M. Knorr

Paraproteinemic Corneal Deposits in Plasma Cell Myeloma

We treated two patients who had IgG-kappa-light chain monoclonal gammopathy with partially crystalline and partially amorphic corneal deposits. Impairment of vision made keratoplasty necessary for each patient. Histologic examination of the corneal specimens showed deposits that stained positively for Massons trichrome in all corneal cells. Immunohistochemical tests identified these deposits as IgG-kappa-light chain immunoglobulin fragments. Electron microscopy showed intracellular, rhomboid-shaped deposits enveloped by a membrane. The same deposits appeared in the conjunctival epithelium, within subconjunctival fibrocytes, and in the plasma cells of the bone marrow. Immunoelectrophoresis showed IgG-kappa-light chain fragments in the blood serum, the lacrimal film, and the aqueous humor. This suggests that the intracellular immunoglobulin fragments may have entered the corneal and conjunctival epithelium by way of the lacrimal film, the keratocytes by way of the corneo-scleral limbus vasculature, and the endothelial cells from the aqueous humor.

Effect of growth factors on the activation of human Tenon’s capsule fibroblasts

Purpose. To investigate stimulatory effects of PDGF-AA, PDGF-AB, PDGF-BB, bFGF, IL-1ß, TGF-ß1 and TGF-ß2 on the proliferation and myofibroblast transformation of cultured human Tenon’s capsule fibroblasts and to characterize expression of PDGF- and TGF-ß-receptors in these cells. Methods. To determine cell proliferation, cell number of 2nd passage cultured human Tenon’s capsule fibroblasts was measured before and after addition of growth factors using a computer-based cell counter system. Immunoblotting was used to detect and quantitate a-smooth-muscle actin (a-SMA) expression. Expression of PDGF- and TGF-ß-receptor mRNA was detected by RT-PCR, expression of the corresponding protein was demonstrated using Western blot. Results. A significant increase in proliferation (p = 0.05) was detected after exogenous stimulation with PDGF-AA (10ng/ml and 100ng/ml), PDGF-AB (10ng/ml and 100ng/ml), PDGF-BB (10ng/ml and 100ng/ml), bFGF (100 ng/ml), IL-1ß (1 ng/ml and 10 ng/ml), TGF-ß1 (0.5 ng/ml) and TGF-ß2 (0.5 ng/ml). Both TGF-ß1 and TGF-ß2 stimulated expression of a-SMA in a dose dependent manner with peak activity at a concentration of 50 ng/ml (TGF-ß1) and 500 ng/ml (TGF-ß2). Protein and mRNA of PDGF-receptor type a and type ß and TGF-ß-receptors type I, II and III are expressed in cultured human Tenon’s capsule fibroblasts. Conclusions. The present investigation strongly supports the hypothesis that PDGF-isoforms are major stimulators of proliferation of Tenon’s capsule fibroblasts after glaucoma filtering surgery while TGF-ß-isoforms are essential for the transformation of Tenon’s capsule fibroblasts into myofibroblasts.

Lens epithelial cell response to isoforms of platelet-derived growth factor

It has been reported previously that platelet derived growth factor (PDGF) may play an important role in the regulation of lens growth and differentiation. To evaluate PDGF-induced effects at the cellular level, we investigated the response of cultured bovine lens epithelial cells (BLEC) to PDGF-AB, -AA, and -BB isoforms at the cellular level. Stimulation of BLEC with PDGF isoforms showed no increase in cell proliferation under the culture conditions of this study. In contrast, measurement of cytosolic free calcium concentration ([Ca2+]i), which has been shown to be an important second messenger for controlling multiple cellular processes in the lens, revealed a dose-dependent rise in [Ca2+]i upon stimulation with PDGF-AB and -BB isoforms. PDGF-AA used in similar concentrations was not effective. Our data suggest that PDGF-AB and -BB may play a role in the regulation of cellular functions in BLEC via modulation of intracellular calcium homeostasis.

Proliferative response of cultured human tenon's capsule fibroblasts to platelet-derived growth factor isoforms

Abstract• Background: Although platelet-derived growth factor (PDGF) has been thought to be critical in the wound-healing response of Tenons capsule fibroblasts after glaucoma filtration surgery, no information is currently available concerning the proliferative effect of PDGF isoforms on this cell type. The aim of the present study was to evaluate the proliferative effect of PDGF-AB heterodimer and PDGF-AA and -BB homodimers on cultured human Tenons capsule fibroblasts. • Methods: Human Tenons capsule fibroblasts, cultured under serum-free conditions, were stimulated with PDGF-AA, -AB and -BB isoforms in concentrations ranging from 1 to 100 ng/ml. Cell numbers were determined on days 1, 3, 5 and 7, using a cell counter. • Results: Addition of PDGF-AB and -BB led to a dosedependent increase in cell proliferation. A maximal response (79.9% over control) was obtained after 7 days with 30 ng/ml of PDGF-BB, with an EC50 of 8.9 ng/ml. The maximal increase in cell proliferation caused by PDGF-AB (30 ng/ml) was 54.9%, with an EC50 of 12.5 ng/ml. Stimulation with PDGF-AA revealed a significant effect only with concentrations higher than 30 ng/ml. • Conclusion: Our results indicate that PDGF-AB and -BB isoforms are potent stimulators of proliferation of human Tenons capsule fibroblasts, suggesting that PDGF-AB and -BB isoforms play an important role in the wound-healing response after glaucoma filtration surgery.

The in vitro effect of platelet-derived growth factor isoforms on the proliferation of bovine corneal stromal fibroblasts depends on cell density

Abstract• Purpose: Recently, it has been shown that corneal stromal fibroblasts express the mRNA for PDGF-β-type receptors, while corneal epithelial cells express the mRNA for the PDGF B-chain, suggesting a role of PDGF isoforms in the regulation of corneal homeostasis and wound healing via an unidirectional epithelial to stromal paracrine interaction. The purpose of this study was to characterize the proliferative response of cultured bovine corneal stromal fibroblasts to PDGF isoforms. • Methods: Bovine corneal stromal fibroblasts were seeded at a cell density of 60 cells/mm2 (low density) and 120 cells/mm2 (high density) and were cultured under serum-free conditions. Except for corresponding controls, PDGF AA, BB and AB (obtained by separate expression of cloned genes inE. coli) were added in concentrations ranging from 3.125 to 100 ng/ml. Cell numbers were determined after an incubation period of 6 days using a cell counter. • Results: Stromal fibroblasts, when cultured at a high density, revealed constant cell numbers during the whole incubation period. Under these culture conditions, stimulation with PDGF AA, BB and AB led to a significant dose-dependent increase in cell proliferation. When cultured at a low cell density, stromal fibroblasts revealed a significant reduction of cell numbers after 6 days of incubation. This reduction was prevented by PDGF AA and AB isoforms in a dose-dependent manner. In contrast, PDGF BB was not effective. • Conclusion: The results of the “high-density” assays suggest that PDGF isoforms act as mitogens for stromal fibroblasts during wound healing, when density of fibroblasts is high. The results of the “low-density” assays support the idea that PDGF AA and AB can prevent cell loss during corneal homeostasis when density of keratocytes is low.

Effect of heparin on human corneal fibroblast proliferation in vitro with and without growth factor stimulation

Abstract · Background: The outcome of keratorefractive procedures such as PRK and LASIK is limited by the wound-healing process in the corneal stroma, which gives rise to complications such as haze formation and regression. The proliferation and matrix synthesis of corneal stromal fibroblasts is the central element of the wound-healing process. In order to develop new therapeutic strategies to reduce wound-healing intensity, we investigated the effect of heparin on the proliferation of cultured human corneal stromal fibroblasts (HCF) alone and in the presence of growth factors. · Methods: Primary cultures of HCF were established using epithelium and endothelium-free explants. Secondary cultures of HCF (first passage), cultured in WM/F12 supplemented with 10 µg/ml transferrin and 10 µg/ml thyroglobulin (LR-1 medium), 1% fetal calf serum (FCS) and 10% FCS were used to determine the effect of heparin on the proliferation of HCF in concentrations ranging from 12.5 µg/ml to 5000 µg/ml. Cell number was determined using the CASY 1 cell counter system. Modulation of HCF proliferation by heparin (50 µg/ml and 2000 µg/ml) was also investigated under serum-free conditions and in the presence of bFGF, EGF and PDGF-BB. · Results: Addition of heparin led to a dose-dependent inhibition of proliferation after 6 days of incubation, which was statistically significant for 500–5000 µg heparin/ml (FCS 1%) and for 200–5000 µg heparin/ml (FCS 10%). IC50 values for this effect were determined to be approximately 700 µg heparin/ml. When cultured under serum-free conditions (LR-1), a significant reduction of cell number was only observed with 5000 µg heparin/ml. There was no significant modulation of PDGF-BB-, bFGF-, or EGF-stimulated cell proliferation by heparin at concentrations of 50 µg/ml and 2000 µg/ml after 6 days of incubation. · Conclusion: Our observations indicate that heparin can inhibit proliferation of HCF effectively. The results of the present study could eventually pave the way to prevent anterior stromal haze formation and regression after keratorefractive surgery.

Argon laser therapy of benign tumors of the eyelid

PURPOSE To report the therapy of benign eyelid tumors with an argon laser as an alternative to surgery. METHODS Forty-one patients with 47 benign tumors of the eyelid were included in this study. In all patients, the eyelid tumor was eliminated by argon laser. In 24 cases the lower eyelid and in 23 cases the upper eyelid was involved, including the lid margin in 17 cases. Laser spot size ranged from 150 to 500 microm. Argon laser power density varied between 4.1 and 61.1 W/mm2. Spots were counted between 40 and 1204. Twenty-seven of 41 patients treated were followed up; the mean follow-up period was 5.8 months (range, 3 to 24 months). Postoperatively, histologic confirmation was obtained in 42 cases of the 47 treated tumors. RESULTS All patients were satisfied regarding the laser therapy and the cosmetic result. Remarkably, the wounds were dry after argon laser therapy. No infections of the wounds occurred; wounds were epithelialized after 2 to 3 weeks by a normal-appearing epithelium. The area of argon laser treatment was less pigmented than the surrounding skin and showed no obvious remarkable notches. As a complication, one patient developed a viral conjunctivitis. No relapses occurred during follow-up. CONCLUSIONS Argon laser therapy of benign eyelid tumors may result in very satisfactory wound healing. Taking the short follow-up and the limited number of cases into account, it seems to be a useful alternative to traditional surgery, especially for tumors positioned close to the lacrimal papillae.

Serum-free cultivation of bovine lens epithelial cells

Mammalian cells in culture have individual nutritional requirements which are mainly fulfilled by the addition of fetal calf serum to the basic culture medium. Since many of the serum components are as yet poorly understood or even completely unknown, a number of difficulties arise in the evaluation of the effect of exogenously added factors or drugs on the growth of the cells. This paper presents data demonstrating the successful adaptation, routine cultivation and cryopreservation of bovine lens epithelial cells (BLEC) under serum-free culture conditions by use of the commercially available serum substitutes BMS, BM 86 Wissler and Ultroser G. Moreover, a culture medium especially designed for cell quiescence, named LR-1, is presented. Cells cultivated in culture medium containing 10% fetal calf serum served as controls. While BM-86 Wissler caused significantly reduced growth rates within 2 days and, finally, cell death after 12 days of incubation, the use of BMS resulted in growth rates which did not differ from the corresponding controls. Ultroser G resulted in a significant increase of proliferative activity of BLEC. LR-1 medium caused cell quiescence and kept the cells alive for a number of days. Thus, LR-1 allowed evaluation of the response of the cells to a mitogenic mixture from bovine brain mainly containing endothelial cell growth factor. The results demonstrate that cultivation of BLEC is possible under serum-free culture conditions. Moreover, the medium LR-1, which causes cell quiescence, is useful for the evaluation of growth factor-induced effects in vitro.

Sind filtrierende Eingriffe bei Glaukompatienten mit ausgedehnten Gesichtsfeldausfällen mit einem größeren funktionellen Risiko verbunden

ZusammenfassungHintergrund: Untersucht wurde die Inzidenz eines Visusverlustes durch einen Ausfall des zentralen Gesichtsfeldes und der foveolaren Fixation in der ersten Woche nach einer filtrierenden Glaukomoperation. Patienten: In die Studie wurden 408 Patienten eingeschlossen, bei denen von Januar 1993 bis April 1997 an der Unversitätsaugenklinik Tübingen eine filtrierende Glaukomoperation durchgeführt wurde und deren Verlauf bis 12 Monate (± 3 Monate) postoperativ bekannt war. Die Auswertung der präoperativen, intraoperativen und postoperativen Daten erfolgte retrospektiv. Ausgeschlossen wurden alle Patienten, bei denen keine ausreichende Verlaufskontrolle durchgeführt werden konnte, bei denen gleichzeitig eine Kataraktextraktion durchgeführt und bei denen weitergehende operative Eingriffe (z.B. Moltenoimplantat) eingesetzt wurden. Ergebnisse: Bei 404 Patienten (99,3%) fand sich kein Visusverlustes durch einen Ausfall des zentralen Gesichtsfeldes und der foveolaren Fixation in der ersten Woche nach einer filtrierenden Glaukomoperation. Von diesen Patienten wiesen 11 eine deutliche postoperative Visusreduktion (>2 dB) auf, die durch eine Zunahme der Linsentrübung erklärt war, bei einem Patienten zeigte sich eine progressive altersbedingte Makulopathie. Bei einem Patienten (0,2%) fand sich ein progredienter relativer Gesichtsfeldausfall im zentralen Bereich. Bei 2 Patienten (0,5%) ließ sich bereits unmittelbar nach der Operation ein Verlust der Fixation und ein Verlust des zentralen Gesichtsfeldes nachweisen. Schlussfolgerung: Ein Verlust der Sehschärfe durch Einbruch des zentralen Gesichtsfeldes und der zentralen Fixation unmittelbar nach einer filtrierenen Operation ist eine seltene Komplikation. Selbst fortgeschrittene Gesichtsfeldausfälle sind daher keine Kontraindikation für eine filtrierende Glaukomoperation.SummaryBackground: We evaluated the prevalence of the loss of visual acuity due to loss of the central portion of the visual field and foveolar fixation in the first week after glaucoma filtering surgery. Patients and methods: We included 408 patients, in whom glaucoma filtering surgery was performed between January 1993 and April 1997 at the University Eye Clinic in Tübingen and who had completed 1-year follow-up examinations. The retrospective evaluation included preoperative, intraoperative and postoperative data. We excluded all patients who did not complete 1-year follow-up examinations (12±3 months), who have died during the 1-year follow-up, who had combined glaucoma and cataract surgery or in whom the Molteno implant procedure was performed. Results: A total of 404 patients (99.3%) did not suffer loss of the central visual field and foveolar fixation in the first week after glaucoma filtering surgery. In 11 cases, loss of visual acuity >2 dB was due to progressive lens opacification. One patient suffered from postoperative progression of his age-related maculopathy. In one patient (0.2%) progression of a preexisting relative central scotoma occurred immediately after the operation. Two patients (0.5%) suffered from loss of fixation and the central visual field immediately after glaucoma filtering surgery. Conclusions: Loss of the central visual field and central fixation immediately after glaucoma filtering surgery is a rare complication. Therefore, glaucoma filtering surgery can also be recommended for patients with advanced visual field defects.

Influence of a lymphocyte-conditioned medium on the expression of smooth-muscle α-aktin in lens epithelial cells in situ

SummaryPurpose: Contraction of the capsule of the ocular lens is based upon proliferation and contraction of transformed lens epithelial cells. It is assumed that these processes can be assisted by postoperative intraocular inflammation. Previously, we reported that lens epithelial cell proliferation is enhanced by lymphocyte-conditioned medium (LCM). In this study we investigated the effect of LCM as well as of a culture medium conditioned by pigmented ciliary epithelial cells (CBCM) on the expression of the smooth-muscle alpha-actin of the contractile cytoskeletal elements. Methods: Explants of the anterior lens capsule of freshly enucleated bovine eyes were cultured in serum-free LCM and CBCM for 3 days, followed by fixation. Smooth-muscle alpha-actin was identified by indirect immunofluorescence. Explants cultured in serum-free bFGF-containing and TGF-β-containing medium served as control. Results: Lens epithelial cells expressed smooth-muscle alpha-actin under the influence of LCM or TGF-β. No smooth muscle alpha-actin could be detected under the influence of CBCM or bFGF. Conclusion: Our results demonstrate that secreted molecules of activated lymphocytes are able to induce the transformation of lens epithelial cells into contractile myofibroblasts and may be involved in the postoperative contraction of lens capsules.ZusammenfassungHintergrund: Zellbiologische Grundlage der posterioren Kapselfältelung sowie der sog. vorderen Kapselschrumpfung nach Kataraktextraktion ist die Proliferation und Kontraktion der postoperativ im Kapselsack verbliebenen Linsenepithelzellen. Vermutet wird, daß postoperativ persistierende Entzündungen diese Veränderungen begünstigen. Nachdem wir früher zeigen konnten, daß ein Lymphozyten konditioniertes Medium (LKM) die Proliferation von Linsenepithelzellen stark stimuliert, wurde in der vorliegenden Arbeit untersucht, inwieweit die Expression des kontraktilen Zytoskelettelements alpha-Aktin durch das LKM begünstigt wird. Zusätzlich wurde der Einfluß eines durch pigmentierte Ziliarkörperepithelzellen konditionierten Mediums (ZKKM) untersucht. Material und Methode: Anteriore Kapselexplantate schlachtfrischer Rinderaugen wurden 3 Tage in serumfreiem LKM bzw. ZKKM kultiviert. Im Anschluß daran erfolgte an fixierten Kapselexplantaten der Nachweis glattmuskulären alpha-Aktins mittels indirekter Immunfluoreszenz. Als Kontrollen dienten in serumfreiem, bFGF-haltigen oder TGF-β-haltigem, aber unkonditioniertem Medium kultivierte Explantate. Ergebnisse: Unter dem Einfluß des LKM bzw. TGF-β exprimierten Linsenepithelzellen glattmuskuläres alpha-Aktin. Der Nachweis dieser Zytoskelettkomponente fiel dagegen bei Linsenepithelien, die in bFGF-haltigem, unkonditioniertem Medium oder in ZKKM inkubiert worden waren, negativ aus. Schlußfolgerung: Die vorliegenden Untersuchungen zeigen, daß Sekretionsprodukte aktivierter Lymphozyten die Expression glattmuskulären alpha-Aktins als zelluläres Zeichen einer myofibrillären Transformation stimulieren und somit an der Induktion einer Linsenkapselschrumpfung beteiligt sein können.