a feature that is typical in human dry eye syndrome (DES). To complement animal experiments we collected tear fluid from human dry eye patients as well as healthy control subjects. Increased tear fluid PLTP activity was observed among DES patients. In conclusion, the presence of PLTP in tear fluid appears to be essential for maintaining a healthy and functional ocular surface. Increased PLTP activity in human tear fluid in DES patients suggests an ocular surface protective role for this lipid transfer protein., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

RP2 is a ubiquitously expressed protein encoded by a gene associated with X-linked retinitis pigmentosa (XLRP), a retinal degenerative disease that causes severe vision loss. Previous in vitro studies have shown that RP2 binds to ADP ribosylation factor-like 3 (Arl3) and activates its intrinsic GTPase activity, but the function of RP2 in the retina, and in particular photoreceptor cells, remains unclear. To begin to define the role of RP2 in the retina and XLRP, we have conducted biochemical studies to identify proteins in retinal cell extracts that interact with RP2. Here, we show that RP2 interacts with N-ethylmaleimide sensitive factor (NSF) in retinal cells as well as cultured embryonic kidney (HEK293) cells by mass spectrometry-based proteomics and biochemical analysis. This interaction is mediated by the N-terminal domain of NSF. The E138G and DeltaI137 mutations of RP2 known to cause XLRP abolished the interaction of RP2 with the N-terminal domain of NSF. Immunofluorescence labeling studies further showed that RP2 colocalized with NSF in photoreceptors and other cells of the retina. Intense punctate staining of RP2 was observed close to the junction between the inner and outer segments beneath the connecting cilium, as well as within the synaptic region of rod and cone photoreceptors. Our studies indicate that RP2, in addition to serving as a regulator of Arl3, interacts with NSF, and this complex may play an important role in membrane protein trafficking in photoreceptors and other cells of the retina.

In cells, one of the main roles of ceramide-enriched membrane domains is to recruit or exclude intracellular signaling molecules and receptors, thereby facilitating signal transduction cascades. Accordingly, in model membranes, even low contents of ceramide segregate into lateral domains. The impact of the N-acyl chain on this segregation and on the morphology of the domains remains to be explored. Using Langmuir monolayers, we have systematically studied binary mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and ceramide (2:1, molar ratio) and varied the N-acyl chain length of ceramide from 2 to 24 carbon atoms (Cer2 to Cer24). Fluid Cer2, Cer6, and Cer8/DMPC mixtures were miscible at all surface pressures. Longer ceramides, however, formed surface pressure-dependent immiscible mixtures with DMPC. The domain morphology under fluorescence microscopy after including a trace amount of fluorescent NBD-phosphatidylcholine into DMPC/Cer mixtures was found to be very sensitive to the N-acyl chain length. Shorter ceramides (Cer10-Cer14) formed flower-like (seaweed) domains, whereas longer ceramides (N-acyl chain length>14 carbon atoms) formed round and regular domains. We attribute the formation of the flower patterns to diffusive morphological instabilities during domain growth.

In the case of local anesthetic intoxication, intravenous administration of lipid-based Intralipid dispersion (Fresenius Kabi) can be used for the entrapment of hydrophobic drugs. Our long-term aim is to develop a sensitive, efficient, and non-harmful lipid-based formulation to specifically trap harmful substances. In this study liposome electrokinetic capillary chromatography (LEKC) was used to study the interactions between local anesthetics and Intralipid or liposome dispersions. Intralipid dispersion and extruded liposomes with different concentrations of 1-palmitoyl-2-oleyl-sn-glycerophosphatidylcholine (POPC), phosphatidylglycerol, cardiolipin, cholesterol, oleic acid, and linoleic acid were used as a pseudostationary phase in LEKC and their interactions with lidocaine, prilocaine, and bupivacaine were studied. POPC liposomes containing 1mol% of palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine as a fluorescent marker were used for the first time in LEKC connected with laser-induced fluorescent detection in order to calculate the retention factor for anesthetics.

Purpose: To assess the long-term refractive results, subjective parameters, and late sequelae of LASIK., Methods: A retrospective follow-up study was conducted on 38 eyes of 21 patients (17 with bilateral treatment and 4 with unilateral treatment) who had LASIK surgery between 1999 and 2000. Laser ablations were done with an excimer laser (VISX STAR and STAR S2). Follow-up was 2 months, 2 years, and > 7 years postoperatively. Uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), manifest refraction, indirect ophthalmoscopy, and wavefront analysis were measured at the last follow-up and a questionnaire was answered., Results: Postoperatively, BSCVA < or = 0.0 (logMAR) was obtained in 91%, 100%, and 89% of eyes at 2 months, 2 years, and at last follow-up, respectively. Postoperative spherical equivalent refraction within +/- 0.50 diopters (D) was obtained in 75%, 63%, and 42% of eyes at 2 months, 2 years, and 7 to 8 years, respectively. At 2 months and 2 years, 83% of eyes were within +/- 1.00 D, which decreased to 42% at 7 to 8 years. Mean spherical equivalent refraction at 2 months was -0.41 D, at 2 years -0.57 D, and at 7 to 8 years continued to decrease to -1.38 D. Patient satisfaction was high--100% of patients would have LASIK again., Conclusions: LASIK outcomes tend to shift toward undercorrection over time. Postoperative ectasia was not noted 7 to 8 years after LASIK. All patients were satisfied with the postoperative results although emmetropia was not reached.

Purpose: To evaluate the long-term effects of excimer laser treatment for ametropia after surgical treatment of rhegmatogenous retinal detachment (RRD) with scleral buckles., Setting: Helsinki University Eye Hospital, Helsinki, Finland., Methods: Ten patients (10 eyes) who had 1 or more surgeries for RRD followed by refractive surgery for myopia were retrospectively enrolled in this study and were examined after excimer laser refractive surgery. Photorefractive keratectomy (PRK) or laser in situ keratomileusis (LASIK) was performed using a Star S2 excimer laser system (Visx). The best spectacle-corrected visual acuity (BSCVA), refraction, degree of anisometropia, and topographical changes were evaluated before and after the surgeries., Results: All patients were free of asthenopic symptoms after refractive surgery. At the end of the follow-up, a mean of 67 months +/- 14 (SD) after refractive surgery, 6 patients were within +/-1.0 diopter of the intended correction. Compared with previously reported cohorts of myopic patients, the achieved refraction in patients who previously had a scleral buckling procedure was worse. The postoperative refraction was stable, and corneal topography did not show induced scar formation, keratectasia, or irregular astigmatism. After refractive surgery, the BSCVA improved 1 Snellen line in 3 patients and 2 lines in 1 patient. One patient lost 1 Snellen line and another patient lost 2 lines. Four patients showed no changes. New retinal complications were not observed., Conclusions: In the long-term, PRK and LASIK were safe methods for the treatment of myopia in RRD patients after scleral buckling. The predictability of myopic LASIK/PRK may be worse than generally reported in myopic cohorts.

Purpose: To test the hypothesis that anisometropic adults without significant amblyopia suffer from mild visual impairment probably due to aniseikonia, which might be improved by corneal refractive surgery., Methods: Fifty-seven patients presenting with myopic anisometropia > or = 3.25 diopters (D) and 174 myopic controls appropriate for refractive surgery were included. Photorefractive keratectomy (PRK) or LASIK was performed on 57 anisometropic eyes. As 43 of the 174 myopic control patients had bilateral surgery, PRK or LASIK was performed on 217 myopic control eyes. Best spectacle-corrected visual acuity (BSCVA), refraction, and refractive correction were measured preoperatively and at 1, 3, 5 to 7, 8 to 13, and 25 months following surgery., Results: Preoperative mean spherical equivalent was -7.20 +/- 2.40 D for anisometropic patients and -6.40 +/- 1.90 D for myopic patients. At 8 to 13 months postoperatively, when 23 (40%) anisometropic eyes and 94 (43%) myopic eyes were examined, the mean spherical equivalent refractions were -0.80 +/- 1.60 D and -0.30 +/- 0.60 D, respectively. Preoperatively, the mean BSCVA on a logMAR scale was -0.0143 +/- 0.0572 (Snellen 0.98 +/- 0.12) in the anisometropic group and 0.0136 +/- 0.0361 (Snellen 1.04 +/- 0.09) in the control group (P = .001). Eight to 13 months postoperatively, these values were 0.0076 +/- 0.0659 (Snellen 1.03 +/- 0.15) and 0.0495 +/- 0.0692 (Snellen 1.13 +/- 0.18) and this difference remained statistically significant (P = .012). For the myopic patients, the improvement in BSCVA reached almost maximum at 3 months, and this improvement was found to be highly significant 3 months after surgery (P = .001). The improvement in BSCVA was significantly slower for anisometropic patients and became statistically significant only after 8 to 13 months postoperatively (P = .041)., Conclusions: Anisometropia reduces visual acuity in the more myopic eye and can be at least partially reversed by refractive correction. The slower improvement in BSCVA for anisometropic patients suggests plastic changes in the visual cortex following refractive surgery.

Purpose: Because of the regulatory role of neuropeptide Y (NPY) in angiogenesis, we set out to determine the presence of the leucine 7-proline 7 (Leu7Pro) polymorphism in exudative age-related macular degeneration (AMD) patients and to analyse its implications., Methods: Genotype analysis of the Leu7Pro polymorphism in the signal peptide region of the human prepro-NPY was performed in blood samples from exudative AMD patients (n = 240) and control subjects (n = 79)., Results: In all, 11% of exudative AMD patients and 14% of control subjects exhibited the NPY signal peptide Leu7Pro polymorphism. There were no statistically significant differences in Leu7Pro polymorphism frequency between the exudative AMD and control cases, as analysed by Fisher's exact two-sided test., Conclusions: Leu7Pro polymorphism in the signal peptide region of the human prepro-NPY is not a risk factor for exudative AMD.

Purpose: To study the changes in irregular astigmatism in patients with recurrent corneal erosion syndrome due to map-dot-fingerprint degeneration and to demonstrate that both symptomatic and asymptomatic map-dot-fingerprint degeneration/recurrent corneal erosion syndrome may be related to irregular astigmatism, which can effectively be corrected by phototherapeutic keratectomy (PTK) or standard spherical photorefractive keratectomy (PRK)., Methods: Eleven eyes with irregular astigmatism related to map-dot-fingerprint degeneration/recurrent corneal erosion syndrome with and without changes at the slit-lamp microscope were treated with PTK or spherical PRK, which was performed with a VISX S2 or S4 excimer laser. All eyes underwent videokeratography performed pre- and postoperatively using either the Tomey TMS-2N Topographic Modeling System or EyeSys Technologies Corneal Analysis System 2000., Results: Mean pre- and postoperative best spectacle-corrected visual acuity (BSCVA) was -0.18 +/- 0.14 logMAR and 0.04 +/- 0.04 logMAR, respectively. This improvement in BSCVA was statistically significant. The mean gain in Snellen lines was 2.4 +/- 2.1. Preoperative corneal videokeratography showed irregular astigmatism with an elevation pattern that was corrected in all eyes despite the fact that no astigmatic photorefractive correction was performed., Conclusions: Following PTK, eyes with irregular astigmatism related to map-dot-fingerprint degeneration showed significant improvement in BSCVA and correction of irregular astigmatism as revealed by videokeratography. This study shows that irregular astigmatism can be exclusively of epithelial origin and in some eyes abnormal corneal epithelium may create optical aberrations. This possibility should be taken into account when, for example, wavefront-guided stromal photoablation procedures are being planned.

Tumors express MMP-2 and MMP-9 gelatinases, which are involved in the formation of tumor vasculature. This suggests that a tumor and its surrounding neovasculature can be visualized by a sensitive gelatinase recognition method. We have studied tumor radioimaging using a gelatinase inhibitory peptide CTTHWGFTLC (CTT), which in a mouse model targets the tumor site following an intravenous injection. We determined a solution NMR structure of CTT and its retro-inversion peptide, and prepared 125I and 99mTc-labelled CTT peptide derivatives. Radiolabelled CTT inhibited gelatinases in vitro, and homed to a tumor xenograft in mice. In normal mice, CTT was instead rapidly cleared from the circulation mainly through the kidney and, after 24 h, no significant radioactivity was accumulated in healthy tissues. Gamma camera imaging of a primary tumor in live mice was obtained with double-labelled liposomes, which were coated with 99mTc-CTT and encapsulated with 125I albumin. CTT also targeted liposomes to the lungs of tumor-bearing mice, which may indicate the existence of non-visible lung micrometastases. Our studies suggest that selective gelatinase-targeting compounds could be useful in the early detection and imaging of primary tumors and metastases.

Purpose: To evaluate the time course of changes in corneal sensitivity to mechanical and chemical stimuli produced by laser in situ keratomileusis (LASIK) in humans., Methods: We performed a cross-sectional study of 17 LASIK-operated eyes (VisX S2, equipped with version 2.50-3.10 software) and 15 control eyes of 17 individuals to evaluate regeneration of corneal sensitivity after LASIK. Gas pulses of variable flow and compositions were applied to the cornea by a non-contact gas esthesiometer. Mechanical stimuli consisted of air puffs at flows from 0 to 200 ml/min. Chemical stimulation was made with gas pulses containing 0% to 80% CO2 in air at subthreshold flow. Mechanical and chemical thresholds and intensity-response curves for the evoked sensations were determined prior to surgery, and 7 to 9 days, 3 to 5 months, and 1.5 to 2.5 years after surgery., Results: Corneal sensitivity to mechanical stimulation was enhanced 7 to 9 days after surgery but subsequently dropped markedly and remained significantly below control levels 3 to 5 months after LASIK. Sensitivity to both mechanical and chemical types of stimuli was close to normal 2 years postoperatively., Conclusions: Corneal sensitivity decreased immediately after LASIK but mechanical sensitivity showed a transient hyperesthesia 7 to 9 days afterward. Subsequently, a long-lasting and deep hypoesthesia to mechanical and chemical stimuli was observed. Gas esthesiometry revealed that disturbances of corneal sensation still exist at times when coarse mechanical sensitivity appeared to be normal.

We describe four patients who developed corneal erosion after an exposure to a pepper spray containing toxic carriers. Two of these patients were exposed to a pepper gas containing 5% oleoresin capsicum (OC) as an irritant and 92% trichlorethylene or unknown amount of dichloromethane as a carrier. One patient was exposed to a mock (containing 92% trichlorethylene as a carrier) training pepper gas without OC. The fourth patient was exposed to an unidentified Russian pepper gas spray. Two of the patients were examined by in vivo confocal microscopy to demonstrate the depth and quality of the stromal damage. To test the toxicity of the commercial tear spray, it was analyzed and test sprayed on a soft contact lens and into a plastic cup. Visual acuity was measured and the eyes were examined with a slit-lamp up to 5 months. Physical damage to a soft contact lens was visually acquired. All patients showed a long-lasting, deep corneal and conjuctival erosion, which resolved partly with medical therapy during the following weeks/months. Confocal microscopy revealed corneal nerve damage, and keratocyte activation reaching two-thirds of stroma for one patient. The spray caused serious damage to both the soft contact lens and the plastic cup. The safety of the commercially available pepper sprays should be assessed before marketing, and a list of acceptable ingredients created. The sprays should also have instructions on the use of the compound as well as on the first aid measures after the exposure. Solvents known to be toxic should not be used.

Sphingomyelinase, SMase (EC 3.1.4.12), was coupled onto amino-derivatized acrylate microspheres and was shown to retain its catalytic activity. The immobilized enzyme allows one to carry out topical enzymatic reaction in a controlled manner. Accordingly, these spheres were held with a micropipet and using micromanipulator brought into contact with a giant liposome membrane composed of phosphatidylcholine and sphingomyelin (SOPC/C16:0-SM, 0.75:0.25, molar ratio), representing the substrate for the immobilized enzyme. The macroscopic consequences of the enzyme reaction were visualized using fluorescence microscopy as well as differential interference contrast microscopy. The surface contact of the giant vesicle and immobilized enzyme causes membrane microdomain formation and domain clustering (capping) in the membrane and subsequent shedding of small vesicles from the membrane into the interior of the giant liposome. The method described represents a novel approach to study enzymatic reactions and allows manipulating giant vesicles as well as cultured cells in a spatially controlled manner.

Macroscopic consequences of the formation of diacylglycerol by phospholipase C (PC-PLC) in giant 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) unilamellar vesicles (GUVs, diameter 10-100 microm) were studied by phase contrast and fluorescence microscopy. PC-PLC caused a series of fast stepwise shrinkages of fluid SOPC GUVs, continuing until the vesicle disappeared beyond the optical resolution of the microscope. The presence of N-palmitoyl-sphingomyelin (mole fraction X = 0.25) in the GUVs did not affect the outcome of the PC-PLC reaction. In addition to hydrolysis, PC-PLC induced adhesion of vicinal vesicles. When multilamellar SOPC vesicles were used only a minor decrease in their diameter was evident suggesting that PC-PLC can exert its hydrolytic activity only in the outer monolayer. A series of stepwise shrinkages was observed also for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) GUVs above their main phase transition temperature, T(m), i.e., when the bilayer is in the liquid crystalline state. However, this process was not observed for DMPC GUVs in the gel state, below T(m). These results are supported by the enhanced activity of PC-PLC upon exceeding T(m) of DMPC large unilamellar vesicles (diameter approximately 0.1 microm) used as a substrate. Studies on SOPC monolayers revealed that PC-PLC can exert its hydrolytic activity only at surface pressures below approximately 30 mN/m. Accordingly, the lack of changes in the gel state DMPC GUVs could be explained by the equilibrium lateral pressure in these vesicles exceeding this critical value.

Atherosclerosis is characterized by the accumulation in the arterial intima of mainly low-density lipoprotein (LDL)-derived lipids, together with apolipoprotein B-100 (apoB-100), the protein moiety of LDL. Recent studies indicate aggregation of LDL within the arterial wall to represent a critical step in the initiation of this disease. Aggregation of LDL has been further proposed to involve ceramide, the levels of which are elevated in atherosclerotic plaques as well as in LDL isolated from these lesions. Biophysical studies have shown ceramide to have a pronounced tendency for self-aggregation, presumably driven by intermolecular hydrogen bonding. Importantly, the segregated ceramide-enriched lipid phases have high melting temperatures and are in a gel state at 37 degrees C. The plasma levels of sphingomyelin, which upon enzymatic hydrolysis by sphingomyelinase (SMase) yields ceramide, have been shown to correlate with the severity of coronary heart disease. The formation of ceramide from sphingomyelin could thus represent a critical step in atherosclerosis. We recently showed that LDL itself possesses SMase activity. Moreover, sequence analogy with bacterial enzymes suggests that this activity may be intrinsic to apoB-100. Possible physiological role of this activity is uncertain, yet could be involved in nonreceptor-mediated endocytotic entry of LDL into cells. Importantly, it also opens a possible mechanistic link between elevated plasma levels of LDL, apoptosis (programmed cell death), and atherosclerosis.