NF1 mutations in conjunctival melanoma.

BACKGROUND: Conjunctival melanoma is a potentially deadly eye tumour. Despite effective local therapies, tumour recurrence and metastasis remain frequent. The genetics of conjunctival melanomas remain incompletely understood. METHODS: A large cohort of 63 conjunctival melanomas was screened for gene mutations known to be important in other melanoma subtypes by targeted next-generation sequencing. Mutation status was correlated with patient prognosis. RESULTS: Frequent mutations in genes activating the MAP kinase pathway were identified. NF1 mutations were most frequent (n = 21, 33%). Recurrent activating mutations were also identified in BRAF (n = 16, 25%) and RAS genes (n = 12, 19%; 11 NRAS and 1 KRAS). CONCLUSIONS: Similar to cutaneous melanomas, conjunctival melanomas can be grouped genetically into four groups: BRAF-mutated, RAS-mutated, NF1-mutated and triple wild-type melanomas. This genetic classification may be useful for assessment of therapeutic options for patients with metastatic conjunctival melanoma.

[Good practice procedures for acquisition and preparation of cryopreserved human amniotic membranes from donor placentas]. / Gute fachliche Praxis zur Gewinnung und Herstellung von kryokonservierter humaner Amnionmembran aus Spenderplazenta.

Within the framework of obtaining a valid authorization for tissue preparation of cryopreserved human amniotic membranes at the Paul Ehrlich Institute, pursuant to § 21a paragraph 1 of the German Medicines Act (AMG), parts of the existing good practice procedures for acquisition of cryopreserved human amniotic membranes from donor placentas were reviewed and supplemented by new knowledge. The present good practice procedures were formulated in cooperation with members of the section for tissue transplantation and biotechnology of the German Ophthalmological Society. The current revised version is presented in this article.

[Ocular graft versus host disease : Corneal complications]. / Okuläre Graft-versus-Host-Disease : Korneale Komplikationen.

BACKGROUND: Ocular graft-versus-host disease (GvHD) following allogeneic blood stem cell transplantation leads to immunologically induced alterations in many ocular tissues, particularly at the ocular surface. Within the framework of the main topic, this article focuses primarily on corneal complications in chronic ocular GvHD. OBJECTIVE: This article aims to promote understanding of the influencing factors, diagnostics, and therapeutic options pertaining to corneal complications in ocular GvHD. Furthermore, the possibilities for prevention are discussed. MATERIALS AND METHODS: This analysis is based on a literature review as well as on data from the Ophthalmology Clinic at the University Hospital Essen. RESULTS: Corneal complications often occur secondarily in ocular GvHD, as a consequence of severe inflammatory alterations of the conjunctiva or eyelid. Spontaneous corneal perforations associated with only mild symptoms are less common during the course of disease. From the ophthalmologist's perspective, it is important that the inflammatory activity of all the different ocular tissues is considered. Treatment may follow a stepwise scheme that includes substitution, immunosuppression, and surgical rehabilitation. CONCLUSION: Systematic diagnosis of ocular GvHD helps to prevent corneal complications or support early therapeutic intervention. An interdisciplinary approach to diagnosis and treatment planning is recommended, in order to optimize local and systemic immunosuppressive therapy.

[Clinical, morphological and molecular biological characteristics of the aging eye]. / Klinische, morphologische und molekularbiologische Charakteristika des alternden Auges.

BACKGROUND: The physiological aging of the eye is associated with loss of visual function. Age-related changes of the eye can result in ophthalmological diseases. The aim of this article is to display morphological, histological and molecular biological alterations of the aging eye. MATERIAL AND METHODS: A web-based search and review of the literature for aging of the visual system including cornea, lens, vitreous humor, retina, retinal pigment epithelium (RPE), choroidea and optic nerve were carried out. The most important results related to morphological, histological and molecular biological changes are summarized. RESULTS: Age-related, morphological alterations can be found in preretinal structures, e. g. cornea, lens and vitreous humor, as well as neuronal structures, such as the retina. In addition to negligible clinical signs of the aging eye, there are clinically relevant changes which can develop into pathological ophthalmological diseases. These transitions from age-related alterations to relevant ophthalmological diseases, e. g. age-related macular degeneration and glaucoma are continuous. CONCLUSION: An understanding of aging could provide predictive factors to detect the conversion of physiological aging into pathological conditions. The derivation of physiological markers or new approaches to detection and treatment of disease-related entities associated with the risk factor aging are desirable. Translational approaches in clinical and basic science are necessary to provide new therapeutic options for relevant ophthalmological diseases in the future.

[Long-term results of autologous transplantation of limbal epithelium cultivated ex vivo for limbal stem cell deficiency]. / Langzeitergebnisse zur autologen Transplantation von ex vivo kultiviertem Limbusepithel bei limbaler Stammzellinsuffizienz.

OBJECTIVE: This study reports the long-term clinical outcome of autologous limbal epithelial cells cultivated ex vivo on intact amniotic membranes (AM) for ocular surface reconstruction in limbal stem cell deficiency (LSCD). PATIENTS AND METHODS: A total of 61 eyes from 57 patients (46 males and 11 females) with LSCD were treated by transplantation of autologous limbal epithelial cells cultivated on intact AM. The etiology of the LSCD was chemical and thermal burns (n = 34), recurrent or primary large-sized pterygium (n = 12), mitomycin C and tumor excision-induced LSCD (n = 9), severe infectious keratitis (n = 3), perforating injury, epidermolysis bullosa and contact lens-associated keratopathy (each n = 1). Only eyes with a follow-up time of at least 12 months were included in the analysis. The main outcome end points were restoration of ocular surface integrity and improvement of visual acuity (VA). RESULTS: The mean follow-up time was 50.8 ± 32.7 months. An entirely stable corneal surface was reconstructed in 46 (75.4%) eyes. Visual acuity significantly increased in 40 (65.6 %) eyes, was stable in 12 (19.7%) eyes and decreased in 9 eyes (14.8%). The mean visual acuity significantly increased (p < 0.0001) from 1.4 ± 0.91 LogMAR preoperatively to 0.8± 0.67 LogMAR postoperatively. CONCLUSION: Transplantation of limbal epithelium cultivated ex vivo on intact AM leads to restoration of a stable corneal surface and resulted in a significant increase of visual acuity in most cases of LSCD. Autologous transplantation of cultivated limbal epithelium showed an excellent prognosis and outcome after long-term follow-up.

[Operative therapy and irradiation of conjunctival melanoma]. / Operative Therapie und Bestrahlung konjunktivaler Melanome.

BACKGROUND: Radiotherapy of conjunctival melanoma has gained in importance in recent years compared to less invasive therapeutic approaches. This is due to the high recurrence rates achieved by omitting adjuvant therapy and to the increasing availability of suitable radiotherapeutic methods, so that tumors formerly not amenable to organ-preserving therapy can now be treated. OBJECTIVE: This article presents the current radiotherapeutic options for conjunctival melanoma. The aim is to describe the diagnostic and therapeutic strategies and the course of therapy of malignant conjunctival melanoma. It is the authors' intention to justify the necessity of the adjuvant therapy of conjunctival melanoma and to emphasize the need for interdisciplinary cooperation during the course of tumor therapy. METHODS: The article is based on results published in the literature as well as on data collected and experience gained in our centre.

Evaluation of a new protocol for sterility controls of corneal culture medium.

Careful testing for microbial contamination is essential for corneal transplants. Sterility tests are performed on the antibiotics containing culture medium leaving the problem that antibiotics might compromise the test results. In this study a protocol for the application of the automated BacT/Alert system for sterility testing of corneal cell culture medium was examined. Corneal culture medium in combination with an antibiotics degrading enzyme were injected in resin containing test bottles of the BacT/Alert system named FA plus (intended for aerobic microorganisms) or FN plus (intended for anaerobic microorganisms) depending on their aerobic or anaerobic nature. Additionally i-FA plus(aerobic test bottle for industrial use) bottles were used. Microbial test strains on the basis of the European Pharmacopaea (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Aspergillus brasiliensis and Clostridium sporogenes) with the addition of Propioniobacterium acnes were added to the test bottles. The bottles were incubated at two different temperatures for 14 days. The time to detection (TTD) was monitored for each bottle. Growth of the test strains except European Pharm was detected in the FA and FN Plus bottles. The TTD for the strains was 44 ± 1.5 h (P. aeruginosa), 57.7 ± 2.2 h (B. subtilis), 56 ± 1 h (S. aureus), 26.3 ± 1 h (C. sporogenes), 223 ± 4.6 h (P. acnes), 64.4 ± 10 (C. albicans). A. brasiliensis was detected in i-FA Plus bottles with a TTD of 94.9 ± 3.7 h. The application of BacT/Alert FA Plus and FN Plus resin bottles in combination with a penicillin degrading enzyme is able to detect small scale microbial contamination with different microorganisms in antibiotic containing corneal culture medium. For detection of Aspergillus brasiliensis in the medium the (i-) FA Plus bottles should be used.

Influence of latanoprost on retinal microcirculation in glaucoma.

PURPOSE: To test whether latanoprost has an influence on ocular haemodynamics, considering the general reputation of prostaglandins which is frequently associated with vasoconstriction. The effect of latanoprost on the retinal blood supply of treatment-naïve glaucoma patients was tested. MATERIALS AND METHODOLOGY: 13 patients (7 male, 6 female) who had just recently been diagnosed with primary open-angle glaucoma (POAG) were treated with latanoprost (0.005%). The average age of our study group was 63.8 years (+/- 2.9 years). The drug's effect on retinal autoregulation was assessed by flicker test using the Dynamic Vessel Analyzer (DVA). Examinations took place before initializing treatment, after 4 weeks and once again after 4 to 6 months. RESULTS: In our group of POAG patients, the IOP under treatment was significantly reduced about 25%. No intraindividual differences in systemic blood pressure and heart rate were observed. In DVA measurements of glaucoma patients, the maximum flicker dilation of the arteries was significantly lower than reported for healthy volunteers. Beyond that, POAG patients did not show significant differences in vessel diameters, peak amplitudes as well as maximum dilations of retinal arteries and veins before and under treatment with latanoprost (0.005%). CONCLUSION: Latanoprost markedly lowered the IOP but it did not exert a significant effect on retinal haemodynamics. There was neither a tendency towards vasoconstriction nor towards vasodilation. Sustaining reperfusion damage after topical latanoprost therapy thus seems to be highly unlikely. Further studies must show if sole IOP lowering or a dual positive effect - IOP lowering and improvement of retinal vessel autoregulation - have a more positive impact on the long term follow-up of glaucoma patients.

[New nanofibrous scaffold for corneal tissue engineering]. / Eine neue Nanofaser-Matrix für den kornealen Gewebeersatz.

BACKGROUND: An estimated 10 million people suffer worldwide from vision loss caused by corneal damage. For the worst cases, the only available treatment is transplantation with human donor corneal tissue. However, in numerous countries there is a considerable shortage of corneal tissue of good quality, leading to various efforts to develop tissue substitutes. The present study aims to introduce a nanofibrous scaffold of poly(glycerol sebacate) PGS as a biodegradable implant, for the corneal tissue engineering. MATERIALS AND METHODS: Nanofibrous scaffolds were produced from PGS and poly(ε-caprolactone) (PCL) by a modified electro-spinning process. The biocompatibility of the material was tested in vitro by colorimetric MTT assay on days 3, 5, and 7 to test the cell viability of human corneal endothelium cells (HCEC). To examine a potential immunological reaction of the scaffolds, samples were exposed to mononuclear cells derived from peripheral blood (PBMCs). After an incubation period of 3 days, supernatants were assayed for apoptotic assessment and immunogenic potentials by annexin V FITC//propidium iodide and flow-cytometric analysis. RESULTS: We could successfully demonstrate that cultivation of HCECs on PGS/PCL scaffolds was possible. Compared to day 3, cell density determined by microplate absorbance was significantly higher after 7 days of cultivation (p < 0.0001). According to the MTT data, none of the samples showed toxicity. Apoptotic assessments by FACS analysis showed that no composition stimulated apoptosis or activated PBMCs occurred. All the compositions were inert for native as well as activated T/B/NK cells and monocytes. It can be concluded that leukocytes and their activity was not affected by the scaffolds. CONCLUSION: A tissue-like scaffold mimicking the human stroma could be developed. The results indicate that PGS/PCL scaffolds could be considered as ideal candidates for corneal tissue engineering as they are biocompatible in contact to corneal endothelial cells and blood cells.

[Validation of an automatic test system for sterile testing of amniotic membranes]. / Validierung eines automatischen Testsystems für die Steriltestung von Amnionmembran.

PURPOSE: The use of cryopreserved amniotic membranes for the treatment of diseases and injuries of the surface of the eye is an established procedure in ophthalmological surgery. Before clinical use of cryopreserved amniotic membranes (AM) a careful testing for microbial contamination is essential to ensure a safe application. In this study the use of the BacT/Alert® test system was evaluated for screening of microbial growth in AMs. MATERIALS AND METHODS: Minced fresh and cryopreserved AMs (approximately 5 × 5 cm in size) were injected with 10 ml of balanced salt solution in separate culture media test bottles and 10 ml of cryopreservation medium bacterial and fungal test strains according to European Union (EU) regulations were applied to test the performance of the system. Approximately 10-100 colony forming units were applied on the samples prior to injection in the corresponding test bottles. Bottles were incubated at 37 °C for 7 days. Positive controls contained only balanced salt solution and the test strains while negative controls contained the test material without microbial test strains. RESULTS: Growth of the test strains was detected in all inoculated samples from non-processed and cryopreserved AM within the 7-day incubation period. In samples of the cryopreservation medium only growth of the fungus Candida albicans could be detected. CONCLUSIONS: The automated BacT/Alert test system is suitable for testing of microbial safety of amniotic membranes but not for testing the cryopreservation medium in clinical practice according to EU regulations.

[Short-term and long-term complications after transplantation of cultivated limbal epithelium]. / Kurz- und Langzeitkomplikationen nach Transplantation von kultiviertem Limbusepithel.

Recent advances in tissue engineering have facilitated the development of new strategies in ocular surface reconstruction. Limitations and possibilities of ex vivo cultivation and limbal epithelium cell culture techniques as well as the short and long-term complications after transplantation of ex vivo expansion of cultivated limbal epithelium for the treatment of limbal stem cell deficiency are summarized in this review.

TERT promoter mutations in ocular melanoma distinguish between conjunctival and uveal tumours.

BACKGROUND: Recently, activating mutations in the TERT promoter were identified in cutaneous melanoma. We tested a cohort of ocular melanoma samples for similar mutations. METHODS: The TERT promoter region was analysed by Sanger sequencing in 47 uveal (ciliary body or choroidal) melanomas and 38 conjunctival melanomas. RESULTS: Mutations of the TERT promoter were not identified in uveal melanomas, but were detected in 12 (32%) conjunctival melanomas. Mutations had a UV signature and were identical to those found in cutaneous melanoma. CONCLUSION: Mutations of TERT promoter with UV signatures are frequent in conjunctival melanomas and favour a pathogenetic kinship with cutaneous melanomas. Absence of these mutations in uveal melanomas emphasises their genetic distinction from cutaneous and conjunctival melanomas.

[The biological basis of limbal stem cell deficiency]. / Biologische Grundlagen der Limbusstammzellinsuffizienz.

Limbal stem cell deficiency results from the loss of tissue regenerating stem and progenitor cells. Corneal epithelial regeneration is maintained by stem and progenitor cells which reside in the schlerocorneal limbus. They possess stem cell characteristics and can be stimulated to proliferate by external signals. The limbus is the stem cell niche for corneal epithelial stem cells and forms a unique microenvironment in which stem cell characteristics are conserved. Regulation of limbal epithelial stem cells is produced by a network of signals within the niche which governs cell fate decisions with regards to proliferation, differentiation or maintenance of a quiescent status.

[Ocular surface reconstruction in limbal stem cell deficiency : Transplantation of cultivated limbal epithelium]. / Oberflächenrekonstruktion bei Limbusstammzellinsuffizienz : Transplantation von Limbusgewebe - Kultivierungsverfahren.

Various ocular surface diseases are caused by loss of corneal epithelial stem cells or dysfunction of the limbal stem cell niche. Besides conventional transplantation of autologous or allogenic limbal tissue, recent advances in tissue engineering have led to the development of new culture and expansion techniques of human limbal stem and progenitor cells (LSPC) as a new strategy to successfully treat limbal stem cell deficiency (LSCD). From a small autologous limbal biopsy with a limited amount of LSPC an epithelium ready for transplantation is achieved. Autologous grafting of cultured limbal epithelium led in most of the treated cases to a successful reconstruction of the corneal surface. Alternative methods which have recently been introduced to treat LSCD use other stem cell sources including the transplantation of oral mucosal epithelium. In this article the challenges and controversies associated with these stem cell culture techniques for ocular surface reconstruction are reviewed.

[Gene therapy in ophthalmology: state of the art]. / Gentherapie in der Ophthalmologie: Stand der Forschung.

The term "gene therapy" denotes the treatment of diseases or gene deficiencies by introduction of genes into cells. To achieve this goal, vectors are used to transfer the genetic information into the cells. Thus, the protein of interest can be overexpressed or silenced. On account of its easy accessibility, the good compartmentalisation and the separation from the main bloodstream by the blood-retina barrier, the eye represents a very attractive target to treat ocular diseases by gene therapy. In this work, we provide an overview of the progress in ocular gene therapy over the last decade and give an outlook on future developments.

The effect of long-term storage on the biological and histological properties of cryopreserved amniotic membrane.

PURPOSE: Cryopreserved amniotic membrane (AM) is widely used in ophthalmology because of its anti-angiogenic, anti-inflammatory, and wound healing promoting capabilities. A common method to conserve the tissue is the storage in cryo-medium containing 50% glycerol at -80°C. The aim of this study was to examine the influence of storage time on the sterility as well as the histological and biological properties of cryopreserved AM. METHODS: Amniotic membrane from different donors was stored in cell culture media containing 50% glycerol for different time periods, on average 4 months (group 1), 15 months (group 2), and 24 months (group 3), at -80°C. Samples of the tissue and cryo-medium were examined for bacterial and fungal contamination. Tissue samples were incubated in 0.5 ml/cm(2) serum-free medium at 37°C. The medium was changed after 1, 2, and 3 days. The proteins released by AM were TCA-precipitated and the presence of the proteins TIMP-1 and IL-1ra was analyzed using Western blotting and semi quantified by means of image analysis. Integrity of the amniotic epithelium and the basement membrane components collagen IV, collagen VII, laminin, laminin 5, and fibronectin were examined by haematoxylin eosin stain and immunohistochemistry in cryosections of AM. RESULTS: None of the examined samples showed bacterial or fungal contamination. The soluble proteins TIMP-1 and IL-1ra were found in all samples of medium incubated for all time periods. The examined proteins were detectable after one-day incubation but the staining signal diminished significantly in the second and third wash after 48 hr and 72 hr. Differences in the intensity of the Western blot signal between the three particular groups were statistically not significant. The epithelia of all samples were intact. The basement membranes of all samples showed a similar distribution of collagen IV, collagen VII, laminin, laminin 5, and fibronectin. CONCLUSIONS: Long-term storage of amniotic membrane in cell culture media with 50% glycerol does not significantly impair sterility, histology, or biological properties of AM.

[Procedural guidelines. Good practice procedures for acquisition and preparation of cryopreserved human amniotic membranes from donor placentas]. / Arbeitsrichtlinien: Gute fachliche Praxis zur Gewinnung und Herstellung von kryokonservierter humaner Amnionmembran aus Spenderplazenta.

A cornea/tissue bank must have an organizational structure in which responsibility and authority to issue directives are clearly defined. It must also use a documented quality management system on the basis of good practice procedures which is maintained to the current standards. The personnel of a cornea/tissue bank must be present in sufficient numbers and be suitably qualified. A cornea/tissue bank must be in possession of appropriate facilities which are suitable for the main purpose of preparation of cryopreserved human amniotic membranes from donor placentas. All equipment must be designed and maintained corresponding to the intended purpose. Deviations from the stipulated quality and safety standards must give rise to documented investigations which include decisions on options for correctional and preventive measures. Acquisition of donors and tissue sampling must be strictly controlled and documented. This also applies to entry of donor tissue in the cornea/tissue bank. Cryopreserved human amniotic membranes can only be preserved from donors undergoing caesarean section and who did not present any known infection of the abdominal cavity or any systemic blood borne infection. Contamination of media used for cryopreservation of donor placenta must be ruled out at least once. Measures must be taken to keep the risk of contamination as low as possible. Cryopreserved human amniotic membranes from donor placentas can only be released if defined criteria are fulfilled. Any suspicion of severe undesired reactions and events for the recipient of an amniotic membrane transplant must be registered with the authorities. The activities of a cornea/tissue bank must maintain and adapt to the state-of-the-art with respect to scientific progress.