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Purpose: The purpose of the study was to compare the anatomical and functional results including reading ability after epiretinal membrane (ERM) surgery in patients with and without the use of autologous platelet concentrate (APC)., Methods: Design: Prospective, comparative non-inferiority series., Setting: Institutional., Patients: 51 eyes of 51 patients, who underwent pars-plana vitrectomy (PPV) for ERM surgery. 29 eyes additionally received intraoperative APC, 22 eyes underwent standard procedure without APC use., Observations: anatomical and functional outcome parameters (central retinal thickness (CRT), best corrected visual acuity (BCVA) and reading ability (RA)) were compared between the two groups at 6 weeks and 6 months postoperatively. Subjective assessment of visual acuity and reading ability was also analysed., Main Outcome Measures: BCVA, RA and CRT., Results: Both groups showed significant CRT reduction and RA improvement, while BCVA improvement was significant only in eyes with intraoperative APC use during the follow-up time of 6 months. There was no statistically significant difference between CRT reduction, BCVA and RA improvement between the groups., Conclusion: Intraoperative APC use for ERM surgery results in similar anatomical and functional outcomes compared with standard ERM surgery without APC use., (© 2024 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Competing Interests: Einhaltung ethischer Richtlinien. Interessenkonflikt: K. Engelmann, V. Kakkassery, G. Stolze, J. Weidner und M. Sedlmayr geben an, dass kein Interessenkonflikt besteht. Für diesen Beitrag wurden von den Autor/-innen keine Studien an Menschen oder Tieren durchgeführt. Für die aufgeführten Studien gelten die jeweils dort angegebenen ethischen Richtlinien.

Background: Evidence-based treatment recommendations are helpful in the corresponding discipline-specific treatment but can hardly take data from real-world care into account. In order to make better use of this in everyday clinical practice, including with respect to predictive statements about disease development or treatment success, models with data from treatment must be developed in order to use them for the development of assistive artificial intelligence., Goal: The aim of the Use Case 1 of the medical informatics hub in Saxony (MiHUBx) is the development of a model based on treatment and research data for a treatment algorithm supported by biomarkers and also the development of the necessary digital infrastructure., Material and Methods: Step by step, the necessary partners in hospitals and practices will be brought together technically or through research questions within Use Case 1 "Ophthalmology meets Diabetology", a regional digital progress hub in health, the medical informatics hub in Saxony (MiHUBx ) of the nationwide medical informatics initiative (MII)., Results: Based on joint studies with diabetologists, robust serological and imaging biomarkers were selected that provide evidence of the development of diabetic macular edema (DME). In the future, these and other scientifically proven prognostic markers will be incorporated into a treatment algorithm that is supported by artificial intelligence (AI). For this purpose, model procedures are being developed together with medical informatics specialists. At the same time, a data integration center (DIZ) was established., Conclusion: In addition to the structured and technical combination of the previously disseminated and partially heterogeneous treatment data, the Use Case 1 defines the chances and hurdles for using such real-world data to develop artificial intelligence., Competing Interests: Einhaltung ethischer Richtlinien. Interessenkonflikt: G. Stolze, V. Kakkassery, D. Kowerko, M. Bartos, K. Hoffmann, M. Sedlmayr und K. Engelmann geben an, dass kein Interessenkonflikt besteht. Für diesen Beitrag wurden von den Autor/-innen keine Studien an Menschen oder Tieren durchgeführt. Für die aufgeführten Studien gelten die jeweils dort angegebenen ethischen Richtlinien., (© 2024. The Author(s).)

Purpose: To analyze changes in demographic parameters and retreatment patterns over a 10-year period in a clinical routine setting of infants with retinopathy of prematurity (ROP) requiring treatment documented in the German Retina.net ROP registry., Design: Multicenter, noninterventional, observational registry study recruiting patients treated for ROP., Subjects: A total of 692 eyes of 353 infants treated for ROP were documented in the Retina.net ROP registry over a 10-year period between 2011 and 2020. These cases cover about 15% of all infants treated for ROP in Germany., Methods: The Retina.net ROP registry was established in 2012 to jointly collect information on infants treated for ROP. The database collects information on demographic parameters (gestational age [GA], birth weight, neonatal comorbidities) as well as treatment parameters (type of treatment, weight and age at treatment, and stage of ROP). A total of 19 centers contributed to the analysis. This is the 10-year analysis of data from 2011 to 2020, in which we focus on changes over time regarding the respective parameters., Main Outcome Measures: Changes over time in demographic parameters and treatment patterns for ROP in Germany., Results: The overall incidence of treatment requiring ROP was 3.5% of all infants screened for ROP at participating centers. Gestational age, weight at birth, and weight at treatment remained stable over the 10-year period, whereas postmenstrual and postnatal age at treatment increased moderately but statistically significantly over the years. The most prevalent ROP severity stage at treatment was stage 3+ in zone II (76.6% of all treated eyes). Treatment patterns changed considerably from predominantly laser treatments in 2011 (75% of all treated eyes) to predominantly ranibizumab treatments in 2020 (60.9% of all treated eyes). The overall retreatment rate was 15.6%. Retreatment rates differed between initial treatment modalities (14.1% after laser coagulation, 12% after bevacizumab and 24.5% after ranibizumab). Treatment-associated systemic or ophthalmic complications were rare., Conclusions: This data analysis represents one of the largest documented cohorts of infants treated for ROP. The data on demographic parameters and treatment patterns provide useful information for further improvement of ROP management., Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article., (Copyright © 2023 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

In ophthalmology, intravitreal operative medication therapy (IVOM) is a widespread treatment for diseases related to the age-related macular degeneration (AMD), the diabetic macular edema, as well as the retinal vein occlusion. However, in real-world settings, patients often suffer from loss of vision on time scales of years despite therapy, whereas the prediction of the visual acuity (VA) and the earliest possible detection of deterioration under real-life conditions is challenging due to heterogeneous and incomplete data. In this contribution, we present a workflow for the development of a research-compatible data corpus fusing different IT systems of the department of ophthalmology of a German maximum care hospital. The extensive data corpus allows predictive statements of the expected progression of a patient and his or her VA in each of the three diseases. For the disease AMD, we found out a significant deterioration of the visual acuity over time. Within our proposed multistage system, we subsequently classify the VA progression into the three groups of therapy "winners", "stabilizers", and "losers" (WSL classification scheme). Our OCT biomarker classification using an ensemble of deep neural networks results in a classification accuracy (F1-score) of over 98%, enabling us to complete incomplete OCT documentations while allowing us to exploit them for a more precise VA modelling process. Our VA prediction requires at least four VA examinations and optionally OCT biomarkers from the same time period to predict the VA progression within a forecasted time frame, whereas our prediction is currently restricted to IVOM/no therapy. We achieve a final prediction accuracy of 69% in macro average F1-score, while being in the same range as the ophthalmologists with 57.8 and 50 ± 10.7 % F1-score., (© 2024. The Author(s).)

Low-density lipoprotein (LDL) apheresis is effective and safe for patients with diabetes, proteinuria, and dyslipidemia. Diabetes mellitus is accompanied by ocular microvascular complications like retinal neovascularization or diabetic macular edema. These are leading causes of blindness and can be mediated by abnormal vessel growth and increased vascular permeability due to elevated levels of vascular endothelial growth factor (VEGF) in diabetic patients. In this study, we established methods to study the expression of different VEGF isoforms in human retinal and endothelial cells. The VEGF-A 165a isoform is much higher expressed in retinal cells, compared to endothelial cells. Stimulation with glyoxal as a model of oxidative stress under diabetic conditions lead to a pronounced induction of VEGF-A 165a in human retinal and endothelial cells. These data suggest that diabetes and oxidative stress induce VEGF-A isoforms which could be relevant in regulating the ingrowths of novel blood vessels into the retina in diabetic patients., (© 2022 The Authors. Therapeutic Apheresis and Dialysis published by John Wiley & Sons Australia, Ltd on behalf of International Society for Apheresis and Japanese Society for Apheresis.)

Background: Retrospective research on real-world data provides the ability to gain evidence on specific topics especially when running across different sites in research networks. Those research networks have become increasingly relevant in recent years; not least due to the special situation caused by the COVID-19 pandemic. An important requirement for those networks is the data harmonization by ensuring the semantic interoperability. Aims: In this paper we demonstrate (1) how to facilitate digital infrastructures to run a retrospective study in a research network spread across university and non-university hospital sites; and (2) to answer a medical question on COVID-19 related change in diagnostic counts for diabetes-related eye diseases. Materials and methods: The study is retrospective and non-interventional and runs on medical case data documented in routine care at the participating sites. The technical infrastructure consists of the OMOP CDM and other OHDSI tools that is provided in a transferable format. An ETL process to transfer and harmonize the data to the OMOP CDM has been utilized. Cohort definitions for each year in observation have been created centrally and applied locally against medical case data of all participating sites and analyzed with descriptive statistics. Results: The analyses showed an expectable drop of the total number of diagnoses and the diagnoses for diabetes in general; whereas the number of diagnoses for diabetes-related eye diseases surprisingly decreased stronger compared to non-eye diseases. Differences in relative changes of diagnoses counts between sites show an urgent need to process multi-centric studies rather than single-site studies to reduce bias in the data. Conclusions: This study has demonstrated the ability to utilize an existing portable and standardized infrastructure and ETL process from a university hospital setting and transfer it to non-university sites. From a medical perspective further activity is needed to evaluate data quality of the utilized real-world data documented in routine care and to investigate its eligibility of this data for research.

Purpose: To report a case with unusual initial manifestation of uveal melanoma in a 46-year-old adult., Observations: A 46-year-old man demonstrated a mid-peripheral temporal partially hyperpigmented lesion in his left eye. Initial clinical appearance as well as multimodal imaging approaches were similar to a chorioretinitic disease. Over the course of 9 months, a massive tumor growth and exudative retinal detachment could be observed. The diagnosis of choroidal melanoma was histopathologically confirmed after endoresection., Conclusions and Importance: Classical choroidal melanoma manifest as pigmented, prominent tumors with >2 mm tumor thickness, exudative retinal detachment, and orange pigment. In our case, none of these classical clinical characteristic signs of melanoma were present at initial presentation, but the tumor underwent progressive development with significant tumor growth and exudative retinal detachment during the following 9 months., Competing Interests: No conflict of interest exists., (© 2022 The Authors.)

Purpose: The aim of this study was to evaluate the feasibility and the accuracy of a secondary, metachronous ultrasound (US)-guided marking of the stereotactic vacuum-assisted breast biopsy (ST-VABB) area., Materials and Methods: The institutional ethics committee approved the study. The retrospective study included 98 patients. In ST-VABB of 45 women, no tissue markers were deployed at the biopsy site, even if no residual calcifications remained. After histology proved the necessity for a subsequent operation, the biopsy site was marked under US guidance using a coil marker. All interventions were technically successful. No complications occurred. Mammography was done to visualize the coil deployment. The distances from the center of the lesion and the biopsy cavity to the coil location were measured in both planes to evaluate the accuracy of the marking procedure., Results: In 24 of the 46 cases, the whole lesion was biopsied without residual elements. The mean time between ST-VABB and sonographic marking of the lesion was 9.7 days (median 6.5). The biopsy cavity could be detected in 40 (87%) cases and thus marked exactly. The mean time of US-guided marking was 12.5 min. The mean distance between the coil and the target lesion was 0.6 ± 1.5 cm in the craniocaudal (cc) view and 0.5 ± 1.5 cm in the mediolateral (ml) view for all markings. The mean delta value from the distance nipple-original lesion and from the distance nipple-coil was 0.85 ± 1.2 cm (median 0.5) in the cc view and 0.88 ± 1.2 cm (median 0.6) in the ml view for all cases. Clip migration was not observed., Conclusion: Our study demonstrates the feasibility and the technical success of secondary metachronous coil marking of the biopsy site under US guidance after receipt of histology. This approach seems to be a cost-effective alternative to the standard procedure of the primary coil marking especially in all completely removed lesions. It may offer advantages for allergic patients., Competing Interests: Prof. Markus Müller-Schimpfle receives a royalty of Cook Medical, Bloomington, IN, USA, for the co-development of the MReye® breast localization coil. The other authors have no conflicts of interest to declare., (Copyright © 2020 by S. Karger AG, Basel.)

Background: To evaluate if a combination therapy with micropulse diode laser (MPL) shows non-inferiority on visual acuity (BCVA) within 12 months in comparison to standard therapy, i.e. intravitreal injection of ranibizumab alone., Setting: Institutional. Prospective randomized single-center trial., Methods: Patients with diabetic macular edema (DME) received three intravitreal injections of 0.5 mg ranibizumab during the upload phase and were then randomised 1:1 to receive either the same dosage of ranibizumab (0.5 mg) injections pro re nata alone (IVOM-Group; n = 9), or with two additional treatments with micropulse diode laser (IVOM+Laser-Group; n = 10). The primary endpoint was change in BCVA after 12 months. Secondary endpoints were change in central macular thickness and overall number of ranibizumab injections., Results: BCVA increased significantly in both groups (IVOM: + 5.86, p < 0.001; IVOM+Laser: + 9.30; p < 0.001) with corresponding decrease in central macular thickness (IVOM: - 105 μm, p < 0.01; IVOM+Laser: - 125 μm; p < 0.01). Patients with additional laser treatment had better visual improvement (group comparison p = 0.075) and needed fewer ranibizumab injections (cumulative proportion of injections 9.68 versus 7.46 in IVOM-Group and IVOM+Laser-Group, respectively)., Conclusion: Non-inferiority of combination therapy in comparison to standard therapy alone could be demonstrated. Patients with additional laser therapy needed fewer ranibizumab injections., Trial Registration: Registered 10 February 2014 on ClinicalTrials.gov; NCT02059772 .

Purpose: A malfunction of the corneal endothelium leading to corneal opacity is one of the main causes of impaired vision. Currently, keratoplasty is the one and only donor cornea-dependent treatment, and this calls for alternatives because of the worldwide lack of donor corneas. Recently, the topography of Descemet membrane (DM) has been discovered as a feasible stem cell differentiation tool. With this study, we further confirm this mechanotransductive system by using preinduced Wharton jelly-derived mesenchymal stem cells (WJ-EPCs)., Methods: To measure the mechanotransductive potential of Descemet-like topography (DLT), WJ-EPCs were cultivated on collagen imprints with DLT. Changes in the gene and protein expressions of corneal endothelial cells (CECs), typical markers such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured. In addition, CEC functionality has been evaluated by measuring the relative potential differences in a 2-compartment system and by measuring corneal transparency in an ex vivo rabbit cornea model. To confirm the activity of WJ-EPCs, rabbit CECs were restless deleted by collagen digestion of a thin layer of rabbit Descemet membrane., Results: The proper CEC-typical hexagonal morphology of WJ-EPCs in combination with a significant expression of ZO-1, Na/K-ATPase, PITX2, and COL-8 could be demonstrated. In addition, the WJ-EPCs were able to build up a relative potential difference of 40 mV and to keep corneas clear and transparent., Conclusions: These data indicate that a well-characterized, functional CEC monolayer was developed by using a DLT-mediated mechanotransductive differentiation of WJ-EPCs.

Purpose: To evaluate the outcome of macular surgery with ILM- and epiretinal membrane peel associated with significant dry age-related macular degeneration (AMD) as defined by the Age-Related Eye Disease Study (AREDS)., Patients and Methods: Institutional. Retrospective case-control study. A total of 42 pseudophacic eyes of 39 patients (7 with full thickness macular hole and 35 with epiretinal membrane) with coexisting dry AMD underwent pars plana vitrectomy (PPV) with internal limiting membrane (ILM) and epiretinal membrane peel. Preoperative and postoperative data including best corrected visual acuity (BCVA), AMD grade according to AREDS, central retinal thickness (CRT), development of choroidal neovascularization (CNV), and central retinal atrophy have been evaluated. Twenty-eight fellow eyes with dry AMD of the included 39 patients served as a control group., Results: A significant improvement in the visual acuity could be observed after surgery (initial BCVA 0.47±0.31 logarithm of the minimal angle of resolution (logMAR) vs 0.33±0.29logMAR 9 months postoperatively; p =0.006). CRT decreased significantly after surgery ( p <0.001). In the surgery group, there were 4 eyes (9.5%) with CNV and 1 eye (2.5%) with new central retinal atrophy development after surgery. All these eyes had preoperative AREDS 3 (4 eyes) or AREDS 4 (1 eye) AMD category. In the control group, there was 1 eye (4%) with CNV and 4 eyes (14%) with new central retinal atrophy development during the follow-up of 9 months. These eyes had initially AREDS 2 (1 eye), AREDS 3 (3 eyes) or AREDS 4 (1 eye) AMD category., Conclusion: Eyes with dry AMD of AREDS 3 and AREDS 4 with coexisting VMI abnormalities improve significantly after PPV with membrane peel. While there is a higher risk of CNV development after surgery (9.5%) in these eyes, the vitrectomy does not seem to accelerate central retinal atrophy progression compared to the fellow eyes course., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Furashova and Engelmann.)

The German Cancer Consortium ('Deutsches Konsortium für Translationale Krebsforschung', DKTK) is a long-term cancer consortium, bringing together the German Cancer Research Center (DKFZ), Germany's largest life science research center, and the leading University Medical Center-based Comprehensive Cancer Centers (CCCs) at seven sites across Germany. DKTK was founded in 2012 following international peer review and has positioned itself since then as the leading network for translational cancer research in Germany. DKTK is long term funded by the German Ministry of Research and Education and the federal states of each DKTK partner site. DKTK acts at the interface between basic and clinical cancer research, one major focus being to generate suitable multisite cooperation structures and provide the basis for including higher numbers of patients and facilitate effective collaborative forward and reverse translational cancer research. The consortium addresses areas of high scientific and medical relevance and develops critical infrastructures, for example, for omics technologies, clinical and research big data exchange and analysis, imaging, and clinical grade drug manufacturing. Moreover, DKTK provides a very attractive environment for interdisciplinary and interinstitutional training and career development for clinician and medical scientists., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Purpose: Loss of corneal endothelial cells (CECs) bears disastrous consequences for the patient, including corneal clouding and blindness. Corneal transplantation is currently the only therapy for severe corneal disorders. However, the worldwide shortages of corneal donor material generate a strong demand for personalized stem cell-based alternative therapies. Because human mesenchymal stem cells are known to be sensitive to their mechanical environments, we investigated the mechanotransductive potential of Descemet membrane-like microtopography (DLT) to differentiate human mesenchymal stem cells into CEC-like cells., Methods: Master molds with inverted DLT were produced by 2-photon lithography (2-PL). To measure the mechanotransductive potential of DLT, mesenchymal stem cells were cultivated on silicone or collagen imprints with DLT. Changes in morphology were imaged, and changes in gene expression of CEC typical genes such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured with real-time polymerase chain reaction. At least immunofluorescence analysis has been conducted to confirm gene data on the protein level., Results: Adhesion of MSCs to DLT molded in silicone and particularly in collagen initiates polygonal morphology and monolayer formation and enhances not only transcription of CEC typical genes such as ZO-1, Na/K-ATPase, PITX2, and COL-8 but also expression of the corresponding proteins., Conclusions: Artificial reproduction of Descemet membrane with respect to topography and similar stiffness offers a potential innovative way to bioengineer a functional CEC monolayer from autologous stem cells.

Background: Retinopathy of prematurity (ROP) is one of the main reasons for childhood blindness. The number of infants requiring treatment, however, is low for individual centers. The Retina.net ROP registry has been founded to allow a joint analysis of treatment patterns and courses post treatment., Objective: This paper reports treatment patterns over 5 years., Material and Methods: All infants born between January 2011 and December 2015 who were entered into the treatment registry by one of the 12 participating centers were analyzed., Results: The data of 150 infants (292 eyes) were analyzed and ROP 3+ in zone II was the most prevalent treatment indication. Gestational age and birth weight remained stable over the years. The treatment patterns, however, changed with anti-VEGF treatment (bevacizumab or ranibizumab) accounting for only 10% of treated eyes in 2011 but for 56% and 30% in 2014 and 2015, respectively. Almost all eyes with AP-ROP or zone I disease received anti-VEGF treatment. Zone II disease was predominantly treated with laser photocoagulation. Recurrences were more common and appeared later in the anti-VEGF group compared to the laser group (23%/interval 60 days vs. 17%/interval 23 days). Perioperative complications were evenly distributed across treatment groups., Conclusion: The data in this analysis represent about 10-15% of treated infants in Germany. The results provide evidence for an increasing use of anti-VEGF agents for ROP. The data reflect a selection bias for anti-VEGF treatment in eyes with a more aggressive disease. This needs to be considered when interpreting data such as disease recurrence rates. The risk for late recurrences after anti-VEGF treatment is of particular clinical significance.

Background/aims: To evaluate the efficacy, safety and tolerability of enteric-coated mycophenolate sodium (EC-MPS) in combination with low-dose corticosteroids compared with a monotherapy with low-dose corticosteroids in subjects with non-infectious intermediate uveitis (IU)., Methods: Open-label, prospective, controlled, randomised multicentre trial. Patients were randomised in a 1:1 ratio to either the treatment group (prednisolone plus EC-MPS) or control group (prednisolone monotherapy). Patients in the control group who relapsed within 6 months changed to the crossover group (prednisolone plus EC-MPS). Maximum treatment duration was 15 months. The primary endpoint was the time to first relapse in the treatment group and control group., Results: Forty-one patients at eight sites were analysed. Twenty-two patients were allocated to the treatment group, with 19 patients in the control group. A first relapse occurred in 9 patients (40.9%) in the treatment group and 15 patients (78.9%) in the control group (p=0.03). The median time to the first relapse was >15 months for the treatment group and 2.8 months for the control group (p=0.07). The probability of relapse-free survival at month 15 was estimated to be 52.9% in the treatment group and 19.7% in the control group (p=0.01). 15 patients changed to the crossover group. Of these, only four patients developed a second relapse. No safety concerns arose during the trial. Only one patient had to discontinue EC-MPS due to increased liver enzymes., Conclusion: EC-MPS can be considered an effective and well-tolerated immunosuppressive drug to prevent relapses in patients with chronic IU., Trial Registration Number: EUDRACT number: 2009-009998-10, Results., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Increased levels of systemic vascular endothelial growth factors (VEGFs) in patients with diabetes are associated with increased risk of microvessel disease. On the other hand, low VEGF levels after intravitreal antibody application may be associated with acute cardiovascular complications and treatment failure. Individual levels of systemic VEGF vary in a wide range depending on analytical methods and quality of diabetes control. So far only limited information exists on intraindividual fluctuations over longer periods and circadian rhythms. We analysed the intraindividual variance of VEGF-A, VEGF-C and placental growth factor (PLGF) in CTAD (citrate-theophylline-adenine-dipyridamol) plasma as well as VEGF-A in serum over a period of 6 months in patients with stable controlled type 2 diabetes (10 M, 10 F) and age and sex matched subjects with normal glucose tolerance (NGT). Furthermore, circadian levels of VEGFs were controlled hourly from 7:30 a.m. to 7:30 p.m. under standardized metabolic ward conditions. In addition, the relationship to metabolic, hormonal and inflammatory biomarkers was analyzed. VEGF-A, VEGF-C and PLGF remained stable in plasma and VEGF-A in serum over 6 months in both groups. No circadian change was observed in VEGF-A serum and plasma concentrations. A minor decrease of VEGF-C plasma levels was evident after 5 p.m. in both groups and a significant peak of PLGF concentrations occurred after lunch, which was more pronounced in T2DM. In multivariate analysis, only serum VEGF-A correlated to diabetes duration, whereas VEGF-C only correlated to HbA1c and fasting blood glucose. We did not observe significant intraindividual variances for VEGF-A in serum and VEGF-A, VEGF-C and PLGF in CTAD plasma over a period of 6 months. Taken together, a single morning measurement of systemic VEGF levels after 7:30 am appears to be a reliable parameter for the individual risk associated with abnormal VEGF concentrations in blood., Trial Registration: NCT02325271.

The success of corneal transplantation highly depends on the quality of the used graft. Various factors play a crucial role such as a perfect stroma without optically relevant, centrally located cloudiness or changes (e. g. scars) or an adherent Descemet membrane. One of the most important parameters is the quality of the endothelial cell layer with a sufficiently large endothelial cell count. An open question is so far whether the donor age affects corneal quality and therefore has an impact on the success of transplantation. A comprehensive review of the available literature revealed that a large amount of scientific data on the influence of donor age exist to answer this question. In a variety of studies, no significant dependence of graft quality of donor age could be detected. Rather the studies prove that graft survival depends primarily on the state of the endothelial cell layer, and postoperative endothelial cell loss must be considered as a major cause of graft failure. Extensive quality assurance procedures in tissue preparation and cornea processing in the eye banks in Germany (Europe) ensures that only corneas with tested high quality are allocated for transplantation regardless of the donor age. Against the background of an aging population, the use of grafts from older donors should not be waived.

Levels of vascular endothelial growth factors (VEGF) are regulated in a complex network of adipokines, glucose control, and low grade inflammation together with activated platelets, leucocytes, and endothelial dysfunction. Increased levels of VEGF are associated with enhanced angiogenesis and impaired repair mechanisms of vascular lesions in endorgans. Little is known about the interaction of systemic VEGF levels with quality of diabetes control, biomarkers of inflammation, and diabetic nephropathy. Moreover, it is unclear, whether serum and plasma VEGF levels are similarly suited to reflect risk associated with VEGF.In this case control study, we analyzed these parameters in serum and plasma of age and sex matched controls without diabetes (n=99) and type 2 diabetes (n=302). Serum VEGF-A was significantly increased in patients with T2DM while plasma levels were in the same range as for controls. Individual levels varied in a wide range. Serum levels were 4.9 times higher in controls and 7.3 times higher in T2DM as compared to plasma levels. T2DM was associated with significantly higher levels of hsCRP, ALAT, and albumin/creatinine ratio. When calculated for tertiles of HbA1c, we observed a highly significant increase from tertile one to the upper tertile for serum VEGF-A but not for plasma VEGF-A. Correlation analysis revealed a significant relationship between VEGF-A, HbA1c, inflammation, and diabetic nephropathy. Our results indicate that increased VEGF-A levels in T2DM significantly depend on quality of HbA1c control. Serum levels of VEGF-A, with a strong contribution of platelet derived VEGF, better reflect the glycemic burden than plasma levels of VEGF-A. Mechanistic studies are needed to explore links to inflammation and diabetic nephropathy., (© Georg Thieme Verlag KG Stuttgart · New York.)

Purpose: To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system., Methods: The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS)., Results: The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system., Conclusions: The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

Background: The macular hole (MH) is a disorder of the visual center of the retina in humans. An untreated MH leads to loss of central visual acuity and reading ability. Surgery for early-stage macular holes has been very successful for many years and leads to very good anatomical and functional results. Despite continuous improvement of surgical procedures, the outcome for the later stages of MH is still unsatisfactory., Method: In a retrospective analysis, we investigated the effect of autologous platelet concentrates in patients presenting later stages of MHs (stage III-IV) with respect to anatomic success (hole closure) and recovery of vision. The application of platelets was performed during retinal surgery (pars plana vitrectomy, ppV). In addition, selected platelet concentrates were qualitatively analysed for growth factors and platelet adhesion., Results: In the first group, 74% of the patients showed a good anatomical macular hole closure. The analyses of the platelet concentrates indicated a possible wound-healing effect due to growth factors (e.g. the platelet-derived growth factor, PDGF) and lesser to the ability of the platelets to adhere after ristocetin administration. Further optimization of the production process of platelet concentrates and of the surgical procedure in the second group of patients showed an increase of the anatomical success (92%) and a very rapid increase of visual acuity within six weeks., Discussion: In the past, the primary goal of MH surgery was to optimize the surgical procedures. Only few concepts focused on wound healing. Based on our data, we postulate the use of autologous platelet concentrates in MH surgery as a healing concept, which helps to increase the functional success of late-stage macular hole surgery.

Background and Purpose: In September 2011 the cornea section of the German Ophthalmological Society (DOG) established the first German Acanthamoeba keratitis registry. The data of this multicenter survey are being collected, compiled and evaluated at the Department of Ophthalmology at the Saarland University. The aim of this article is to present an intermediate report., Patients and Methods: Data from 172 eyes with Acanthamoeba keratitis were collected during the last 10 years. For this interim report we actually evaluated 121 eyes (60.2 % female patients, average age 41.3 years) and collected the following data: date of onset of symptoms, date and method of diagnosis, initial diagnosis, anamnestic data, clinical symptoms and signs at diagnosis and during follow-up, conservative and surgical therapy. Criteria for inclusion in the Acanthamoeba registry was the established diagnosis of an Acanthamoeba keratitis with at least one of the methods described in this article., Results: Acanthamoeba keratitis could be histologically proven in 55.3 % of the cases, via PCR in 25.6 %, with confocal microscopy in 20.4 % and using in vitro cultivation in 15.5 %. Clinical symptoms and signs in Acanthamoeba keratitis were pain in 67.0 %, ring infiltrates in 53.4 %, pseudodendritiform epitheliopathy in 11.7 % and keratoneuritis in 5.8 %. In 47.6 % of the cases the initial diagnosis was herpes simplex virus keratitis followed by bacterial keratitis in 25.2 % and fungal keratitis in 3.9 %. Acanthamoeba keratitis was the correct initial diagnosis in only 23.2 % of cases. The average time period between first symptoms and diagnosis was 2.8 ± 4.0 months (range 0-23 months). A triple therapy with Brolene® Lavasept® and antibiotic eye drops at least 5 ×/day was used in 54.5 % of eyes (n = 66). Penetrating keratoplasty was performed in 40.4 %, in 18 cases in combination with cryotherapy of the cornea. The mean graft diameter was 7.9 ± 1.1 mm (range 3.5-11.0 mm). The final visual acuity (Snellen visual acuity chart at 5 m) was comparable in the two groups of eyes with (5/40 ± 5/25) and without (5/32 ± 5/25) keratoplasty., Conclusion: Acanthamoeba keratitis is a rare and often very late diagnosed disease and two thirds of the cases were initially misdiagnosed. The early recognition of the typical symptoms is crucial for the prognosis of the disease. All ophthalmological departments in Germany are invited to submit further data of all confirmed cases (berthold.seitz@uks.eu), whether retrospectively or prospectively in order to generate an adequate standardized diagnostic and therapeutic approach for this potentially devastating disease.

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly( N -isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na + /K + -ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro . The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.

Amniotic membrane is applied to the diseased ocular surface to stimulate wound healing and tissue repair, because it releases supportive growth factors and cytokines. These effects fade within about a week after application, necessitating repeated application. Generally, amniotic membrane is fixed with sutures to the ocular surface, but surgical intervention at the inflamed or diseased site can be detrimental. Therefore, we have developed a system for the mounting of amniotic membrane between two rings for application to a diseased ocular surface without surgical intervention (sutureless amniotic membrane transplantation). With this system, AmnioClip, amniotic membrane can be applied like a large contact lens. First prototypes were tested in an experiment on oneself for wearing comfort. The final system was tested on 7 patients in a pilot study. A possible influence of the ring system on the biological effects of amniotic membrane was analyzed by histochemistry and by analyzing the expression of vascular endothelial growth factor-A (VEGF-A), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF 2) and pigment epithelium-derived factor (PEDF) from amniotic membranes before and after therapeutic application. The final product, AmnioClip, showed good tolerance and did not impair the biological effects of amniotic membrane. VEGF-A and PEDF mRNA was expressed in amniotic membrane after storage and mounting before transplantation, but was undetectable after a 7-day application period. Consequently, transplantation of amniotic membranes with AmnioClip provides a sutureless and hence improved therapeutic strategy for corneal surface disorders.Trial Registration:ClinicalTrials.gov NCT02168790

Background/aims: In a previous study, we observed a deleterious effect of serum-supplemented Minimal Essential Medium (MEM) on human corneal endothelial cell survival in a cell culture model. Consequently, here we studied the effects of conventional, serum-supplemented MEM and a serum-free medium in combination with two different deswelling substances on cell survival in whole corneas in a mouse model., Methods: Murine corneas were cultured for 4 days in MEM+2% fetal calf serum (FCS) or serum-free Human Endothelial-SFM (SFM), both supplemented with either 6% dextran T500 or 7.5% hydroxyethyl starch (HES) 130/0.4. Cells were examined by differential interference contrast microscopy, H&E staining, immunocytochemistry for cleaved caspase-3, Bcl-2, haem oxygenase-1 and immunoblotting for cleaved caspase-3., Results: In MEM, endothelial cells were almost completely lost after 4 days and the number of epithelial cells was markedly reduced. The remaining cells showed fragmented nuclei and were positive for cleaved caspase-3 and strongly positive for Bcl-2. Corneas cultured in SFM retained an almost closed layer of endothelial cells. Fewer cells were positive for Bcl-2, and only a few cells were positive for cleaved caspase-3 even under staurosporine administration. HES supplementation was well tolerated by corneal cells over 4 days, while a 4-day supplementation with dextran resulted in the loss of endothelial and epithelial cells., Conclusions: Serum-free medium, Human Endothelial-SFM, promoted cell survival during corneal organ culture better than MEM+2% FCS. HES 130/0.4 appeared to be tolerated better by the cells than dextran T500., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Endothelial cells (ECs) play a pivotal role in the allogeneic immune response upon transplantation. Semaphorin 3A (Sema3A) was implicated in the modulation of EC growth, but its effects on immunogenicity were not previously investigated., Study Design and Methods: ECs were transduced with a lentiviral vector encoding for the green fluorescence protein (GFP) sequence under the control of a Class II transactivator (CIITA)-dependent promoter. Upon stimulation of nonmodified ECs with recombinant Sema3A protein, mRNA and protein levels of CIITA, HLA-DR, and Sema3A receptors were evaluated. An enzyme-linked immunosorbent assay was developed to quantify Sema3A levels in the sera of kidney-transplanted patients., Results: Sema3A stimulation of lentiviral vector encoding for the GFP sequence ECs caused a significant up regulation of the transgene expression, indicating an increase in CIITA levels. Stimulation of nonmodified ECs with Sema3A resulted in an up regulation of CIITA expression, which was associated with enhanced HLA-DR levels and an increase in alloreactive CD4+ T-cell proliferation. Sema3A receptor expression was enhanced by CIITA, establishing a positive feedback loop. Higher levels of Sema3A were observed in sera of patients presenting with organ rejection., Conclusion: This study links Sema3A signaling in ECs with increased CIITA levels and higher HLA-DR expression, resulting in CD4+ T-cell activation, which might have important implications for tissue and organ transplantation., (© 2014 AABB.)

Background: To keep the loss of endothelial cell density in donor corneas to a minimum, a storage medium which is adjusted to their nutritional needs is necessary. Different media, used either serum-supplemented or serum-free, are available. The quality of medium- and serum-batches as well as support of endothelial cell viability by the medium are to be tested with a quality assured screening system that allows routine examination., Methods: A screening system was developed which is based on cell-culture tests with the well-established human corneal endothelial cell line HCEC-12, and therefore can be performed without the need for donor corneas. The cells are plated at a defined density in cell-culture dishes, and are cultured for a defined period of time in the test media. Evaluation is carried out by assaying cell count, activity of cell metabolism (resazurin conversion), and determining the number of apoptotic and necrotic cells (combined vital staining with YO-PRO®-1/propidium iodide and subsequent flow cytometry)., Results: Human corneal endothelial cells that are cultured in a medium which is adjusted to their nutritional needs achieve higher cell numbers and show a higher metabolic rate. Simultaneously, the percentage of apoptotic and necrotic cells is lower. The screening system developed in this study allows for easy and reliable detection of slightest differences between different media, different processing steps for same media, and different supplements, as well as different serum batches., Conclusions: The differentiated results show that the screening system is sensitive enough to show even minor quality differences. Therefore, it is more suitable than the hitherto commonly used growth assay with primary, mostly porcine, corneal endothelial cells.

Background and Purpose: The intracerebral hemorrhage (ICH) score is the most commonly used clinical grading scale for outcome prediction after adult ICH. We created a similar scale in children to inform clinical care and assist in clinical research., Methods: Children, full-term newborns to 18 years, with spontaneous ICH were prospectively enrolled from 2007 to 2012 at 3 centers. The pediatric ICH score was created by identifying factors associated with poor outcome. The score's ability to detect moderate disability or worse and severe disability or death was examined with sensitivity, specificity, and area under the receiver operating characteristic curve., Results: The pediatric ICH score components include ICH volume>2% to 3.99% of total brain volume (TBV): 1 point; ICH volume≥4% TBV: 2 points; acute hydrocephalus: 1 point; herniation: 1 point; and infratentorial location: 1 point. The score ranges from 0 to 5. At 3-month follow-up of 60 children, 10 were severely disabled or dead, 30 had moderate disability, and 20 had good recovery. A pediatric ICH score≥1 predicted moderate disability or worse with a sensitivity of 75% (95% confidence interval [CI], 59% to 87%) and a specificity of 70% (95% CI, 46% to 88%). A pediatric ICH score≥2 predicted severe disability or death with a sensitivity and specificity of 90% (95% CI, 55% to 99%) and 68% (95% CI, 53% to 80%), respectively. The area under the receiver operating characteristic curve for classifying outcome as severe disability or death was 0.88 (95% CI, 0.78-0.97)., Conclusions: The pediatric ICH score is a simple clinical grading scale that may ultimately be used for risk stratification, clinical care, and research.

Functional impairment of the human corneal endothelium can lead to corneal blindness. In order to meet the high demand for transplants with an appropriate human corneal endothelial cell density as a prerequisite for corneal function, several tissue engineering techniques have been developed to generate transplantable endothelial cell sheets. These approaches range from the use of natural membranes, biological polymers and biosynthetic material compositions, to completely synthetic materials as matrices for corneal endothelial cell sheet generation. This review gives an overview about currently used materials for the generation of transplantable corneal endothelial cell sheets with a special focus on thermo-responsive polymer coatings.

Amniotic membrane transplantation has been used very successfully in ophthalmology since the 1940s for treatment of surface disorders. Over the course of the years the indications for use have been continuously extended. In contrast the operative technique is predominantly invasive, i.e. the amniotic membrane is surgically sutured onto the surface of the eye. In order to avoid surgical trauma the authors have developed a device which allows the amniotic membrane to be stretched in a ring and the amniotic ring can then be placed suture-free on the eye surface. The authors are hopeful that the biological principle of the amniotic membrane can be better utilized by uncomplicated repetitive application.

Corneal endothelial diseases lead to severe vision impairment, motivating the transplantation of donor corneae or corneal endothelial lamellae, which is, however, impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. The current study presents a novel thermo-responsive carrier based on simultaneous electron beam immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces, which allows one to adjust layer thickness, stiffness, switching amplitude and functionalization with bioactive molecules to meet cell type specific requirements. The efficacy of this approach for HCEC, which require elaborate cell culture conditions and are strongly adherent to the substratum, is demonstrated. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Determination of protein function requires tools that allow its detection and/or purification. As generation of specific antibodies often is laborious and insufficient, protein tagging using epitopes that are recognized by commercially available antibodies and matrices appears more promising. Also, proper spatial and temporal expression of tagged proteins is required to prevent falsification of results. We developed a new series of binary Gateway cloning vectors named pAUL1-20 for C- and N-terminal in-frame fusion of proteins to four different tags: a single (i) HA epitope and (ii) Strep-tagIII, (iii) both epitopes combined to a double tag, and (iv) a triple tag consisting of the double tag extended by a Protein A tag possessing a 3C protease cleavage site. Expression can be driven by either the 35 S CaMV promoter or, for C-terminal fusions, promoters from genes encoding the chloroplast biogenesis factors HCF107, HCF136, or HCF173. Fusions of the four promoters to the GUS gene showed that endogenous promoter sequences are functional and drive expression more moderately and consistently throughout different transgenic lines when compared to the 35 S CaMV promoter. By testing complementation of mutations affected in chloroplast biogenesis factors HCF107 and HCF208, we found that the effect of different promoters and tags on protein function strongly depends on the protein itself. Single-step and tandem affinity purification of HCF208 via different tags confirmed the integrity of the cloned tags.

The related proteins D1 and D2 together build up the photosystem II reaction center. Synthesis of D1 (PsbA) is highly regulated in all photosynthetic organisms. The mechanisms and specific protein factors involved in controlled expression of the psbA gene in higher plants are highly elusive. Here, we report on the identification of a chloroplast-located protein, HCF244 (for high chlorophyll fluorescence244), which is essentially required for translational initiation of the psbA messenger RNA in Arabidopsis (Arabidopsis thaliana). The factor is highly conserved between land plants, algae, and cyanobacteria. HCF244 was identified by coexpression analysis of HCF173, which encodes a protein that is also necessary for psbA translational initiation and in addition for stabilization of this messenger RNA. Phenotypic characterization of the mutants hcf244 and hcf173 suggests that the corresponding proteins operate cooperatively during psbA translation. Immunolocalization studies detected the majority of the two proteins at the thylakoid membrane. Both HCF244 and HCF173 are members of the atypical short-chain dehydrogenase/reductase superfamily, a modified group, which has lost enzyme activity but acquires new functions in the metabolism of the cell.

Purpose: Recently, insertion of immuno-modulatory or anti-apoptotic genes into corneal endothelial cells (HCECs) came into focus. Basic FGF-2 occurs in one secreted (low molecular weight, LMW, 18 kD) and four nuclear (high molecular weight, HMW, 22-34 kD) isoforms. HMW isoforms are known differentiation and survival factors, while LMW FGF-2 is a known mitogen. The effect of FGF-2 overexpression of each of the five known isoforms on HCEC cell survival after lentiviral gene transfer in different culture media was investigated., Methods: Cells were transduced with lentiviral vectors encoding for each of the five FGF-2 isoforms. Transduction efficiency and expression of individual FGF-2 isoforms was assessed by marker gene transfer and western blotting. Primary HCECs were cultured and transduced in four different media previously described for HCEC cultivation or corneal organ cultivation. Cytotoxic effect of virus infection and a possible rescue effect of FGF-2 overexpression were determined by resazurin conversion assay., Results: Transduction with FGF-2 encoding lentiviral vectors resulted in overexpression of the respective isoform in all tested cell populations. Western blotting after total cell lysis proved nuclear localization of transgenic HMW isoforms. Overexpression of HMW FGF-2-especially 34 kD FGF-2-reduced lentiviral cytotoxicity, while overexpression of LMW FGF-2 aggravated viral cytotoxicity., Conclusions: Cytotoxicity of lentiviral gene transfer in corneal endothelial cells may be reduced by using bicistronic vectors that encode for the target gene and the 34-kD isoform of human FGF-2. Such cotransduction of a survival factor may increase cell survival after gene transfer, thereby improving gene therapeutic approaches.

The human cornea is a tri-laminar structure composed of several cell types with substantial mitotic potential. Age-related changes in the cornea are associated with declining visual acuity and the onset of overt age-related corneal diseases. Corneal transplantation is commonly used to restore vision in patients with damaged or diseased corneas. However, the supply of donor tissue is limited, and thus there is considerable interest in the development of tissue-engineered alternatives. A major obstacle to these approaches is the short replicative lifespan of primary human corneal endothelial cells (HCEC). Accordingly, a comprehensive investigation of the signalling pathways and mechanisms underpinning proliferative lifespan and senescence in HCEC was undertaken. The effects of exogenous human telomerase reverse transcriptase expression, p53 knockdown, disruption of the pRb pathway by over-expression of CDK4 and reduced oxygen concentration on the lifespan of primary HCEC were evaluated. We provide proof-of-principle that forced expression of telomerase, when combined with either p53 knockdown or CDK4 over-expression, is sufficient to produce immortalized HCEC lines. The resultant cell lines express an HCEC-specific transcriptional fingerprint, and retain expression of the corneal endothelial temperature-sensitive potassium channel, suggesting that significant dedifferentiation does not occur as a result of these modes of immortalization. Exploiting these insights into proliferative lifespan barriers in HCEC will underpin the development of novel strategies for cell-based therapies in the human cornea., (© 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Previous studies of pediatric intracerebral hemorrhage have investigated isolated intraparenchymal hemorrhage. The authors investigated whether detailed assessment of intraventricular hemorrhage enhanced outcome prediction after intracerebral hemorrhage. They prospectively enrolled 46 children, full-term to 17 years, median age 2.7 years, with spontaneous intraparenchymal hemorrhage and/or intraventricular hemorrhage. Outcome was assessed with the King's Outcome Scale for Childhood Head Injury. Twenty-six (57%) had intraparenchymal hemorrhage, 10 (22%) had pure intraventricular hemorrhage, and 10 (22%) had both. There were 2 deaths, both with intraparenchymal hemorrhage and intraventricular hemorrhage volume ≥4% of total brain volume. Presence of intraventricular hemorrhage was not associated with poor outcome, but hydrocephalus showed a trend (P = .09) toward poor outcome. In receiver operating characteristic curve analysis, combined intraparenchymal hemorrhage and intraventricular hemorrhage volume also showed a trend toward better outcome prediction than intraparenchymal hemorrhage volume alone. Although not an independent outcome predictor, future studies should assess intraventricular hemorrhage qualitatively and quantitatively.

The transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+)-and Mg(2+) permeable cation channel that might be a cellular osmosensor since it is activated upon hypotonic cell swelling. TRPV4 is also thermosensitive and responds to moderate heat (from 24 to 27 °C) as well as to phorbol esters (4α-PDD) and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. The resulting Ca(2+) influx occurring in response to swelling induces regulatory volume decrease (RVD) behavior. As regulation of cell volume is essential for corneal endothelial function, we determined whether human corneal endothelial cells have functional TRPV4 channel activity. RT-PCR identified TRPV4 gene expression in the HCEC-12 cell line as well as two clonal daughter cell lines (HCEC-H9C1, HCEC-B4G12). [Ca(2+)](i) transients were monitored in fura-2 loaded cells. Nonselective cation channel currents were recorded in the whole-cell mode of the planar patch-clamp technique. TRPV4 mRNA was found in HCEC-12 and the clonal daughter cell lines. TRPV4 channel agonists (4α-PDD and GSK1016790A; both 5 μmol/l) as well as moderate heat (<40 °C) elicited [Ca(2+)](i) transients. Hypotonicity increased [Ca(2+)](i) and nonselective cation channel currents in HCEC-12 cells. There is functional TRPV4 expression in HCEC-12 and in its clonal daughter cell lines based on Ca(2+) transients and underlying currents induced by known activators of this channel., (Published by Elsevier Ltd.)

Purpose: To evaluate retroviral vectors as a tool to transduce normal human corneal endothelial cells (HCECs) and to optimize transduction to increase gene transfer efficiency., Methods: Enhanced green fluorescent protein (EGFP) encoding retroviral vectors based on HIV-1 or murine leukemia virus (MLV), pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or a modified foamy virus envelope protein (FV Env), and prototype foamy virus (PFV) were produced. Transduction was performed in four HCEC culture media that were previously described for specific cultivation of HCECs or organ culture of donor corneas, namely enriched HCEC growth medium F99(HCEC), its unsupplemented basal medium F99, MEM + 2% fetal calf serum (FCS) (MEM), and Human Endothelial-SFM (SFM). Transduction efficiency was evaluated by marker gene transfer assay, and cytotoxic effects of virus infection were evaluated by means of resazurin conversion assay., Results: PFV- and HIV-1-based vectors showed superior transduction efficiency compared with MLV-based vectors. Pseudotyping with a modified FV Env increased transduction efficiency compared with pseudotyping with VSV-G. In medium SFM, transduction efficiency of PFV, HIV-1-/FV Env, and MLV-based vectors was markedly reduced compared with the other culture media. When cells were cultured in F99-based media, cell viability was reduced by retroviral transduction compared with uninfected or mock infected controls, but remained unaffected when cells were cultured in SFM and was even increased when cells were cultured in MEM., Conclusions: HIV-1-based vectors pseudotyped with FV Env can efficiently be used to transduce primary HCECs in vitro. However, transduction efficiency is dependent on culture conditions and impairs metabolic activity and viability of HCECs in vitro.

Background: Endothelial keratoplasty is a promising surgical procedure which may replace penetrating keratoplasty in cases of endothelial cell diseases of the cornea. This method may thereby help to prevent postoperative astigmatism and transplant rejection., Methods and Results: A survey of publications reporting about results after endothelial keratoplasty shows that the main problem of this transplantation technique is a postoperative endothelial cell loss which is comparable to or even higher than that observed in penetrating keratoplasty. Improving surgical techniques led to a reduction of the endothelial cell loss, however, cell-based strategies to prevent postoperative cell loss or to enhance the cell densities of donor corneas or endothelial lamellae are rare., Discussion: This review presents an overview of clinical results after endothelial keratoplasty. Current strategies in the field of cell biology and tissue cultivation of corneal endothelial cells, genetic manipulation of the corneal endothelium and tissue engineering strategies aiming at the production of transplantable endothelial cell sheets are described., Conclusion: The limited availability of donor corneas makes it mandatory to develop methods in the field of tissue engineering in order to improve corneal endothelial cell survival or to increase corneal endothelial cell density, using interdisciplinary approaches., (© Georg Thieme Verlag KG Stuttgart · New York.)

Aim: To evaluate the influence of organ culture media on corneal endothelial cell survival., Methods: The human corneal endothelial cell line HCEC-12 was cultured in five different media: human corneal endothelial cell (HCEC) growth medium (F99(HCEC)), standard minimal essential corneal organ culture medium (MEM)+2% fetal calf serum (FCS), MEM+5% FCS, and humanised, endothelial serum-free medium (SFM) (with and without antibiotics). A portion of the cells was treated with 0.5 μmol/l staurosporine and examined for signs of apoptosis by assessing mitochondrial membrane polarisation state (intravital JC-1 staining), by YO-PRO-1 and propidium iodide staining, by determining fragmentation of nuclei by sub-G1 DNA content, by immunocytochemistry for cleaved caspase-3, cleaved caspase-8, Bcl2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2), and by western blotting for cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP)., Results: The number of apoptotic cells in untreated control cultures was significantly higher in MEM compared with F99(HCEC) and SFM. Staurosporine treatment induced apoptosis in all tested cultures to varying degrees. Cells cultured in MEM showed stronger staining for cleaved caspase-3, cleaved caspase-8, Bax, Bcl-2 and cleaved PARP, increased sub-G1 DNA content, more propidium iodide- and YO-PRO-1-positive cells, and more mitochondria with depolarised membranes. All parameters were significantly higher in MEM compared with F99(HCEC) and SFM. SFM cultures were significantly less susceptible to cell stress., Conclusion: SFM is superior to MEM in promoting HCEC survival.

We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α(2) were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α(2) was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α(2) expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α(2)-mediated matrix binding was verified by preincubation with an α(2)-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers., (Copyright © 2011 S. Karger AG, Basel.)

Purpose: To use nanoscopically defined, two-dimensional matrices assembled from aligned collagen type I fibrils as a sheet substratum for in vitro cultivation of human corneal endothelial cells (HCECs). To assess the effect of matrix architecture on HCEC morphology and to characterize integrin-mediated HCEC-matrix interaction., Methods: Cell alignment and cell-matrix interactions of primary HCECs and three different immortalized HCEC populations on native and UV-cross-linked collagen type I matrices were examined by time-lapse microscopy. Specific integrin α(2)β(1) binding to the collagen matrix was demonstrated using a function-blocking α(2) antibody. Integrin α(2) subunit expression levels of the four HCEC populations were analyzed by Western blot analysis., Results: All HCEC populations aligned along the oriented collagen fibrils. Primary HCECs and, to a lesser extent, the other tested HCEC populations exerted high traction forces, leading to progressive matrix destruction. Cross-linking of the collagen matrices considerably increased matrix stability. Integrin subunit α(2) expression levels of the four cell types correlated with the degree of cell alignment and exertion of traction forces. In turn, blocking integrin subunit α(2) reduced cell alignment and prevented matrix destruction., Conclusions: HCECs align directionally along parallel arrays of collagen type I fibrils. The interactions of HCECs with collagen type I are primarily mediated by integrin α(2)β(1). Integrin subunit α(2) levels correlate with matrix contraction and subsequent destruction. Sustained cultivation of HCECs on ultrathin collagen matrices thus requires matrix cross-linking and moderate integrin α(2)β(1) expression levels.

The physiology and transparency of the cornea are dependent on corneal endothelial function. The role of temperature sensitive ion channels in maintaining such activity is unknown. This study was undertaken to probe for the functional expression of such pathways in human corneal endothelial cells (HCEC). We used HCEC-12, an immortalized population derived from whole corneal endothelium, and two morphologically distinct clonal cell lines derived from HCEC-12 (HCEC-H9C1, HCEC-B4G12) to probe for gene expression and function of transient receptor potential (TRP) channels of the vanilloid (V) isoform subfamily (i.e. TRPV1-3) in these cell types. Expression of TRPV isotypes 1, 2 and 3 were detected by RT-PCR. Protein expression of TRPV1 in situ was confirmed by immunostaining of corneoscleral remnants after keratoplasty. TRPV1-3 functional activity was evident based on capsaicin-induced Ca(2+) transients and induction of these responses through rises in ambient temperature from 25 degrees C to over 40 degrees C. The currents underlying Ca(2+) transients were characterized with a novel high throughput patch-clamp system. The TRPV1 selective agonist, capsaicin (CAP) (10-20 microM) increased non-selective cation whole-cell currents resulting in calcium increases that were fully blocked by either the TRPV1 antagonist capsazepine (CPZ) or removal of extracellular calcium. Similarly, heating from room temperature to over 40 degrees C increased the same currents resulting in calcium increases that were significantly reduced by the TRP channel blockers lanthanum chloride (La(3+)) (100 microM) and ruthenium-red (RuR) (10 microM), respectively. Moreover, application of the TRPV channel opener 2-aminoethoxydiphenyl borate (2-APB) (400 microM) led to a reversible increase in intracellular Ca(2+) indicating putative TRPV1-3 channel activity. Taken together, TRPV activity modulation by temperature underlies essential homeostatic mechanisms contributing to the support of corneal endothelial function under different ambient conditions., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The purpose of establishing the RheoNet registry was to evaluate the safety and efficacy of rheopheresis, a specific method of therapeutic apheresis used to treat microcirculatory disorders. Apheresis centers providing rheopheresis therapy and physicians caring for the underlying disease were asked to participate in the registry, and the registry data were analyzed for safety and tolerability. Age-related macular degeneration (AMD) was selected as a model disease to evaluate efficacy. The RheoNet registry was successfully established recording 7722 rheopheresis treatments of 1110 patients, including 833 AMD patients. The mean age of patients was 72 years. Adverse events (AE) were reported in 5.67% of treatments, but termination of the treatment session was only required in 0.48%. Transient hypotension was the most frequently reported AE. No age-related increase in AE was observed. Ophthalmological data of 428 eyes (of 279 treated patients) with dry AMD could be analyzed; 85 eyes of 55 untreated AMD patients served as the control. At 6.75 +/- 5.25 months post-baseline, 42% of the treated eyes had improved visual acuity. The proportion of eyes with a decline in visual acuity was 17%, compared to 40% in the untreated controls (P < 0.01). The RheoNet registry has been successfully established and data analysis revealed that rheopheresis is a safe outpatient treatment for microcirculatory disorders. Moreover, RheoNet currently provides the largest evaluation of the efficacy of rheopheresis for dry AMD. Registry analysis contributes to a safe and appropriate use of rheopheresis in clinical practice.

This is the first differential expression proteomics study on a human syngeneic cellular in vitro progression model of the colorectal adenoma-to-carcinoma sequence, the anchorage-dependent non-tumorigenic adenoma derived cell line AA/C1 and the derived anchorage-independent and tumorigenic carcinoma cell line AA/C1/SB10C. The study is based on quantitative 2-DE and is complemented by Western blot validation. Excluding redundancies due to proteolysis and post-translational modified isoforms of over 2000 protein spots, 13 proteins were revealed as regulated with statistical variance being within the 95th confidence level and were identified by peptide mass fingerprinting in MALDI MS. Progression-associated proteins belong to the functional complexes of anaerobic glycolysis/gluconeogenesis, steroid biosynthesis, prostaglandin biosynthesis, the regulation and maintenance of the cytoskeleton, protein biosynthesis and degradation, the regulation of apoptosis or other functions. Partial but significant overlap was revealed with previous proteomics and transcriptomics studies in colorectal carcinoma. Among upregulated proteins we identified 3-HMG-CoA synthase, protein phosphatase 1, prostaglandin E synthase 2, villin 1, annexin A1, triosephosphate isomerase, phosphoserine aminotransferase 1, fumarylacetoacetate hydrolase and pyrroline-5-carboxylate reductase 1 (PYCR1), while glucose-regulated protein 78, cathepsin D, lamin A/C and quinolate phosphoribosyltransferase were downregulated.

The MUC1 glycoprotein is considered a tumor antigen due to its over expression and aberrant glycosylation in cancer tissues. The latter results in appearance of new antigenic tumor specific glycopeptides not found on normal glycoforms of the mucin. MUC1 glycopeptides can be presented by APCs on MHC class II molecules to activate glycopeptide specific helper T-cells. No study has yet reported presentation of MUC1 glycopeptides on MHC class I molecules as stimulators of cytotoxic T-cells. In this study we show that human immunoproteasomes and cathepsin-L can generate octa to undecameric glycopeptides from the MUC1 repeat domain in vitro. We identified glycosylated fragments of which the decameric glycopeptide SAP10 [SAPDT(GalNAc)RPAPG] containing a single sugar binds with comparable strength to the MHC class I allele HLA A*0201 as predicted high-score binding epitopes of the tandem repeat. The same sequence glycosylated with the disaccharide Gal-GalNAc does not bind. The glycan on SAP10 is predicted by molecular modeling to either protrude out or point into the MHC groove. SAPDTRPAPG peptide and the respective glycopeptide stimulated cytotoxic T-cells in vitro. Our findings suggest that MUC1 tandem repeat glycopeptides are capable of activating both helper and cytotoxic T-cells and thus represent good candidates for further development as vaccines.