Albert J. Augustin

Two-dimensional analysis of tear protein patterns of diabetic patients

In diabetic patients, dry eye and other ocular surface diseases occur more often than in healthy subjects. The aim of this study was to analyze the tear protein patterns of patients suffering from diabetes mellitus type II (dia) and to compare them to the patterns of healthy volunteers (ctrl). Tear proteins of nonstimulated tears of 20 patients (ctrl nu200a=u200a10, dia nu200a=u200a10) were separated using two‐dimensional electrophoretic techniques. The protein patterns of each group were analyzed by a multivariate analysis of discriminance. Furthermore, for all spots of each gel, a 50×50 variables pH/Mr (molecular weight) array was generated and subsequently analyzed by a multivariate analysis of discriminance. Additionally, an artificial neural network was trained using the matrix data as input and a sensitivity analysis was performed to figure out, which spots were the most important to differentiate between the tear protein patterns. In both groups a complex staining pattern could be obtained. In diabetic patients significantly more spots were detected compared to the control group (P<0.02). The analysis of discriminance found a highly significant difference between dia and ctrl (P<0.00001). Using the matrix data, the analysis of discriminance showed a significant difference between the two groups, too (P<0.0001). The sensitivity analysis by means of the artificial neural network revealed several spots that were more expressed or more frequently present in the diabetic group. Our findings reveal that the composition of tear proteins of diabetic patients is different from that of healthy subjects. The use of the two‐dimensional electrophoretic technique could give more insight into the diabetic‐related changes in the tear film composition.

Inflammation and the pathogenesis of age-related macular degeneration

Background: Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. Many changes occur in various areas of the eye as it ages. These include choroidal thinning, thickening of Bruchs membrane and drusen formation. Each of these is associated with the onset of AMD. Methods: Recent findings on how those changes contribute to the pathogenesis of AMD with a focus on inflammation are examined. Results: There is evidence suggesting that all changes identified so far as being involved in the pathogenesis of AMD are not able to cause AMD alone. Instead, susceptibility genes, and in particular a coding variant of a gene on chromosome 1 result in dysfunction of the immune system. This leads to an inappropriate inflammatory response, which then sets the stage for AMD onset. Conclusions: It is now well-known that AMD is a multi-factorial disease, with environmental causes and genetics all playing a role.

Analysis of tear proteins by one- and two-dimensional thin-layer iosoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. Detection of a new component: cystatin C

Abstractu2002· Background: Isoelectric focusing (IEF) of tear proteins has not yet been carried out in a satisfactory way. Two-dimensional (2D) electrophoresis, especially in the combination of IEF with SDS, is able to differentiate between proteins in detail. The purpose of this study was therefore to analyze tear proteins by 1D IEF alone and in combination with a 2D pattern, and by IEF followed by lectin staining. · Methods: Ampholines, covering a broad range from pH 3 to pH 10, were applied. After IEF, semi-dry blotting and incubation with a group II lectin and two group V lectins was performed.u2002· Results: Tear proteins could be separated into 31 single bands. Tear-specific pre-albumin (TSPA), lactoferrin, sIgA, IgG and lysozyme were found to be main components. Isoelectric points (IEPs, pIs) of all proteins separated were determined by comparison with IEF standards. 2D patterns of IEF and SDS electrophoresis were obtained for the main subunit components of lactoferrin, sIgA, TSPA, and lysozyme. An additional new component of considerable concentration was focused at pI 8.6 with a subunit MW of 14 kDa. With s-WGA a component at an IEP of 5.2 was visualized, representing transferrin. With SNA, lactoferrin stained as a sharp main band at pI 5.1 with three additional weaker bands at IEPs from 4.8 to 4.9. At IEPs between 4.4 and 6.1, multiple components of sIgA were stained with MAA. The sugar specificity of transferrin at pI 5.2 was β-GlcNAc. Lactoferrin showed glycation with NANA-α-2–6-Gal or NANA-α-2–6-GalNAc, whereas the sugar specificity of sIgA was NANA-α-2–3-Gal. · Conclusions: The investigative strategy applied here, including IEF alone, in combination with SDS-electrophoresis, and SDS-electrophoresis followed by lectin staining proved to be a reproducible method for tear protein analysis of hitherto unexperienced capacity. Lectin-stained bands of native tear proteins are not uniformly glycated by one sugar residue, but show various sugar specificities. IgA as a whole molecule is specifically glycated with NANA-α-2–3-Gal.

Healon5 viscoadaptive formulation: Comparison to Healon and Healon GV

Purpose: To compare the rheological characteristics of a viscoadaptive viscoelastic formulation with those of 2 standard ophthalmic viscosurgical devices (OVDs). Setting: Department of Ophthalmology, Johannes Gutenberg‐University, and Max Planck‐Institute for Polymer Research, Mainz, Germany. Methods: An independent comparative study of 3 OVDs of sodium hyaluronate (Healon®, Healon GV®, and Healon®5) was performed using the Advanced Rheometric Expansion System to analyze rheologic behavior (eg, dynamic frequency dependence of the complex viscosity) as well as rheological parameters (eg, viscosity at zero shear rate, pseudoplasticity, relaxation time, elastic and viscous modulus). Results: Mean viscosity at zero shear rate was 243 Pas ± 5 (SD) for Healon, 2451 ± 12 Pas for Healon GV, and 5525 ± 14 Pas for Healon5. Mean pseudoplasticity was 173 ± 7, 754 ± 10, and 591 ± 6, respectively. Mean relaxation time was 21 ± 3 sec, 83 ± 4 sec, and 88 ± 6 sec. At low shear rates, viscosity and elasticity (elastic and viscous modulus) increased from Healon through Healon5. Healon5 exhibited a dynamic behavior of the complex viscosity dependent on the duration of the shear rate exposure: At low shear rates, it slowly built up a high viscosity. At higher shear rates, it demonstrated a lower viscosity that decreased further during constant exposure to a specific shear rate. Conclusions: Healon5 had the highest viscosity and elasticity when exposed to low and high shear rates. These characteristics maintain anterior chamber depth. Also, the high viscosity of Healon5 exhibited a dynamic frequency dependence. In the presence of turbulence and phaco power (continuous high shear rates), it became dispersive by fragmentation and formed a cavity with an outer retentive shell. The cohesive and dispersive properties of Healon5 make it the best of the 3 OVDs evaluated for use atall stages of phacoemulsification.

Mechanisms of Ocular Angiogenesis and Its Molecular Mediators

Angiogenesis is defined as the formation of new blood vessels from the existing vasculature. It is a highly coordinated process occurring during development of the retinal vasculature, ocular wound healing, and in pathological conditions. Complex interactions are involved between non-vascular and microvascular cells, such as endothelial cells and pericytes, via several angiogenic growth factors and inhibitors. Of these growth factors, vascular endothelial growth factor (VEGF) has emerged as the single most important causal agent of angiogenesis in health and disease in the eye. During the angiogenic process, endothelial cells shift from a homogeneous quiescent population into a population of heterogeneous phenotypes, each with a distinct cellular fate. So far, three angiogenic specialized phenotypes have been identified: (1) tip cells, which pick up guidance signals and migrate through the extracellular matrix; (2) stalk cells, which proliferate, form junctions, produce extracellular matrix, and form a lumen, and (3) phalanx cells, which do not proliferate, but align and form a smooth monolayer. Eventually, a robust mature new blood vessel is formed which is capable of supplying blood and oxygen to tissues. Pathological angiogenesis is a key component of several irreversible causes of blindness. In most of these conditions, angiogenesis is part of a wound healing response culminating, via an angiofibrotic switch, in fibrosis and scar formation which leads to blindness. Currently, VEGF-A antagonists are standard care in the treatment of exudative age-related macular degeneration, and have been found to be a valuable additional treatment strategy in several other vascular retinal diseases.

Vacuoles in the AcrysofTM Intraocular Lens as Factor of the Presence of Serum in Aqueous Humor

Objective: Acrysof™ (Alcon) foldable lenses (IOLs) have been shown to be highly biocompatible and exhibit a low incidence of posterior capsular opacification. However, minute vacuoles or ‘glistenings’ have been observed in some Acrysof IOLs. The clinical relevance of vacuole formation is presently unclear. To help clarify the influence of factors present under in vivo conditions on vacuole formation, the present experimental study examines the influence of aqueous humor components on the occurrence of vacuoles in Acrysof IOLs. Methods: A total of 12 sterile Acrysof IOLs (Alcon, MA60BM) were incubated at body temperature (37°C) for 3 or 6 months in anterior-chamber aqueous humor with or without human serum. The center portion was cut from two unconditioned, unhydrated, sterile control IOLs and the 12 conditioned IOLs and examined using light microscopy for vacuole formation. A third unconditioned, hydrated control IOL was examined using light and scanning electron microscopy. Measures: The incidence of vacuoles was quantified by two independent investigators using light microscopy. After hydration with balanced salt solution, the surface quality of a control IOL was examined using scanning electron microscopy. Results: The control IOLs exhibited no or very few vacuoles. Scanning electron microscopy revealed that the control IOLs had normal surface texture without any surface vacuoles. IOLs that had been conditioned in aqueous humor without serum exhibited no great increase in the number of vacuoles, whereas IOLs conditioned in aqueous humor with serum exhibited a greater number of vacuoles that increased over time. Conclusions: The number of vacuoles increases with incubation time in aqueous humor containing serum. The addition of serum increased the proportion of lipids and proteins in the solution, which also occurs with a breakdown in the blood-aqueous barrier. The results of the present study point to a physiological factor that may lead to vacuole formation in IOLs and may aid clinicians in identifying risk factors involved in the formation of vacuoles.

Analysis of tear protein patterns by a neural network as a diagnostical tool for the detection of dry eyes.

The electrophoretic patterns of tears from patients with dry‐eye disease (n = 43) and from healthy subjects (n = 17) were analyzed by means of multivariate statistical methods and an artificial neural network (ANN), following sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). From each electrophoretic pattern a data set was created, randomly divided into test (unknown samples) and training patterns (known samples), with ANN training by one of these sets. After training, the performance of the ANN was checked by presenting the test data set to the ANN. Furthermore, the data was classified using multivariate analysis of discriminance. The groups were significantly different from each other (P < 0.05). The statistical procedure yielded 97% (known samples) and 71% (unknown samples) correct classifications. The ANN revealed 89% of correct classifications using the test set (unknown samples). The use of pruning algorithms (optimization procedure which automatically eliminates small weighted neurons) or genetic algorithms (optimization procedure which performs genetically induced changes of the neural net) resulted in a slight decrease of correct classifications compared to those of the nonoptimized neural network. The results reveal significant differences between the two groups. Using the ANN we were able to classify the electrophoretic tear protein pattern for diagnostic purposes.

Oxidative tissue damage after phacoemulsification ☆: Influence of ophthalmic viscosurgical devices

Purpose: To quantify the oxidative tissue damage after phacoemulsification, correlate the damage to the energy applied, and investigate the influence of ophthalmic viscosurgical devices (OVDs). Setting: Department of Ophthalmology, University of Mainz, Mainz, Germany. Methods: The study comprised 130 eyes operated on by 1 surgeon using the same phacoemulsification machine. Some eyes received an OVD before phacoemulsification and some did not. Energy values were expressed as phaco time; that is, ultrasound (US) time (seconds) after conversion to 100% phaco power. Patients were grouped as follows: Group 1, phaco time less than 20 seconds and no OVD; Group 2, phaco time 20 to 40 seconds and no OVD; Group 3, phaco time more than 40 seconds and no OVD; Group 4, phaco time 20 to 40 seconds and hydroxypropyl methylcellulose 2% (HPMC); Group 5, phaco time 20 to 40 seconds and sodium hyaluronate 1%. Aqueous humor from pseudophakic eyes served as a control. At the end of surgery, anterior chamber fluid was analyzed for lipid peroxides using the thiobarbituric acid method. Results: Lipid peroxides were detected in all groups. The values were significantly higher in Group 2 than in Group 1 (P<.01) and in Group 3 than in Groups 1 and 2 (P<.01). The differences in lipid peroxide values between all phaco groups and the control group were statistically significant. Sodium hyaluronate 1% and HPMC 2% produced significantly lower lipid peroxide values than in the respective phaco groups that did not receive an OVD (both P<.01). Conclusions: Oxidative tissue damage occurred during phacoemulsification. The damage, which correlated with the US energy applied, can be reduced by the use of OVDs.

Treatment of neovascular age-related macular degeneration: Current therapies

Choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) is now the leading cause of blindness and severe vision loss among people over the age of 40 in the Western world. Its prevalence is certain to increase substantially as the population ages. Treatments currently available for the disease include laser photocoagulation, verteporfin photodynamic therapy, and intravitreal injections of corticosteroids and anti-angiogenic agents. Many studies have reported the benefits of each of these treatments, although none is without its risks. No intervention actually cures AMD, nor the neovascularization associated with it. However, its symptoms are treated with varying degrees of success. Some treatments stabilize or arrest the progress of the disease. Others have been shown to reverse some of the damage that has already been done. These treatments can even lead to visual improvement. This paper will review the major classes of drugs and therapies designed to treat this condition.

Effect of smoking on tear proteins

AbstractnPurpose. Cigarette smoking is a serious risk factor for many diseases, e.g., cardiovascular and pulmonary diseases. It is also a risk factor in several eye diseases, including macula degeneration, glaucoma, and cataract. Ischemic, toxic, and oxidative effects of cigarettes are thought to play an important role in damaging ocular tissue. Furthermore, smoking can cause symptoms of dry-eye disease. The aim of this study was to analyze and compare electrophoretic patterns in tears of smokers (SP), severs smokers (SSP), and nonsmokers (CTRL).nMethods. We examined 105 eyes (SP: n=29, SSP: n=26, CTRL: n=50). Each patient was asked for subjective symptoms such as burning, itching, and foreign-body sensation. Tear proteins were separated by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Digital image analysis was performed by BioDocAnalyze (Biometra, Göttingen, Germany), with densitometric data files being created for each electrophoretic lane. Data was analyzed by multivariate statistical techniques.nResults. Tear protein patterns in SP (P<0.05) and SSP (P<0.05) were different from those of CTRL. There were significantly more protein peaks in the SSP group (P<0.005) than in CTRL.nConclusion. Electrophoretic analysis of tear protein patterns could detect changes in tear proteins of smokers in comparison to nonsmokers. These changes were correlated with an increase of dry-eye-related subjective symptoms in smokers. Thus, electrophoretic analysis of tear proteins provided greater insight into the pathogenesis of smoking-induced ocular surface diseases.