Lens Epithelial Cell Proliferation in Response to Ionizing Radiation.

Lens epithelial cell proliferation and differentiation are naturally well regulated and controlled, a characteristic essential for lens structure, symmetry and function. The effect of ionizing radiation on lens epithelial cell proliferation has been demonstrated in previous studies at high acute doses, but the effect of dose and dose rate on proliferation has not yet been considered. In this work, mice received single acute doses of 0.5, 1 and 2 Gy of radiation, at dose rates of 0.063 and 0.3 Gy/min. Eye lenses were isolated postirradiation at 30 min up until 14 days and flat-mounted. Then, cell proliferation rates were determined using biomarker Ki67. As expected, radiation increased cell proliferation 2 and 24 h postirradiation transiently (undetectable 14 days postirradiation) and was dose dependent (changes were very significant at 2 Gy; P = 0.008). A dose-rate effect did not reach significance in this study (P = 0.054). However, dose rate and lens epithelial cell region showed significant interactions (P < 0.001). These observations further our mechanistic understanding of how the lens responds to radiation.

[Critical endothelial procedures during posterior lamellar graft preparation and transplantation]. / Endothelkritische Prozeduren während der Herstellung und Transplantation posteriorer lamellärer Hornhauttransplantate.

BACKGROUND: In view of the very low proliferation rate and functional importance of the corneal endothelium in maintaining corneal transparency, safeguarding the integrity of this monolayer plays a central role in posterior lamellar corneal transplantation. Several critical endothelial procedural stages are necessary to carry out such a transplantation. OBJECTIVE: This article presents various preparatory and operative approaches for carrying out the necessary and critical stages within the framework of posterior lamellar corneal transplantation and concentrates on the question of optimization. METHODS: A review of our own studies and studies of other groups is presented. RESULTS: For the performance of critical endothelial procedural steps, a variety of approaches are available. These range from preparation and insertion of the transplant, through the manipulation during centralization up to the effects of postoperative air or gas bubble tamponade. CONCLUSION: Because endothelial damage can permanently impair the integrity of lamellar transplants, a minimal handling and no touch policy should be strived for in all critical procedures. Long-term data on the follow-up course will show which of the procedures favored by various authors lead to the best postoperative results.

[The emerging technology of tissue engineering : Focus on stem cell niche]. / Zukunftstechnologie Tissue-Engineering : Fokus auf die Stammzellnische.

Limbal stem cells reside in a highly specialized complex microenvironment that is known as the stem cell niche, an anatomically protected region at the bottom of the Palisades of Vogt, where the stem cells are located and where their quiescence, proliferation and differentiation are maintained in balance. Besides the epithelial stem and progenitor cell clusters, the limbal niche comprises several types of supporting niche cells and a specific extracellular matrix mediating biochemical and biophysical signals. Stem cell-based tissue engineering aims to mimic the native stem cell niche and to present appropriate microenvironmental cues in a controlled and reproducible fashion in order to maintain stem cell function within the graft. Current therapeutic approaches for ex vivo expansion of limbal stem cells only take advantage of surrogate niches. However, new insights into the molecular composition of the limbal niche and innovative biosynthetic scaffolds have stimulated novel strategies for niche-driven stem cell cultivation. Promising experimental approaches include collagen-based organotypic coculture systems of limbal epithelial stem cells with their niche cells and biomimetic hydrogel platforms prefunctionalized with appropriate biomolecular and biophysical signals. Future translation of these novel regenerative strategies into clinical application is expected to improve long-term outcomes of limbal stem cell transplantation for ocular surface reconstruction.

[Therapy options for malignant eyelid tumors]. / Therapie bei malignen Lidtumoren.

Basal cell carcinomas are the most common malignant tumors of the eyelids. Patient history, clinical symptoms and signs, inspection, palpation and slit-lamp examination usually allow a working diagnosis; however, the clinical diagnosis requires histopathological confirmation and determination of the histopathological type. Squamous cell carcinomas, sebaceous gland carcinomas, melanomas and Merkel cell carcinomas can metastasize usually via the lymph vessels into the regional lymph nodes. Microscopically controlled excision of the primary tumor into healthy tissue is most commonly the first goal. Palpation and ultrasonography of the regional lymph nodes and also computed tomography (CT) with contrast enhancement and magnetic resonance imaging (MRI) for tumors with perineural sheath cell invasion are necessary to define the TNM stage. Non-surgical treatment options are becoming more popular in the further management of malignant eyelid tumors.

Tomo-seq: A method to obtain genome-wide expression data with spatial resolution.

To improve our understanding of pattern formation during development and disease we heavily rely on the identification of novel regulators and pathways. While RNA sequencing yields genome-wide expression data that suit this purpose, it lacks spatial resolution. Such spatial resolution can be obtained by microscopy-based methods like in situ hybridization, but these fail to provide information on more than a few genes at a time. Here, we describe tomo-seq, a technique that combines the advantages of the above-mentioned approaches and provides genome-wide expression data with spatial information. The tomo-seq technique is based on cryosectioning of an embryo or tissue of interest and performing RNA-seq on individual sections. Using this method, we have generated genome-wide transcriptomics with high spatial resolution of the whole zebrafish embryo at various stages of development (Junker et al., 2014) and of adult zebrafish hearts after injury (Wu et al., 2016).

Blockade of CCR7 leads to decreased dendritic cell migration to draining lymph nodes and promotes graft survival in low-risk corneal transplantation.

The chemokine receptor CCR7 is essential for migration of mature dendritic cells (DCs) to the regional lymph nodes, and it has been shown that blocking of CCR7 improves graft survival after high-risk corneal transplantation in vascularized recipient corneas. However, it is so far unknown whether blocking of CCR7 reduces migration of DCs from the avascular cornea to the draining lymph nodes and whether this leads to improved graft survival also in the low-risk setting of corneal transplantation, which accounts for the majority of perforating transplantations performed. Therefore, in this study, pellets containing Freund's adjuvant and bovine serum albumin (BSA) conjugated to Alexa488 fluorescent dye were implanted into the corneal stroma of BALB/c mice to analyze antigen uptake by corneal DCs and their migration to the regional lymph nodes. After pellet implantation, mice were either treated by local administration of a CCR7 blocking fusion protein that consisted of CCL19 fused to the Fc part of human IgG1 or a control-IgG. In vivo fluorescence microscopy showed uptake of Alexa488-conjugated BSA by corneal DCs within 8 h. Furthermore, analysis of single cell suspensions of draining lymph nodes prepared after 48 h revealed that 2.1 ± 0.3% of CD11c(+) cells were also Alexa488(+). Importantly, DC migration was significantly reduced after topical administration of CCL19-IgG (1.2 ± 0.2%; p < 0.05). To test the effect of CCR7 blockade on graft rejection after allogeneic low-risk keratoplasty, corneal transplantations were performed using C57BL/6-mice as donors and BALB/c-mice as recipients. Treatment mice received two intraperitoneal loading doses of CCL19-IgG prior to transplantation, followed by local treatment with CCL19-IgG containing eye drops for the first two weeks after transplantation. Control mice received same amounts of control-IgG. Kaplan-Meier survival analysis showed that in the CCL19-IgG treated group, 76% of the grafts survived through the end of the 8 week observation period, whereas 38% of the grafts survived in the control group (p < 0.05). Taken together, our study shows that blockade of CCR7 reduces the migration of mature corneal DCs to the draining lymph nodes and leads to improved graft survival in low-risk corneal transplantation.

[Pathology and prognostic factors of conjunctival melanoma]. / Pathologie und Prognosefaktoren konjunktivaler Melanome.

BACKGROUND: Conjunctival melanoma is a rare but potentially fatal disease. The 10-year melanoma mortality can be up to 30 %, recurrence rates after treatment up to 50 % and the overall incidence of metastasis is 26 %. Improved treatment options are needed to increase the tumor-free survival of affected patients. OBJECTIVES: The aim of the study was to perform clinical and pathological staging using the TNM classification and to correlate the results with treatment modalities and recurrence rates. MATERIAL AND METHODS: The study included a case series of 80 eyes from 80 patients (42 females and 38 males, age 28-90 years) with histopathologically proven conjunctival melanoma studied by reviewing medical records, pathology reports and color photographs. The main evaluated characteristics were demographic information, tumor size, thickness, pathological diagnosis, BRAF mutation testing, clinical and pathological staging, recurrence, metastasis and duration of follow-up (mean 48 months). RESULTS: The lesions predominantly involved the bulbar conjunctiva (60 %) and other sites that were less often involved were the palpebral conjunctiva (23 %), conjunctival fornix (22 %) and lacrimal caruncle (15 %). Of the tumors 36 % were TNM classified as pTis, 34 % as pT1, 20 % as pT2 (palpebral, fornix and caruncle) and 10 % as pT3. Local recurrences were noted in 36 % of the patients (18 % Tis, 26 % T1, 32 % T2 and 70 % T3) and regional and distant metastasis in 20 % of the patients (0 % Tis, 10 % T1, 15 % T2 and 60 % T3). DISCUSSION: In this study increasing T stages were more often associated with recurrences and metastasis. Future studies correlating the TNM staging with histopathological and genetic predictors may help to improve the management of patients with conjunctival melanoma.

[Immunotherapy of uveal melanoma: vaccination against cancer. Multicenter adjuvant phase 3 vaccination study using dendritic cells laden with tumor RNA for large newly diagnosed uveal melanoma]. / Immuntherapie beim Aderhautmelanom: Vakzination gegen Krebs. Multizentrische adjuvante Phase-III-Impfstudie mit Tumor-RNA-beladenen dendritischen Zellen bei neu diagnostizierten, großen Uveamelanomen.

Uveal melanomas are the most common malignant tumors of the eye. With modern molecular biological diagnostic methods, such as chromosome 3 typing and gene expression analysis, these tumors can be categorized into highly aggressive (monosomy 3, class II) and less aggressive forms. This molecular biological stratification is primarily important for determining the risk of these tumors as no therapy is currently available that is able to prevent or delay metastases. A randomized study of patients with a poor prognosis (monosomy 3) is currently being carried out in order to determine whether a cancer vaccine prepared from autologous (patient's own) dendritic cells and uveal melanoma RNA can prevent or delay progression and further metastases of this extremely aggressive form of cancer. Inclusion in the uveal melanoma study, which hopes to provide a potential therapeutic option for patients, is only possible if patients are referred to an institution that is able to manufacture and provide this vaccination before the patient is operated on or treated with radiation. Untreated tumor material is necessary for producing the vaccine on an individualized patient basis.

[Treatment of severe recurrent symblepharopterygium: combined ipsilateral autologous limbus and homologous amniotic membrane transplantation]. / Behandlung des schweren rezidivierenden Pterygiums mit Symblepharonbildung: Kombinierte ipsilaterale autologe Limbus- und homologe Amnionmembrantransplantation.

BACKGROUND: This retrospective study reports on four patients with severe recurrent symblepharopterygium formation and extensive subconjunctival scarring who were treated with a novel surgical technique combining free limbal autografting and amniotic membrane transplantation. PATIENTS AND METHODS: The surgical technique included symblepharolysis, meticulous removal of subconjunctival scar tissue, ipsilateral free limbal autograft and homologous amniotic membrane transplantation. RESULTS: There were no intraoperative or postoperative adverse events and three patients had no manifestation of recurrence of pterygium, symblepharon or diplopia during a mean follow-up period of 172 ± 18 weeks (39 ± 4 months) postoperatively. Only one patient had persistent symblepharon and experienced a recurrence of pterygium approximately 40 weeks (9 months) after surgery. CONCLUSION: The results suggest that ipsilateral autologous limbal and homologous amniotic membrane transplantation can be an effective therapeutic approach in the treatment of recurrent pterygium with symblepharon formation.

[Pigment dispersion syndrome and pigmentary glaucoma. Morphometric analysis of the anterior chamber segment with SL-OCT]. / Melanindispersionssyndrom und -glaukom. Morphometrische Analyse des vorderen Augenabschnittes mittels SL-OCT.

BACKGROUND: The purpose of this study was to analyze if anterior chamber parameters are risk factors for the development of pigment dispersion syndrome (PDS) and/or for the conversion to pigmentary glaucoma (PG). PATIENTS AND METHODS: This study included a total of 63 eyes from 35 patients with PDS and PG and 65 eyes from 49 unaffected volunteers as the control group. The following parameters were measured by slit lamp optical coherence tomography (SL-OCT): anterior chamber volume (ACV) and depth (ACD), angle opening distance (AOD) and the trabecular iris space area (TISA) at 500 µm and 750 µm from the scleral spur. Comparisons between the following groups were performed: between the PDS/PG and the control group, between PDS and PG and between male and female patients. RESULTS: The results of ACV, ACD, AOD and TISA were significantly higher in PDS/PG patients when compared to the control group. There were no significant differences between PDS and PG. The gender-specific comparison also showed no significant differences. CONCLUSIONS: Significantly higher anterior chamber parameters are a possible risk factor for development of PDS; however, a higher risk of conversion to PG does not seem to correlate with increased anterior chamber parameters. The parameters of the anterior chamber are apparently not associated with the male predominance of PDS and PG.

Anti-CD4 treatment inhibits autoimmunity in scurfy mice through the attenuation of co-stimulatory signals.

A major concept in autoimmunity is that disruption of Foxp3(+) regulatory T cells (Tregs) predisposes to breach of tolerance. This is exemplified by the Foxp3-linked disorder termed IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked) which affects newborn children. There has been considerable clinical interest in the role of non-depleting anti-CD4 antibodies as a means of upregulating the function of Foxp3(+) Tregs in order to control detrimental inflammatory responses such as transplant rejection. However, according to the paradigm of a Treg-dependent mechanism of action, the effectiveness of anti-CD4 antibodies as a therapy for human autoimmune diseases is unclear considering that Treg function might be intrinsically impaired. Specifically, anti-CD4 therapy is expected to fail in patients suffering from the IPEX syndrome due to the lack of functional Foxp3(+) Tregs. Taking advantage of natural Foxp3 mutant scurfy (sf) mice closely resembling the IPEX syndrome, and genetically engineered mice depleted of Foxp3(+) Tregs, we report here that anti-CD4 treatment induces tolerance independent of Foxp3(+) Tregs. This so far undefined mechanism is dependent on the recessive non-infectious tolerization of autoreactive T cells. Treg-independent tolerance alone is powerful enough to suppress both the onset and severity of autoimmunity and reduces clinically relevant autoantibody levels and liver fibrosis. Mechanistically, tolerance induction requires the concomitant activation of autoreactive T cells and is associated with the down-regulation of the co-stimulatory TNF-receptor superfamily members OX40 and CD30 sustaining CD4(+) T cell survival. In the light of ongoing clinical trials, our results highlight an unexpected potency of anti-CD4 antibodies for the treatment of autoimmune diseases. Particularly, CD4 blockade might represent a novel therapeutic option for the human IPEX syndrome.

[Limbal stem cells and their niche: implications for bioengineered tissue constructs]. / Limbusstammzellen und ihre nische: bedeutung für biotechnologischen gewebeersatz.

Regeneration and repair of corneal epithelium rely on a reservoir of unipotent progenitor cells, which is situated within the basal epithelial layer at the corneoscleral limbus. If these cells are lost, corneal surface integrity is disturbed, which may lead to a painful loss of vision. Since the late 1990s cultivated grafts of limbal epithelium are being used therapeutically. Limbal epithelial cells are obtained from the fellow eye or from an allogeneic donor, propagated in culture on different types of carriers, and subsequently transplanted. This process entails removal of progenitor cells from their natural environment. However, surrounding cells and extracellular matrix are widely believed to provide important stimuli for stem cell maintenance and for correct differentiation. Therefore, new approaches aim at providing this so-called stem cell niche ex vivo and following transplantation. Niche factors can also drive transdifferentiation of alternative progenitor cell types towards a corneal phenotype. This permits the use of autologous cells in cases of bilateral limbal stem cell insufficiency. Several biosynthetic substrates have been devised for culture, transdifferentiation and transplantation of donor cells. This work intends to provide an overview of constructs that are currently available and to some extent clinically employed. In addition, a summary is given of novel concepts which aim at integrating putative niche factors into the stem cell carriers to replicate the stem cell niche.

[Cross-linking in an artificial human cornea via induction of tissue transglutaminases]. / Vernetzung einer humanen künstlichen Hornhaut durch Induktion von Tissue-Transglutaminasen.

PURPOSE: In recent years many three-dimensional cornea models have been developed. However, they show poor collagen stability in the stroma. Transglutaminases (Tgases) are calcium-dependent proteins which play an important role in cross-linking of the corneal stroma. The purpose of this study was to find out whether it is possible to induce in vitro cross-linking of the stroma in an artificial hemicornea model with the help of Tgases. MATERIALS AND METHODS: For the construction of the hemicornea, human SV40 adenovector corneal epithelial cells (HCE) and human SV40 adenovector corneal keratocytes (HCK) were cultivated. Confluent HCK cells were treated for 24 h with transforming growth factor beta (TGFb) 1, 2 and 3 at different concentrations as well as with other growth factors and the treated cells were compared to untreated cultivated cells. The quantification of the expression of the Tgases by HCKs was examined with the use of real time PCR, Western blot imaging and immunochemistry. RESULTS: All concentrations of TGFbs used resulted in a significant increase of Tgase-mRNA, Tgase protein level and Tgase activity. The Tgases remained unaffected after treatment with other growth factors in comparison to untreated control cells. Treatment of the hemicornea with TGFb2 showed a very strong contraction and haze in comparison to the untreated hemicornea. CONCLUSION: It has been shown for the first time that TGFb induces a strong expression of Tgases in HCK cells. This effect caused an undesired contraction and haze of the human hemicornea model. Further research is necessary in order to find out whether the induction of Tgases in the HCK cells can be regulated without losing stability of the constructed hemicornea.

[Topical cyclosporine A 0.05% in the treatment of keratoconjunctivitis sicca]. / Ciclosporin A 0,05 % Augentropfen zur Therapie der Keratokonjunktivitis sicca.

BACKGROUND: The current understanding of the pathogenesis of dry eye disease has proceeded to recognition of inflammation as the key pathogenetic mechanism. The purpose of this study was to evaluate the effect of cyclosporine 0.05% eye drops on subjective symptoms and objective signs of patients with keratoconjunctivitis sicca. PATIENTS AND METHODS: In this clinical trial 62 patients with severe keratoconjunctivitis sicca (DEWS grade 3) were included. Over a time period of 3 months all patients received treatment with preservative-free hyaluronic acid artificial tears at one drop 5 times per day and in addition 31 patients received one drop of cyclosporine 0.05% twice daily. Screening parameters were LIPCOF, tear break-up time (BUT), fluorescein and rose bengal staining, the intraocular pressure (IOP) and the OSDI score. RESULTS: In the cyclosporine A group BUT, Jones test and OSDI score improved significantly after 3 months in contrast to the controls. Moreover the values of BUT and Jones test in the cyclosporine A group were significantly higher after 3 months compared to the healthy controls. Fluorescein and rose bengal staining improved only in the cyclosporine A group after 3 months. CONCLUSION: Anti-inflammatory therapy with cyclosporine A 0.05% eye drops as off label use significantly improves subjective symptoms and objective signs in patients with severe dry eye disease providing a good safety profile. These findings suggest a widespread use of cyclosporine A 0.05% eye drops in patients with moderate to severe keratoconjunctivitis sicca.

Influence of atypical retardation pattern on the peripapillary retinal nerve fibre distribution assessed by scanning laser polarimetry and optical coherence tomography.

AIM: To investigate the influence of atypical retardation pattern (ARP) on the distribution of peripapillary retinal nerve fibre layer (RNFL) thickness measured with scanning laser polarimetry in healthy individuals and to compare these results with RNFL thickness from spectral domain optical coherence tomography (OCT) in the same subjects. METHODS: 120 healthy subjects were investigated in this study. All volunteers received detailed ophthalmological examination, GDx variable corneal compensation (VCC) and Spectralis-OCT. The subjects were divided into four subgroups according to their typical scan score (TSS): very typical with TSS=100, typical with 99 ≥ TSS ≥ 91, less typical with 90 ≥ TSS ≥ 81 and atypical with TSS ≤ 80. Deviations from very typical normal values were calculated for 32 sectors for each group. RESULTS: There was a systematic variation of the RNFL thickness deviation around the optic nerve head in the atypical group for the GDxVCC results. The highest percentage deviation of about 96% appeared temporal with decreasing deviation towards the superior and inferior sectors, and nasal sectors exhibited a deviation of 30%. Percentage deviations from very typical RNFL values decreased with increasing TSS. No systematic variation could be found if the RNFL thickness deviation between different TSS-groups was compared with the OCT results. CONCLUSIONS: The ARP has a major impact on the peripapillary RNFL distribution assessed by GDx VCC; thus, the TSS should be included in the standard printout.

[Screening questionnaire for documentation of medical history and diagnostic findings in dry eye disease]. / Erhebungsbogen zur Anamnese und Diagnostik des trockenen Auges.

Keratoconjunctivitis sicca is one of the most common ocular diseases world-wide. These patients suffer from severe symptoms which lead to an extremely reduced quality of life. Dry eye syndrome constitutes a major diagnostic and therapeutic challenge to all ophthalmologists because there is often a discrepancy between objective ocular signs and subjective symptoms of the patients. Furthermore, there exist only few causal therapeutic options. The physician-patient relationship plays an outstanding role in this condition. For the treatment of moderate to severe dry eye syndrome, special dry eye clinics have proved to be extremely useful. For follow-up measurements as well as the realisation of evidence-based medicine and quality control, it is a fundamental necessity to document symptoms, signs and therapy of these patients in order to optimise therapeutic strategies. For this purpose, we have developed special forms and standardised questionnaires for the individual documentation of medical history and diagnostic findings. To objectively assess the patient's complaints we use the "ocular surface disease index" (OSDI score). Only the establishment of standardised diagnostic and therapeutic algorithms with the help of special forms and questionnaires can help in the long run to improve the treatment of these severely affected patients.

[Histopathology of retrocorneal membranes after keratoplasty]. / Histopathologische Untersuchung von retrokornealen Membranen bei irreversiblem Transplantatversagen.

BACKGROUND: This retrospective study examines the histopathological changes, especially the occurrence of retrocorneal membranes, in irreversible graft failure after penetrating keratoplasty. PATIENTS/MATERIALS AND METHODS: 371 corneas of 308 patients were examined. The examination was carried out using a light microscope. RESULTS: 45% of the corneas (167/371) showed a retrocorneal membrane with a thickness of 2-520 micrometers. Re-endothelialisation was detected in 75 cases. In 74% (124/167) cellular infiltration into the stroma could be observed. In 32% (119/371) the graft-host border was visible. CONCLUSIONS: Retrocorneal membranes are a frequent finding in irreversible graft failure after penetrating keratoplasty. Aetiologically the graft-host border as well as the formation of connective tissue seem to play a key role.

[Complications after posterior lamellar keratoplasty (DSAEK): prevention, detection and treatment]. / Komplikationen nach posteriorer lamellärer Keratoplastik (DSAEK): Vermeiden, Erkennen und Behandeln.

PURPOSE: The aim of this study was to outline typical complications after Descemet's stripping automated endothelial keratoplasty (DSAEK) and to discuss their prevention and management. METHODS: Our own clinical results and PUBMED literature search were evaluated. RESULTS: Postoperative flap dislocation, which can be effectively treated by re-bubbling the graft, is the most common and typical complication after DSAEK. CONCLUSIONS: Careful preoperative indication, surgery and postoperative care make DSAEK a safe and effective new therapeutic option for patients with endothelial corneal disease.