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Background: It has been suggested that variations in the quality of organ culture preservation media are responsible for variations in early postoperative graft morphology. Spates of such variations have been observed repeatedly for short periods. This paper reports the results of a series of grafts with low postoperative clearing observed during a period of 6 weeks. Simultaneously, preoperative phase-contrast microscopy evaluation of the corneal endothelium revealed that an unusually large proportion of donor corneae were unsuitable for transplantation., Methods: The corneal storage media were therefore rigorously screened, paying particular attention to specific components and properties of the medium, including L-glutamine, amphotericin B, water quality, pH, and the glassware used. Possible toxic effects were identified by means of a sensitive growth assay performed using isolated human corneal endothelial cells., Results: The evaluation demonstrated that both the water quality and the L-glutamine which had been used for preparation of the medium were substandard during the period in which poor clinical results were obtained., Conclusion: It is recommended that cornea banks undertaking long-term organ culture use standardized protocols and carefully monitored equipment. The quality of the basal media and supplements should be routinely checked.

Several methods for isolation and cultivation of human corneal endothelial cells have been described during the last few decades. In contrast to the situation in vivo, the cultured cells show mitogenic activity but often lose their typical morphological appearance. In this paper, we describe a technique to isolate and cultivate morphologically unchanged endothelium from the human cornea. This method revealed different characteristics of endothelial cells according to their position within the human cornea. Endothelial cells isolated from the central part have a morphology similar to that of cells in vivo (i.e., they are densely packed and show no mitogenic activity). In contrast, endothelial cells derived from the peripheral part of the cornea are characterized by mitogenic activity but their cell-to-cell attachment seems to be less tight than in vivo. The significance of these two different endothelial cell types for wound healing in the human cornea is discussed.

Background/aims: Bacterial endotoxin is a potent inflammatory stimulator, the local and systemic responses thereby elicited being mediated via the release of cytokines from diverse cell types. Under physiological conditions, the corneal endothelium is protected from these toxins by the epithelial and vascular barriers, but in organ culture these safeguards are no longer operative, and such substances will therefore have ready access to this cell layer. The consequences of such exposure may take the form of overt damage to the endothelium and/or a more discreet influence on the cornea's immunological status, the effects of which may be realised only after transplantation, by its poor performance. The media bathing organ cultured donor corneas were monitored for the presence of various cytokine mediators of the inflammatory response before and after incubation with endotoxin, and these data compared with those pertaining to endothelial cell morphology and numerical density., Methods: Six pairs of fellow donor corneas were cultured for an initial equilibration period of 10 days and then transferred to fresh medium; thereafter, one of each pair was incubated in the absence, and the other in the presence, of endotoxin (50 micrograms/ml = 25,000 units/ml), and culturing continued for a further 10 days. Samples of medium were withdrawn at regular intervals throughout the 20 days and screened for the cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, GM-CSF, AND TNF by ELISA; endothelial cell morphology and area density were assessed on days 0, 10, and 20., Results: Spiking of organ culture media with endotoxin led to a substantial increase in the level of IL-8, and a smaller one in that of IL-6, but none of the other cytokines were detected. In five of the six stimulated corneas, these changes coincided with an increased incidence of endothelial cell loss, compared with that incurred by the fellow control, and the surviving population also evinced signs of degeneration not seen in the latter., Conclusion: Endotoxin induced increases in the levels of IL-6 and IL-8 appear to be correlated with endothelial cell loss. Since no adverse effects of this toxin on long term cultured monolayers of human corneal endothelial cells have been previously observed, the damage incurred in corneal organ culture may well be attributable to the influence of cytokines produced by other corneal cells or a non-intrinsic (passenger) cell population, such as macrophages, Langerhans cells or lymphocytes present under these latter conditions.

Purpose: To assess whether the biological quality of corneoscleral tissue dissected in situ is, after organ culture, comparable to that harvested after enucleation., Methods: Corneoscleral discs were prepared from 23 donor eyes, either after enucleation, under laminar flow conditions (right eyes; group 1) or by direct excision in situ (left eyes; group 2). Endothelial cell counts were made and the degree of tissue contamination assessed both prior to and upon termination of organ culture., Results: Microbial growth was found in 12/22 conjunctival swabs collected from group 1 eyes and in 14/22 of those obtained from group 2 globes (p = 0.76). Bacterial growth was detected in four primary culture media, two from each group, at low colony densities. No significant difference in endothelial cell counts were encountered between the two groups, either immediately after dissection [group 1: 2940 +/- 308 (2100-3500) c/mm2; group 2: 2947 +/- 345 (2200-3700) c/mm2; p = 0.945] or upon termination of organ culture [group 1: 2646 +/- 321 (1895-3200); group 2: 2723 +/- 312 (2100-3650); p = 0.413]., Conclusion: Dissection of corneoscleral discs in situ may serve as an alternative to the conventional technique if consent is obtained to remove only the cornea. The risk of contamination is no higher and endothelial cell viability no lower than in tissue derived from enucleated globes, provided that the excision is performed by a skilled surgeon and a rigorous disinfection protocol is instigated.

Purpose: In a comparative study we investigated the effects of epidermal growth factor, acidic and basic fibroblast growth factor, transforming growth factor-beta1, and vascular endothelial growth factor on proliferation and migration of cultured bovine corneal endothelial cells during wound healing., Methods: Cell proliferation was determined by incorporation of 5-bromo-2'-deoxy-uridine and by cell counting. To investigate cell migration, we established a method to produce identically shaped cell-free areas in monolayers of cultured cells and documented the wound-healing process., Results: Concerning the five tested growth factors, only epidermal growth factor as well as basic and acidic fibroblast growth factor stimulated DNA synthesis, cell proliferation, and migration during wound healing. Vascular endothelial growth factor stimulated migration during wound healing without influencing DNA synthesis or cell proliferation., Conclusion: Whereas epidermal growth factor and the basic and acidic forms of fibroblast growth factor enhance proliferation as well as migration during wound healing, vascular endothelial growth factor seems to be a stimulatory agent specific for corneal endothelial cell migration.

Background: Donors who have suffered a traumatic death are, on average, younger than those who have died from other causes, and the time from death to enucleation (DET) also tends to be shorter. Corneae obtained from such donors are therefore considered particularly suitable for grafting. One of the reasons for excluding a donor cornea from transplantation is the occurrence of endothelial cell necrosis during organ culture, and we investigated whether the incidence of this phenomenon bears a relationship to death by traumatic or nontraumatic means., Methods: Data from 2125 donor corneae were collected using standardized evaluation protocols between January 1991 and December 1995 and included information on cause of death, age and DET, as well as endothelial cell loss and necrosis. Traumatic deaths were recorded in 346 cases, nontraumatic deaths in the other 1779 cases. Since differences in age (P = 0.006) but not in DET occurred within each of these groups, a more refined comparison, with matched data (< 35 years), was also undertaken., Results: Forty (11.6%) of the 346 corneae derived from traumatic death donors manifested total or partial endothelial cell death in organ culture. The corresponding figure in the nontraumatic death group was only 105/1779 (5.8%; P = 0.0002). After matching for age, endothelial cell death during culturing was revealed in 18 (13.5%) of the 133 of the traumatic death corneae and in 3 (2.6%) of the 115 nontraumatic death ones (P = 0.004); the overall incidence of endothelial cell death (total or partial) during organ culture was 6.8% (145/2125). Endothelial cell loss during culturing of the 227 age-matched donor corneae which still had an intact endothelial monolayer at the end of the incubation period was 340 +/- 388 cells/mm2 in traumatic death corneae (n = 115) and 255 +/- 318 cells/mm2 in nontraumatic death ones (n = 112; P = 0.051)., Conclusion: Corneae obtained from traumatic death donors were more liable to undergo total or partial endothelial cell death during organ culture than were those procured from nontraumatic death ones. However, in corneae which survived the period of culture, there was no significant difference in endothelial cell loss between the two groups. Whilst the mechanism underlying this increased susceptibility of traumatic death corneae to cell death remains elusive, the data gleaned from this investigation nonetheless emphasize the potential importance of being able to perform meaningful in vitro viability tests on donor corneae; this is possible only under organ culture conditions.

Background: Gyrate atrophy is a form of choroidal dystrophia characterized by a genetic defect of the mitochondrial matrix enzyme ornithine ketoacidoaminotransferase on chromosome 10. Apart from rare myopathic problems, cases of choroidal, iridal atrophy, complicated subcapsular cataract, optic atrophy, macular edema, reticular retinoschisis, vasculitis and perivasculitis could be observed so far. To our knowledge, rheumatogenous retinal detachment has been described before. We present a 34-year-old female subject with a retinal detachment of the right eye over 3 quadrants with 3 small round foramina in the area 10 to 11 o'clock., Patients: Concentric defects of the visual field had been known since the age of 4; the patient was slightly myopic. At the age of 31, the patient had undergone cataract extraction with implantation of posterior chamber lenses in both eyes. The ophthalmological examination revealed large areas of choroidal atrophia in both eyes. A determination of visual acuity resulted in hand movements in the right eye and 0.1 in the left eye. The ERG was extinguished., Results: The retina could be fully reattached by pars plana vitrectomy and instillation of silicone oil. At the same time, we performed an excision of secondary cataract. On demission, the visual acuity of the right eye was finger counting. Because a brother of the patient had also suffering from a progressive choroidal atrophy since his childhood, an autosomal recessive disease was suspected. The only abnormality detected was strongly elevated plasma levels of ornithin (7.5 to 14 mg/dl, compared to levels of 0.6 to 2.0 in controls). Urinary excretion of ornithin was also strongly increased., Discussion: To our knowledge there was no previous description of a case of atrophia gyrata in association with rhegmatogenous retinal detachment after intraocular surgery. As the pigment epithelium is missing, intraoperative endolaser-coagulation would be useless. For this reason, and since a tendency for reattachment is lacking consistent with one of the characteristics of this disease, the only approach to this case is intraocular tamponade with silicone oil.

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.

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.

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.

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.

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.

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.

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.

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%.

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.

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.

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.

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.

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.

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.

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.

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.

Highly purified virulent poliovirus preparations harbour an endogenous protein kinase. Enzyme activity increases significantly upon purification of infectious virus particles from infected HeLa cells. Enzyme activity is stimulated by divalent cations. The substrate specificity and the degree of stimulation of the kinase are dependent on the nature of the divalent cations included in the assay. The preferred substrates for this kinase are the viral capsid proteins. Exogenously added proteins such as alpha-casein, phosvitin and protamine are also phosphorylated by the kinase. Moreover, these proteins enhance the phosphorylation of viral proteins. In the presence of Mg++ VP 2 and VP 0 are highly phosphorylated, while in the presence of Zn++ only VP 2 and VP 4, but not VP 0 or exogenous proteins are phosphorylated. Poliovirus associated protein kinase exhibits optimal activity at pH 7.9 in the presence of 10 mM Mg++. The Km for ATP is shown to be 40 microM. By testing different nucleotides as phosphate donors a specificity of the phosphorylation reaction for ATP is demonstrated. Phosphoamino acid analysis of hydrolysates of the substrates phosphorylated in the presence of Mg++ by thin layer electrochromatography and HPLC yielded phosphoserine and phosphothreonine from viral capsid proteins while hydrolysates of protamine yield only phosphoserine. Destabilization of the viral capsid, e.g. by preincubation at 42 degrees C for 20 minutes results in a stimulation of kinase activity. Moreover, phosphorylation of the poliovirus capsid proteins itself results in destabilization of the viral capsid. These findings suggest that phosphorylation of the viral coat proteins triggers or enhances the uncoating of poliovirus leading to the release of viral RNA.

Human corneal endothelial cells (HCEC) were isolated by means of enzymatic treatment of excised corneas. The corneas were incubated for 1.5 hr together with a high concentration of collagenase (0.5%), followed by a long-term incubation (up to 16 hr) using a low concentration of the enzyme (0.04%). Endothelial cells were enriched against contaminating fibroblasts by using a selective L-valine-free medium which inhibited fibroblast growth during the first passages. Subcultures of HCEC were passaged for more than 20 generations without showing signs of senescence. Laminin and chondroitin sulfate functioned as a substrate for HCEC, promoting proliferation and allowing the cells to grow in monolayer formation. The inclusion of fibroblast growth factor (FGF) as well as chondroitin sulfate in the medium led to an additional increase in the rate of proliferation.