Wolfgang Göttinger

Effective methods for the investigation of human tear film proteins and lipids

The investigation of human tear film proteins and lipids is of value for the elucidation of contact lens incompatibilities, tear film instabilities, dry eye syndrome and various other eye diseases. Improved efficient methods for the investigation of human tear film proteins and lipids are presented in this paper. Tear proteins were examined by ultrathin sodium dodecyl sulfate-polyacrylamide gel electrophoresis, celluloacetate gel, isoelectric focusing, and high-performance liquid chromatography. The methods differ in sensitivity, resolution, convenience and reproducibility. Tear lipids were examined by high-performance thin-layer chromatography, and good separation into the major lipid classes was achieved. With this method it is possible to examine the lipids present in tears of an individual subject and not just in pools made up from the tears of several persons.

Histological and immunohistochemical findings after laser in situ keratomileusis in human corneas

Purpose: To describe histopathological and immunohistochemical findings in human corneas after myopic laser in situ keratomileusis (LASIK) followed by iatrogenic keratectasia and after hyperopic LASIK. Setting: Department of Ophthalmology, University of Innsbruck, Innsbruck, Austria. Methods: Clinical, histological, and immunohistochemical investigations were performed of 1 human cornea with iatrogenic keratectasia following myopic LASIK and 1 human cornea with irregular astigmatism and central scar formation after hyperopic LASIK. Corneal buttons were obtained during penetrating keratoplasty in both patients. Results: Histopathological examination showed thinning of the central stroma with a posterior residual thickness of 190 &mgr;m in the patient with iatrogenic keratectasia after myopic LASIK and significant midperipheral thinning in the patient who had hyperopic LASIK. However, this characteristic ablation profile of the stroma after hyperopic LASIK was partially mitigated and compensated by the epithelium, which was significantly thinned in the center and markedly thickened in the midperiphery. Traces of wound healing with minimal scar tissue were present at the flap margin after myopic and hyperopic LASIK. In a few sections of the cornea with keratectasia after myopia LASIK, only a few collagen lamellae were visible crossing between the posterior residual stroma and the superficial flap. Immunohistochemical examination revealed minimally increased staining of dermatan sulfate proteoglycan within the stroma adjacent to the interface of the microkeratome incision. Increased staining of hepatocyte growth factor was found on keratocytes/fibroblasts at the flap margin in both corneas. Conclusions: The wound‐healing response is generally poor after LASIK, which may result in significant weakening of the tensile strength of the cornea after myopic LASIK, probably due to biomechanically ineffective superficial lamella. After LASIK in patients with high hyperopia, compensatory epithelial thickening in the annular midperipheral ablation zone might be partly responsible for regression.

Analysis of human tear proteins by different high-performance liquid chromatographic techniques

A comparison of the efficiencies of hydrophobic interaction chromatography, ion-exchange chromatography, reversed-phase chromatography and gel permeation chromatography in the separation of tear proteins was made using a variety of different buffers. Separation of immunoglobulins, lactoferrin, albumin, PMFA (protein migrating faster than albumin) and lysozyme was accomplished by gel permeation chromatography in less than 30 min using a TSK-type SW3000 column equilibrated with ammonium acetate buffer (pH 4.1) with a high reproducibility. When gel permeation chromatography was used as a completely automated diagnostic method, only minute volumes (1.0 microliter) of tear samples were necessary for the quantitative analysis of proteins. The other three methods proved to be more suitable for the preparation of individual tear proteins but were less suitable for their quantitation.

Retinal Pigment Epithelial Phagocytosis and Metabolism Differ from Those of Macrophages

The purpose of this study is to compare primary human retinal pigment epithelium (RPE) cells with respect to particle uptake and further processing steps with immunological phagocytes for a better understanding of the possible role of RPE cells in triggering autoimmune diseases in the eye. We investigated the similarities of human RPE and monocytes/macrophages studying the uptake of fluorescein- and europium-labeled synthetic microparticles and microbial pathogens by human and bovine RPE cultures and a permanent RPE cell line (CRL). The uptake was monitored by laser scanning microscopy, flow cytometry and time-resolved fluorescence analysis; for comparison, macrophages and a macrophage-like cell line (MonoMac6) were used. A size-dependent uptake was seen in primary RPE cultures as well as in CRL, showing a preferential uptake of smaller beads followed by Staphylococcus aureus and Escherichia coli. Opsonization with serum caused a modest increase in bacteria uptake, but in contrast to macrophages, the classical complement receptors were not found on RPE cells. Living bacteria were also ingested in a time-dependent manner, but, as no intracellular overgrowth was observed, we further investigated the oxidative ability of RPE as a possible mechanism for microbial suppression. Unlike macrophages/granulocytes, no respiratory burst was detected in RPE cells, but, comparable to MonoMac6, IFN-γ induced neopterin in the human RPE. Interestingly a diurnal rhythm of phagocytosis was observed which was influenced by light exposure suggesting that RPE cells maintain their circadian rhythm also in cell culture to a certain extent. This study further demonstrates that in addition to similar phagocytic properties the RPE still shows substantial metabolic differences in comparison to blood-derived phagocytes.

Susceptibility of human retinal pigment epithelial cells to different viruses

Abstract• BackgroundDifferent viruses have been reported to be involved in retinal diseases in animalsystems. In humans, herpes simplex virus and cytomegalovirus have been found to cause retinal disease. Most of the studied viruses are neurotropic. In this study, the in vitro susceptibility of human retinal pigment epithelial cells (RPEC) to representative members of different groups of human pathogenic viruses was investigated.• MethodsEarly cultures of RPE C — after two or three passages — were infected with the following viruses: herpes simplex virus (HSV) type 1, human herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus types 1 and 7, measles virus, parainfluenza virus and coxsackie virus B3.• ResultsCultures of RPE C could be infected with neurotropic viruses like HSV or measles virus as well as with typical respiratory viruses like parainfluenza or adenoviruses. Coxsackievirus, an enterovirus, replicated as well as human CMV whereas EBV and HHV-6, two lymphotropic viruses, failed to infect RPE.• ConclusionThese findings suggest that a variety of viruses, including those causing rather common illnesses, might be capable of inducing retinal lesions under certain circumstances due to haematogenous spread during the course of viraemia.

Fibroblast growth-promoting activity in proliferative vitreoretinopathy: antagonism by acetylsalicylic acid

Proliferative vitreoretinopathy is a severe reactive process which leads to the formation of cellular membranes on the surface of the retina and in the vitreous. We determined the fibroblast growth-promoting activity of intraocular fluid from patients suffering from proliferative vitreoretinopathy, retinal detachment or cataract and further evaluated the effect of acetylsalicylic acid on growth-stimulated fibroblasts. The results demonstrated a significant enhancement of growth-promoting activity of intraocular fluid in proliferative vitreoretinopathy as compared to that of control samples. We showed that the augmented growth-promoting activity of intraocular fluid in proliferative vitreoretinopathy was significantly antagonized by inhibition of cyclooxygenase with acetylsalicylic acid (ID50 approximately 5 microM). In contrast, no significant effect was seen in corresponding control experiments. The findings suggest that metabolites of the cyclooxygenase pathway are involved in the regulation of enhanced intraocular fluid-induced fibroblast proliferation in proliferative vitreoretinopathy and that acetylsalicylic acid might be useful as an antiproliferative agent in intraocular fibrogenesis.

Intraocular lens power calculation after decentered photorefractive keratectomy

A 59-year-old patient who had photorefractive keratectomy (PRK) to correct high unilateral myopia developed a progressive nuclear cataract. Phacoemulsification and intraocular lens (IOL) implantation were performed. However, determination of IOL power using automated keratometry and computerized videokeratography was not successful in this case of high axial myopia because of a decentered ablation zone, resulting in too-steep keratometric readings. Postoperative hyperopia could only be corrected by an IOL exchange. Because it may not be possible to determine the exact keratometric values for IOL calculation after PRK, subtracting the change in refraction induced by PRK from the preoperative keratometric readings might have been more accurate in this patient.

Cost-benefit analysis of diabetic eye disease.

Diabetic retinopathy is the main cause of blindness in adults 25-74 years of age in Western countries. At 100% diagnosability and 100% treatability, with laser photocoagulation vision can be retained in at least one eye in 73% of patients with proliferative retinopathy and in 67% of patients with diabetic maculopathy. The cost-benefit analysis draws a comparison of the costs incurred through benefits granted to a blind diabetic and those incurred through proper screening, examination and treatment to avoid blindness as much as possible. These calculations are valid for the State of Tyrol in Austria. The anticipated annual costs for blindness are thus ATS 19,000,000, of which ATS 14,600,000 could be avoided through an optimal screening, examination and therapy program. The maximum costs for examination and therapy amount to ATS 10,700,000, thus giving a minimum saving of ATS 3,900,000 in favor of preventive medicine.

Elevated levels of substance P in intraocular fluid in proliferative vitreoretinopathy

Proliferative vitreoretinopathy is the most common reason for failure in retinal reattachment surgery. Since both substance P (SP) and SP receptors were found to be present in the human eye, and as pharmacological studies suggest an importance of SP for ocular functions, we investigated intraocular fluids for the presence of SP in eyes elected for cataract surgery, retinal detachment surgery and retina surgery for severe proliferative vitreoretinopathy (PVR) as well as in eyes with proliferative diabetic retinopathy (PDR). High performance liquid chromatography and radioimmunoassay (RIA) for SP immunoreactivities were performed. The SP mean concentration in intraocular fluid (IOF) of patients for cataract surgery (CS) was 2.2 fmol/ml, for retinal detachment (RD) was 2.7 fmol/ml and for PDR was 1.9 fmol/ml; significantly higher levels (mean concentration of 26.9 fmol/ml) were measured in eyes with PVR. HPLC analysis revealed two immunoreactive peaks coeluting with synthetic SP and SP-sulfoxide, indicating that RIA values represent authentic SP. We conclude that SP may play an important role in PVR. Since SP antagonists are known to inhibit a variety of SP effects in the eye, there might be a useful tool to reveal the importance of SP in this disease and, in this instance, a new possible treatment.

Phagocytosis of Human Retinal Pigment Epithelial Cells: Evidence of a Diurnal Rhythm, Involvement of the Cytoskeleton and Interference of Antiviral Drugs

Retinal pigment epithelial (RPE) cells provide crucial functions for the maintenance of the retinal environment. We investigated the phagocytotic mechanisms of RPE cells evaluating the question whether particle uptake underlies a diurnal rhythm. Additionally, a possible connection of volume regulation and the phagocytotic function of RPE cells was studied. As antiviral nucleoside analogues influence cell-volume-regulating mechanisms, we tested several antiviral drugs. Cultured primary RPE cells and a permanent cell line (ARPE-19) were tested for uptake of europium-labeled microspheres quantified by time-resolved fluorometry. Cells were also exposed to cyclic illumination or continuous light and dark culture conditions. Inhibitors of cytoskeleton (microtubuli, actin) and osmotic swelling were also tested. Ingested FITC-labeled microparticles were found in phagosomes strongly associated which the cytoskeleton as they could not be easily moved by laser tweezer microscopy. Phagocytosis was observed predominately during dark intervals and was reduced by continuous light exposure. The diurnal rhythm of unsynchronized RPE cultures was abolished by microtubule inhibitors although no inhibition of overall particle uptake by cytoskeletal blockers was observed. Hypoosmotic swelling of RPE also decreased phagocytosis. Acyclovir was found inhibitory in ARPE-19 cells, whereas azidothymidine showed a protracted inhibiting activity on primary RPE cells and ganciclovir was inactive in both cell types. The presence of a diurnal rhythm also in culture indicates genetic determination of light-regulated particle uptake. This mechanism appears to be influenced by the regulation of cell volume and microtubule function. Inhibition of RPE function by antiviral drugs is a novel finding and in accordance with interferences of the tested drugs with cellular chloride channels described earlier. It may give a hint towards possible ocular side effects in the long-term use of nucleoside-analogous substances.