F. H. Grus

Intraocular pressure and ocular pulse amplitude using dynamic contour tonometry and contact lens tonometry

BackgroundThe new Ocular Dynamic Contour Tonometer (DCT), investigational device supplied by SMT (Swiss Microtechnology AG, Switzerland) allows simultaneous recording of intraocular pressure (IOP) and ocular pulse amplitude (OPA). It was the aim of this study to compare the IOP results of this new device with Goldmann tonometry. Furthermore, IOP and OPA measured with the new slitlamp-mounted DCT were compared to the IOP and OPA measured with the hand-held SmartLens®, a gonioscopic contact lens tonometer (ODC Ophthalmic Development Company AG, Switzerland).MethodsNineteen healthy subjects were included in this study. IOP was determined by three consecutive measurements with each of the DCT, SmartLens®, and Goldmann tonometer. Furthermore, OPA was measured three times consecutively by DCT and SmartLens®.ResultsNo difference (P = 0.09) was found between the IOP values by means of DCT (mean: 16.6 mm Hg, median: 15.33 mm Hg, SD: +/- 4.04 mm Hg) and Goldmann tonometry (mean: 16.17 mm Hg, median: 15.33 mm Hg, SD: +/- 4.03 mm Hg). The IOP values of SmartLens® (mean: 20.25 mm Hg, median: 19.00 mm Hg, SD: +/- 4.96 mm Hg) were significantly higher (P = 0.0008) both from Goldmann tonometry and DCT. The OPA values of the DCT (mean: 3.08 mm Hg, SD: +/- 0.92 mm Hg) were significantly lower (P = 0.0003) than those obtained by SmartLens® (mean: 3.92 mm Hg, SD: +/- 0.83 mm Hg).ConclusionsDCT was equivalent to Goldmann applanation tonometry in measurement of IOP in a small group of normal subjects. In contrast, SmartLens® (contact lens tonometry) gave IOP readings that were significantly higher compared with Goldmann applanation tonometer readings. Both devices, DCT and SmartLens® provide the measurement of OPA which could be helpful e.g. for the management of glaucoma.

Changes in the tear proteins of diabetic patients.

BackgroundPrevious studies have shown a significant increase in tear protein peaks in the tears of diabetic patients suffering from dry eye. The aim of this study was to analyze the tear protein patterns from patients with diabetes mellitus who do not suffer from ocular surface diseases (DIA).MethodsA total of 515 patients were examined in this study (255 healthy subjects (controls) and 260 patients suffering from diabetes mellitus). Tear proteins were separated by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis. After digital image analysis densitometric data files were created and subsequently used for multivariate statistical procedures.ResultsA significant increase in the number of peaks was detected in diabetic patients compared to controls (P < 0.0003). The analysis of discriminance revealed a highly significant discrimination between diabetic patients and controls (Wilks lambda: 0.27; P < 0.000001). Furthermore, a significant difference in the protein pattern of diabetic patients could be detected between those suffering from dry eye or not (P < 0.002). The changes in protein patterns of diabetic patients increased with the duration of the diabetic disease. In diabetic patients with a disease duration longer than 10 years the changes were significantly more expressed than in patients with a shorter diabetic history (P < 0.003) and in healthy subjects (P < 0.0001).ConclusionsThe tear protein patterns of diabetic patients are very different in the number and intensity of spots from those of healthy subjects. Furthermore, it could be demonstrated that the differences found in the tear patterns of diabetic patients are not equal to those found in previous studies in patients suffering from dry-eye disease. The alterations in the diabetic tears were correlated with the duration of the diabetic disease. With longer disease, history changes in the tear protein patterns increased. With the course of the disease some protein peaks appeared that are not present in healthy persons. Our study shows that the analysis of electrophoretic tear protein patterns is a new non-invasive approach in the early diagnosis and analysis of the pathogenesis of diabetes induced ocular surface disease.

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.

Enhanced Characterization of Serum Autoantibody Reactivity Following HSP 60 Immunization in a Rat Model of Experimental Autoimmune Glaucoma

Purpose: Antibodies against heat shock proteins have been identified in sera of human glaucoma patients in several studies and immunization with heat shock protein 60 (HSP 60) causes retinal ganglion cell (RGC) loss in an animal model of experimental autoimmune glaucoma. The aim of this study was to observe the time course of increased anti-retina antibody appearance in the serum and characterize the identification of prominent autoantibodies that accompany HSP 60 immunization in a rat model of experimental autoimmune glaucoma. Methods: Eight weeks after immunization with HSP 60 retinal flatmounts were prepared and RGCs were counted in eight predefined areas and compared to controls. Serum collected before, as well as four and eight weeks after, immunization was used to detect antibody patterns against bovine retinal antigens using Western blotting techniques. These patterns were analyzed by multivariate statistical methods. Autoantibodies that were prominently increased were further identified through mass spectrometry. Intraocular pressure was measured throughout the study. Results: After eight weeks, animals immunized with HSP 60 showed significant RGC loss of retinal flatmounts (P = 0.02), which was intraocular pressure independent. Early changes in antibody profiles, many of them significant upregulations, were detected. Antigens with significantly upregulated antibody reactivity after four weeks were identified as histone H2B type 1, S-arrestin, glial fibrillary acidic protein, vimentin, and heat shock protein 60. These upregulated autoantibodies returned to normal levels four weeks following their initial upregulation. Antibodies against retinaldehyde binding protein 1 on the other hand became upregulated after eight weeks. Conclusion: This is the first study to identify the appearance and disappearance of retinal autoantibodies in the serum of rats at several time-points following their initial upregulation in response to HSP 60 immunization in a model of experimental autoimmune glaucoma.

Analysis of the Antibody Repertoire in Tears of Dry-Eye Patients

Purpose: It has recently been suggested that dry-eye disease has an underlying autoimmune mechanism. This hypothesis is further supported by the successful treatment of the disease with immunomodulatory drugs such as cyclosporin A. Although it is known that tears contain antibodies, very little is known about the antibody repertoires in tears. It was the aim of this study to analyze the IgA antibody repertoire against ocular antigens in the tears of patients suffering from dry-eye disease and compare it to those of healthy volunteers. Methods: Two groups were examined: 20 healthy volunteers (controls) and 28 patients suffering from dry-eye disease. The patients were grouped according to the results of the basic secretory test. Patients with values ≤10 and subjective symptoms were classified as dry-eye patients. All tears were tested against Western blots of ocular antigens. For each Western blot, a densitograph was built by digital image analysis, and subsequently a multivariate discriminate analysis was performed. Results: A complex staining pattern was found in the tears of both dry-eye patients and healthy controls. However, the number of peaks was statistically significantly increased (p < 0.05) in the tears of dry-eye patients. The discriminant analysis found a statistically significant difference between the antibody repertoires of both groups (Wilks’ λ = 0.11; p < 0.001). Conclusions: In this study, it could be shown that the complex antibody repertoires in the tears of patients suffering from dry-eye disease are different from those found in the tears of healthy volunteers. Thus, our findings support the hypothesis that the dry eye disease has an autoimmune mechanism.

Detection of eicosanoids in epiretinal membranes of patients suffering from proliferative vitreoretinal diseases.

AIM Arachidonic acid is metabolised via lipoxygenase to 15-HETE (15-hydroxyeicosatetraenoic acid) and 15-HPETE (15-hydroperoxyeicosatetraenoic acid), which are believed to influence proliferation in tissue culture. 15-HETE is the reduction product of 15-HPETE. Cell proliferation is believed to be decreased by 15-HPETE and increased by 15-HETE. The aim of this study was to investigate epiretinal membranes for the presence of these lipoxygenase products and to compare membranes from different disease processes. METHODS Epiretinal membranes of 15 patients suffering from proliferative vitreoretinopathy (PVR, n=7) and proliferative diabetic retinopathy (PDR; n=8) were removed during vitrectomy and analysed by means of thin layer chromatography. The plates were evaluated by digital image analysis. RESULTS Both 15-HETE and 15-HPETE were identified in membranes from eyes of patients with PVR and PDR with HETE values significantly higher (p<0.05) than HPETE values (HETE/HPETE ratio = 5.2). CONCLUSION This study demonstrates that eicosanoids are present in the epiretinal membrane tissue of patients with PVR and PDR. Considering that HETE increases cell proliferation while HPETE inhibits it, it is conceivable that eicosanoids are an additional factor contributing to the regulation of membrane growth in proliferative retinal disorders. Thus, inhibition of lipoxygenase could be a therapeutic approach in these diseases.

Arzneimittelrechtliche Erlaubnis bei der Herstellung von Serum-Augentropfen

According to European Union and German legislation, the production of medicines such as eye drops from autologous serum requires a license from an appropriate authority. However, an exemption is granted to medical doctors who produce and apply medications under their immediate responsibility. If doctors do not actually produce such medications themselves, they must select appropriate personnel to do so. These individuals have to be sufficiently qualified and reliable and must be carefully instructed on the standard quality and manufacturing procedures, although the doctors are responsible for controlling the production and quality of the final product. The treating physicians are also responsible for the appropriate application of the produced medications. To dispense medication to patients without an appropriate license is considered a criminal offense. We describe how two German hospitals have established the production of serum eye drops in full compliance with legal regulations.

[Legal regulations to produce serum eye drops : when is it necessary, and how can it be obtained?].

According to European Union and German legislation, the production of medicines such as eye drops from autologous serum requires a license from an appropriate authority. However, an exemption is granted to medical doctors who produce and apply medications under their immediate responsibility. If doctors do not actually produce such medications themselves, they must select appropriate personnel to do so. These individuals have to be sufficiently qualified and reliable and must be carefully instructed on the standard quality and manufacturing procedures, although the doctors are responsible for controlling the production and quality of the final product. The treating physicians are also responsible for the appropriate application of the produced medications. To dispense medication to patients without an appropriate license is considered a criminal offense. We describe how two German hospitals have established the production of serum eye drops in full compliance with legal regulations.

Repeated Intraocular Pressure Measurement in Awake Lewis Rats Does Not Bias Retinal Ganglion Cell Survival

Purpose: The TonoPen applanation tonometry is an established method for intraocular pressure (IOP) measurement. The IOP is one of the main variables affecting retinal ganglion cell (RGC) loss in experimental animal models in ophthalmology and the main risk factor for human glaucoma. In this study, we examined if IOP measurements with the TonoPen itself lead to retinal ganglion cell loss or any other possible retina damages, such as intraocular bleedings or ablation, in Lewis rats. Methods: Three groups of rats (n = 5 each) were formed. IOP monitoring, using a TonoPen XL, was performed on groups 1 and 3. Animals in groups 1 and 2 received funduscopies before and after one and two weeks of the study, in order to detect possible abnormalities. After two weeks, retinal flatmounts were stained to detect ganglion cells. RGCs were manually counted in eight predefined areas to compare mean RGC densities between groups 1 and 2 (IOP readings vs. no readings), using student t-test. Results: No significant difference in RGC density between animals that underwent IOP readings and controls could be observed (p = 0.8). As expected, no IOP alterations were monitored in groups 1 and 3 throughout the study. No retinal abnormalities, such as bleeding or retina ablation, were detectable. Conclusion: We could detect no effects on retinal ganglion cell survival in Lewis rats or any other damages to the retina caused by IOP measurements using a TonoPen XL. This study proposes that repeated applanation tonometry does not affect RGC numbers, one of the main monitored variables in most glaucoma model studies. Therefore, the use of a TonoPen XL for repeated IOP monitoring in Lewis rats can be considered harmless.

Vom Genom zum Proteom

Im Sommer 2000 wurde das humane Genom vollständig sequenziert. Die DNA-Technologien wurden immer besser und schneller, immer einfacher anzuwenden, teilweise sogar automatisierbar. Das Genom wird v. a. durch 4 chemisch ähnliche Bestandteile aufgebaut, die in ihrer linearen Anordnung die codierte Information der DNA enthalten. Beim Sequenzieren der DNA wird diese in Fragmente aufgebrochen und die Sequenz kann dann durch einen automatisierten repetitiven Prozess untersucht werden. Die automatisierte Analyse des Genoms wurde auch dadurch erleichtert, dass unabhängig vom Ursprung des Gewebes, wegen der Ähnlichkeit der DNA verschiedener Spezies, dieselbe Technologie angewandt werden konnte. Dies erklärt die exponentielle Geschwindigkeit bei der Entwicklung von automatisierten Prozessen zur Sequenzierung des Genoms. Mit etwa 30.000–40.000 Genen ist das menschliche Genom mehr als doppelt so groß wie das einer Fruchtfliege.