Biomedical subjects

K Engelmann

Publications and source records attributed to K Engelmann.

At least 73 records · Page 4Linked to original sources

PubMed1996

[HLA typing of donor corneas with extended post mortem time].

In case of high-risk patients cornea transplantation should be carried out using HLA-matched donor corneas to minimize the risk of rejection. HLA typing using blood lymphocytes of the donor is impossible because of the long post-mortem times. Alternatively, HLA typing can be performed using retinal pigment epithelium (RPE). Nevertheless, this method is limited by increasing post-mortem times. The aim of the present study was the optimization of culture conditions for RPE cells isolated from donor eyes with long post-mortem times. The HLA type should be evaluated during an acceptable period of organ preservation of the corresponding cornea. In different steps the method for isolation of the cells was optimized and a growth medium for RPE cells was established. Various supplements, including uvea-conditioned medium, were assessed using growth assays. The optimization of culture conditions led to an increase in the estimation of the complete HLA I and HLA II antigens from 36% to 74%. The time needed for the typing-procedure could be reduced from 38 to 17 days (average). At present 30% of the donor tissue with long post-mortem times can be typed in less than 14 days.

Cell Division↗

PubMed1996

[Effect of differentiation on expression of genes for growth factors and growth factor receptors in human corneal endothelial cells].

In earlier publications we described a method for the isolation and long-term cultivation of human corneal endothelial cells. In several cultures we observed dedifferentiation of these cells, shown by loss of their normal polygonal shape and assumption of a fibroblastlike morphology. To reveal the role of growth factors for this dedifferentiation we analyzed the expression of genes encoding growth factors and growth factor receptors by differentiated and dedifferentiated human corneal endothelial cells. By means of northern blot analysis we demonstrated expression of the genes encoding basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF beta 1), vascular endothelial growth factor (VEGF), FGF receptor-1 (flg-type) and epidermal growth factor receptor (EGF receptor) by both differentiated and dedifferentiated human corneal endothelial cells. In addition, expression of VEGF was stimulated by supplementation of growth medium by bFGF. In contrast, expression of the gene encoding flt-1, a receptor for VEGF, was only observed in dedifferentiated and not in differentiated human corneal endothelial cells. Despite this expression of flt-1, the dedifferentiated cells showed no mitogenic response to VEGF. The role of growth factors for dedifferentiation of human corneal endothelial cells is discussed.

Blotting, Northern↗

PubMed1996

Influence of vascular endothelial growth factor on bovine corneal endothelial cells in a wound-healing model.

In this study we determined the influence of vascular endothelial growth factor (VEGF) on bovine corneal endothelial cell proliferation and wound healing. Proliferation was determined by measurement of DNA replication as well as by counting of the number of cells present after a defined growth period. In a wound-healing model, reproducible cell-free areas were created within monolayers of cultured bovine corneal endothelial cells and the migration of the cells into these areas was analyzed. The DNA replication and cell proliferation of bovine corneal endothelial cells were not influenced by VEGF. In contrast, in the wound-healing model, VEGF supplementation at concentrations of 1 and 10 ng/ml increased the cell density of the wounded area by 20% and 50%, respectively, as compared with the cell density of wounds left untreated by VEGF. Furthermore, no increase in DNA replication was found in cells involved in wound healing. Our results demonstrate that healing of bovine corneal endothelial cell layers after wounding is predominantly performed by cell migration rather than by proliferation. This migration can be stimulated by the addition of exogenous VEGF.

Animals↗

PubMed1996

Differences in proliferation and migration of corneal endothelial cells [correction of epithelial cells] after cell transplantation in vitro.

For regular function the human cornea requires an intact endothelial cell layer with a sufficiently high cell density. One approach used to compensate endothelial cell loss is transplantation of cultured corneal endothelial cells. Using a previously described transplantation protocol, we observed topographic differences after transplantation of cultured human corneal endothelial cells to recipient corneas previously denuded of their own endothelium. The results presented in this paper suggest different interactions of the transplanted endothelial cells with the central or the peripheral part of the corneal matrix, respectively. Furthermore, cells isolated from the center of a human cornea differ from those isolated from the periphery in terms of their mitogenic capacity. The significance of these observations for corneal endothelial cell transplantation is discussed.

Animals↗

PubMed1996

Detection of endotoxin in media from sterile corneal organ cultures.

Lipopolysaccharides (LPS) are elements of the cell wall of gram-negative bacteria. They are also called endotoxins and are known to stimulate various inflammatory reactions by interaction with cytokines and macrophages. Additionally, they may have a direct effect on other cells. As contamination of sterile organ-culture media with bacterial substances may influence the donor-tissue prognosis, we investigated a series of culture media drawn from organ culture for the presence of endotoxin. A total of 341 samples of sterile organ-culture media from 3 different cornea banks using either organ culture or the refrigerated-storage technique were tested for endotoxin. The assay was performed using the Limulus amebocyte-lysate test. A level of endotoxin above the background threshold was found in 99/341 (29%) culture media. The incidence of endotoxin ranged from 14% up to 50%, depending on the cornea bank and culture system used. Endotoxin detected in sterile corneal organ cultures probably derives from nonreplicating bacterial postmortem donor-tissue contamination. The presence of endotoxin-positive cultures varied between the eye banks but was not related to a given storage method. As endotoxin may directly influence graft viability or trigger inflammatory host responses, these findings may have significance for the clinical results of corneal grafting.

Cornea↗

PubMed1995

The role of exogenous/endogenous basic fibroblast growth factor (FGF2) and transforming growth factor beta (TGF beta-1) on human corneal endothelial cells proliferation in vitro.

Adult human corneal endothelial cells (HCEC) have extremely low turnover rates but undergo rapid division in vitro when stimulated with soluble growth factors. We have investigated the role played by FGF2 and TGF beta-1 in the regulation of HCEC growth stimulation. HCEC from donors who were over 30 years old were cultured and experiments performed on cultures between the 2nd and the 6th passage in the presence of 5% NCS. Cell counts revealed a maximal stimulation of 2.1x for FGF2 and 1.9x for TGF beta-1 compared to control cultures. When both factors were added, a synergistic effect was noticed with a maximal stimulation of the proliferation rate of 4.5x over controls. In addition, endogenous FGF2 produced by HCEC was quantitated in a sensitive EIA assay. After 5 days in culture, 10(6) cells contained 150 ng FGF2 and 35 ng was extracted from trypsin-digested ECM. Two molar NaCl washes of ECM released 15.6 ng FGF2, which induced a slight mitogenic activity (1.5x over control) in HCEC cultures, which was partially inhibited by an anti-FGF2 antibody. Northern blot analysis of HCEC extracts revealed the presence of FGF receptors R1 and R2 mRNA. The bioactive FGFRs were demonstrated by the toxic effect of a mitotoxin FGF2-SAP. These results suggest that FGF2 could participate in the autocrine regulation of HCEC proliferation and survival. The synergy between exogenously added FGF2 and TGF beta demonstrates that a combination of different growth factors may be important to stimulate proliferation of these cells in vivo.

Adult↗

PubMed1995

Expression of genes coding growth factors and growth factor receptors in differentiated and dedifferentiated human corneal endothelial cells.

Growth and differentiation of human corneal endothelial cells are shown to be influenced by growth factors and by the extracellular matrix. Under standard culture conditions, these cells tend to dedifferentiate to a fibroblast-like phenotype. The aim of this study was to examine the expression of genes encoding growth factors and growth factor receptors in differentiated and dedifferentiated human corneal endothelial cells and in keratocytes (stromal fibroblasts) by means of Northern blot analysis. Expression of genes coding basic fibroblast growth factor (FGF), FGF receptor-1 (flg-type), epidermal growth factor receptor, and transforming growth factor-beta 1 in keratocytes and human corneal endothelial cells could be confirmed. In contrast, significant expression of the gene encoding acidic FGF was not detected. However, expression of the genes encoding vascular endothelial growth factor (VEGF) and the high-affinity tyrosine kinase receptor for VEGF (flt-1) in human corneal endothelial cells could be demonstrated for the first time. Moreover, exogenous basic FGF seemed to have a positive influence on the VEGF gene expression level. The flt-1 gene was expressed in dedifferentiated, but not in differentiated human corneal endothelial cells. The results indicate a possible role of VEGF in the differentiation of human corneal endothelial cells.

Animals↗

PubMed1995

Incidence of bacterial and fungal contamination of donor corneas preserved by organ culture.

We reviewed the results of sterility testing from culture media of 1,134 donor corneas preserved by organ culture at 37 degrees C in our eye bank. All corneas were stored in minimal essential medium containing 2% fetal calf serum, 0.1 mg/ml penicillin G, 0.1 mg/ml streptomycin, and 2.5 micrograms/ml amphotericin B. After removal of ocular adnexal tissue, donor globes were rinsed with sterile saline solution, incubated in 3% polyvinylpyrrolidone-iodine solution for 3-5 min, and subsequently rinsed again with sterile saline solution. Samples for microbiological evaluation were obtained from the initial evaluation medium, at every medium change (every 10 days), and from the medium used for deswelling of the individual cornea 1 day before transplantation. Incidence of contamination was 0.53% (6 of 1,134 corneas). Three corneas were contaminated by Micrococcus species, three by fungi. We conclude from our study that a combination of rinsing donor globes with sterile saline solution, the initial use of a disinfectant, and the employment of penicillin/streptomycin and amphotericin B in the organ culture medium, which have been commonly used in cell culture for decades, result in a low incidence of bacterial and fungal contamination of corneas preserved by organ culture.

Bacteria↗

PubMed1995

Corneal and external disorders and refractive surgery.

A review of disorders of the anterior segment of the eye may show new avenues of research as well as clinical perspectives arising from recently accumulated data. It will have to select new theoretical and clinical findings, which may most probably be apt to survive the test of time, thus providing a long-standing stimulus in science. This body of scientific work in corneal and external disorders may be divided into infectious and noninfectious entities as well as refractive and tectonic or structural problems. In this review, we will focus on the inflammatory implications of anterior segment ocular disease, and mention some evolving diagnostic features and new treatment modalities, as well as opinions on refractive surgery complicated by inflammation.

Animals↗

PubMed1995

Growth of human corneal endothelial cells in a serum-reduced medium.

A new medium composition was established for growth of adult human corneal endothelial cells (HCEC) under serum-reduced conditions. Growth assays were performed in clonal densities with HCEC using serum-reduced culture conditions. The growth-promoting effect of different substances was tested step by step. The new serum-reduced medium was compared with the generally used medium supplemented with conventional amounts of serum. A culture medium recently described (Engelmann K, Böhnke M, Friedl P. Optimization of culture conditions for human corneal endothelial cells. In Vitro Cell Dev Biol 1989;25:1065-72) for the long-term cultivation of human corneal endothelial cells derived from adult donors was used as the starting point for the development of a new low-serum medium. It was found that the addition to the supplements ascorbic acid, insulin, selenium, transferrin, lipids, and fibroblast growth factor to the basal medium F99 (a 1:1 mixture of Ham's F12 and M199) allowed reduction of the serum content to as low as 2%. The new medium formula, called F99sr, showed an improved dose-response curve of cell growth to serum content over the range 2-40%. To ensure the maintenance of a typical endothelial cell morphology at confluence, the serum content should be 5%.

Aged↗

PubMed1993

[Quality control in the corneal bank--a necessary measure?].

BACKGROUND: During a period of 3 months 20% of patients who received a corneal transplant showed a prolonged postoperative swelling, of the stroma and an increased number of folds in the epithelium was prolonged. These findings were atypical because there were no complications during the operation and the preoperative examination of the donor cornea showed a normal corneal endothelium. All donor corneas were stored in organ culture before transplantation using minimal essential medium supplemented with 2% fetal calf serum (FCS). This method has been used in the university eye clinic of Hamburg since 1981. In order to establish any correlation between the used organ culture conditions and the clinical findings the different components of the culture medium were checked. We noticed that the serum batch used for organ preservation had been changed at the beginning of the above mentioned period. PATIENTS: From April 20th to June 23rd, 28 penetrating keratoplasties were performed at the department of ophthalmology of the University Clinic of Hamburg. No complications during the surgical procedure or postoperative infection were detectable. Four weeks after transplantation all transplants showed a normal swelling. METHODS: The quality of media or sera is not guaranteed by the company where the substances were purchased. We performed clonal growth assays using human or porcine corneal endothelial cells to evaluate the quality of different charges of fetal calf serum. RESULTS: It could be demonstrated that the acticity of the serum charge used for organ preservation during the described period had a significantly reduced activity in a clonal growth assay compared with various FCS-charges from different companies. CONCLUSIONS: After changing the serum batch the problems described above disappeared. We conclude that quality testing of all supplements of the organ culture medium is necessary in a cornea bank to guarantee a reliable standard of corneal preservation. A clonal growth assay is a sensitive test which is suggested to be useful for quality testing.

Adolescent↗

PubMed1993

[Endothelial cell transplantation in a model].

Human donor corneas that were unsuitable for transplantation were denuded of their endothelium and then used as substrates for seeding in vitro cultured human or porcine corneal endothelial cells. Only cells that expressed an endothelial-like morphology in vitro formed a monolayer on the denuded matrix whereas fibroblast-like cells showed no redifferentiation. The maximal achieved cell density was 3,500 cells/mm2 if porcine cells were used and 1,800 cells/mm2 for human cells. Adhesion of the cells was best if the subendothelial matrix was carefully prepared mechanically. Moreover, cell seeding was improved by slight centrifugation. Fibroblast growth factor (FGF), which enhanced endothelial cell proliferation in vitro, did not influence proliferation after cell transplantation, but seemed to allow better regeneration of the newly formed monolayer on the corneas.

Animals↗

PubMed1992

A comparative immunohistochemical study of human corneotrabecular tissue.

Using in situ immunohistochemical techniques and a broad panel of antibodies directed against intermediate filament proteins, vascular endothelial markers, neuroectodermal/neuroendocrine markers, and monoclonal antibodies raised against human corneal endothelial cells (HCECs), a comparative phenotypical analysis was performed on HCECs, keratocytes, trabecular cells, and cells lining the canal of Schlemm. The coexpression of cytokeratins and neurofilaments by HCECs argues in favor of a neuroectodermal origin, which is further supported by the fact that they stain positive for neuron-specific enolase (NSE) and that they express neural cell adhesion molecules (N-CAM) at their surface. The expression of NSE and N-CAM also applies to the trabecular cells. The cells lining the canal of Schlemm were found to share many immunophenotypical features with vascular endothelial cells (i.e., factor-VIII-related antigen and BMA-120), rather than with HCECs. This was further supported by the reactivity of two monoclonal antibodies (i.e., 9.3 E and 5.52 H) that were raised against HCECs, and which labelled vascular endothelium and cells lining Schlemm's canal.

Animals↗

PubMed1992

[Prerequisites for the use of preserved donor tissue in perforating and lamellar keratoplasty].

The longterm tissue culture is performed at the University Eye-Hospital of Hamburg since 1985 with a standardized procedure. The number of donor material and operations in the first 7 months of 1991 are reported. Preparation, storing and HLA-typing of the tissue are considered. The technical working up of the stored tissue for penetrating and lamellar keratoplasties will be explained.

Corneal Transplantation↗

PubMed1991

Proliferative capacity of mononuclear cells in the human lung.

In pulmonary sarcoidosis, a chronic granulomatous disorder with different stages of activity, the proliferative capacity of the alveolar mononuclear cells is unknown. To get a closer look at this proliferation we combined pulse labeling (by means of tritium thymidine incorporation and autoradiography) with an immunocytochemical staining assay. This assay revealed on one single slide simultaneously blue CD4+ lymphocytes, brown CD8+ lymphocytes and red macrophages. We were able to show that CD4+ as well as CD8+ lymphocytes were radiolabeled only in the active state of the disease. But macrophage proliferation occurred independently of the activity of the disease. In other words with this combination of techniques, it is possible to differentiate, on a single slide, three subsets of mononuclear cells, in combination with their proliferative behavior.

Autoradiography↗

PubMed1989

Optimization of culture conditions for human corneal endothelial cells.

Long-term cultivation of human corneal endothelial cells (HCEC) was optimized with respect to different components of the culture system: 25 different nutrient media, different sera, 6 mitogens and various substrates were tested in their ability to influence clonal growth and morphology of HCEC. F99, a 1:1 mixture of the two media M199 and Ham's F12, was the most effective basal medium in promoting clonal growth of HCEC. Among various sera, human serum and fetal bovine serum showed optimal growth promoting activities in combination with F99, whereas newborn bovine serum (NBS) was by far superior for the development of a typically corneal endothelial morphology. Crude fibroblast growth factor (FGF), or alternatively endothelial cell growth supplement, was absolutely essential for clonal growth of HCEC at low serum concentrations, for example 5% NBS. Formation of a monolayer with a morphology similar to corneal endothelium in vivo was observed only on culture dishes coated with basal membrane components such as collagen type IV, laminin, or fibronectin. The most pronounced effect on morphologic appearance was obtained by culturing the cells on the extracellular matrix (ECM) produced by bovine corneal endothelial cells. Moreover, ECM could substitute for crude FGF in clonal growth assays.

Animals↗

PubMed1989

Life span of human corneal endothelial cells in long-term cultures.

Human corneal endothelial cells (HCEC) were isolated from excised corneas of adult donors in the age range of 28-70 years, and successfully grown for 10-12 passages. Moreover, one cell line, that had been established from a 45-year-old donor, was further passaged at defined split ratios in order to determine the proliferative life span of the HCEC isolated from donors of this age group.

Adult↗

PubMed1989

[Growing human corneal endothelium in cell culture].

Enzyme treatment of the corneal endothelium with collagenase was employed in a new method of isolating human corneal endothelial cells (HCEC) from the corneas of adult donors (30-70 years). It was possible to isolate 5-10 X 10(4) endothelial cells from each cornea, and primary cultures were established. During the first 3-5 passages, the growth of contaminating fibroblast like cells was inhibited by the use of an L-valine-free selective culture medium. Proliferation of the cells was stimulated by supplementation of the culture medium with fibroblast growth factor and also by coating the culture dishes with a mixture of laminin and chondroitin sulfate. After 3-6 passages, the cell number had increased 150-200 times. During the passage the cells changed with regard to their typical morphology. This change depended on the amounts of serum and mitogen, respectively, in the culture medium, as well as on the in vitro age of the cells.

Adult↗