White Centered Retinal Hemorrhages in Vitamin B12 Deficiency Anemia

Background: To report a case of severe vitamin B12 deficiency anemia presenting with white centered retinal hemorrhages. Methods: Interventional case report. Results: A 40-year-old man, general practitioner himself, presented with a 1-day history of diminished left visual acuity and a drop-shaped central scotoma. The corrected visual acuities were 20/20, OD and 20/100, OS. Ophthalmic examination revealed bilaterally pale tarsal conjunctiva, discretely icteric bulbar conjunctiva and disseminated white centered intraretinal hemorrhages with foveal involvement. OCT imaging through these lesions revealed a retinal thickening caused by a sub-ILM accumulation of hyperreflective and inhomogeneous deposits within the nerve fiber layer. Immediate laboratory work-up showed severe megaloblastic anemia caused by vitamin B12 deficiency requiring erythrocyte transfusions. Discussion: Most reports of white centered retinal hemorrhages have been described in patients with leukemic retinopathy and bacterial endocarditis. It is interesting that this case of vitamin B12 deficiency anemia retinopathy has a clinically indistinguishable fundus appearance. This is probably due to the common pathology of capillary disruption and subsequent hemostatic fibrin plug formation. In megaloblastic anemia, direct anoxia results in endothelial dysfunction. The loss of impermeability allows extrusion of whole blood and subsequent diffusion from the disrupted site throughout and above the nerve fiber layer. Therefore the biomicroscopic pattern of white centered hemorrhages observed in anemic retinopathy is most likely due to the clot formation as the reparative sequence after capillary rupture

Calcium Electroporation Reduces Viability and Proliferation Capacity of Four Uveal Melanoma Cell Lines in 2D and 3D Cultures

Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes and permeabilization. Although EP represents an established therapeutic option for solid malignancies, recent advances shift to the investigation of non cytotoxic agents, such as calcium, which can also induce cell death. The present study aims to evaluate the cytotoxic effect, the morphological changes in tumor spheroids, the effect on the cell viability, and the cell-specific growth rate following calcium electroporation (CaEP) in uveal melanoma (UM) 2D monolayer cell cultures as well as in 3D tumor spheroid models. The experiments were conducted in four cell lines, UM92.1, Mel270, and two primary UM cell lines, UPMD2 and UPMM3 (UPM). The 2D and 3D UM cell cultures were electroporated with eight rectangular pulses (100 µs pulse duration, 5 Hz repetition frequency) of a 1000 V/cm pulse strength alone or in combination with 0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin. The application of calcium chloride alone induced an ATP reduction only in the UM92.1 2D cell cultures. Calcium alone had no significant effect on ATP levels in all four UM spheroids. A significant decrease in the intracellular adenosine triphosphate (ATP) level was documented in all four 2D and 3D cell cultures for both CaEP as well as ECT with bleomycin. The results suggest a dose-dependent ATP depletion with a wide range of sensitivity among the tested UM cell lines, control groups, and the applied settings in both 2D monolayer cell cultures and 3D tumor spheroid models. The colony formation capacity of the cell lines after two weeks reduced significantly after CaEP only with 0.5 mg/mL and 1.1 mg/mL, whereas the same effect could be achieved with both applied bleomycin concentrations, 1.0 µg/mL and 2.5 µg/mL, for the ECT group. The specific growth rate on day 7 following CaEP was significantly reduced in UM92.1 cell lines with 0.5 and 1.1 mg/mL calcium chloride, while Mel270 showed a similar effect only after administration of 1.1 mg/mL. UM92.1 and Mel270 spheroids exhibited lower adhesion and density after CaEP on day three in comparison to UPM spheroids showing detachment after day 7 following treatment. CaEP and bleomycin electroporation significantly reduce cell viability at similar applied voltage settings. CaEP may be a feasible and inexpensive therapeutic option for the local tumor control with fewer side effects, in comparison to other chemotherapeutic agents, for the treatment of uveal melanoma. The limited effect on normal cells and the surrounding tissue has already been investigated, but further research is necessary to clarify the effect on the surrounding tissue and to facilitate its application in a clinical setting for the eye

The Paediatric Glaucoma Diagnostic Ability of Optical Coherence Tomography: A Comparison of Macular Segmentation and Peripapillary Retinal Nerve Fibre Layer Thickness

Paediatric glaucoma leads to a decreased thickness of the peripapillary retinal nerve fibre layer (pRNFL) and of the macula. These changes can be precisely quantified using spectral domain-optical coherence tomography (SD-OCT). Despite abundant reports in adults, studies on the diagnostic capacity of macular SD-OCT in paediatric glaucoma are rare. The aim of this study was to compare the glaucoma discriminative ability of pRNFL and macular segment thickness in paediatric glaucoma patients and healthy children. Data of 72 children aged 5–17 years (glaucoma: 19 (26.4%), healthy: 53 (73.6%)) examined with SD-OCT (SPECTRALIS®, Heidelberg Engineering) were analysed retrospectively. The thickness of pRNFL sectors and of macular segment subfields were compared between diseased and healthy participants. Areas under the receiver-operating characteristic curves (AUC), sensitivity, and specificity from logistic regression were used to evaluate the glaucoma discriminative capacity of single and combined pRNFL and macular segments’ thickness. The results revealed a reduced thickness of the pRNFL and of the three inner macular layers in glaucoma patients, which correlates highly with the presence of glaucoma. The highest glaucoma discriminative ability was observed for the combination of pRNFL sectors or inner macular segments (AUC: 0.83 and 0.85, respectively), although sensitivity remained moderate (both 63% at 95% specificity). In conclusion, while confirmation from investigations in larger cohorts is required, SD-OCT-derived pRNFL and macular thickness measurements seem highly valuable for the diagnosis of paediatric glaucoma

Microvascular and Morphologic Changes of the Macula over Lifetime.

In ocular, neurologic, and cardiovascular diseases, macular segmentation data from spectral-domain optical coherence tomography (SD-OCT) provide morphologic, and OCT-angiography (OCTA) results give microvascular information about the macula. Age was shown to influence both methods' measurements. To further characterize this association, macular SD-OCT and OCTA changes were investigated in a population of juvenile, adult, and older individuals. Macular segment thickness and superficial (SCP) and deep plexus (DCP) vascular density (VD) of 157 healthy individuals aged 10-79 years were analyzed retrospectively. One-way analysis of variance (ANOVA) was used to compare age groups. The association between macular segmentation and OCTA parameters and between these and age was evaluated using linear regression. ANOVA and linear regression analysis showed a thickness decrease in the whole macular and in the ganglion cell and inner plexiform layers with age. While the foveal avascular zone area remained constant between age groups, VD of the SCP and DCP also decreased with age. In multiple linear regression, SCP and DCP VD were associated with inner macular segment thickness in an age-independent way. To conclude, the age-related microvascular and morphological changes in the macula described in this study can contribute to improving the understanding of macular aging processes and better interpreting OCT(A) results in healthy individuals and patients suffering from various retinal diseases

Chick Chorioallantoic Membrane as a Patient-Derived Xenograft Model for Uveal Melanoma : Imaging Modalities for Growth and Vascular Evaluation

Background: Patient-derived tumor xenografts (PDXs) have emerged as valuable preclinical in vivo models in oncology as they largely retain the polygenomic architecture of the human tumors from which they originate. Although animal models are accompanied by cost and time constraints and a low engraftment rate, PDXs have primarily been established in immunodeficient rodent models for the in vivo assessment of tumor characteristics and of novel therapeutic cancer targets. The chick chorioallantoic membrane (CAM) assay represents an attractive alternative in vivo model that has long been used in the research of tumor biology and angiogenesis, and can overcome some of these limitations. Methods: In this study, we reviewed different technical approaches for the establishment and monitoring of a CAM-based uveal melanoma PDX model. Forty-six fresh tumor grafts were acquired after enucleation from six uveal melanoma patients and were implanted onto the CAM on ED7 with Matrigel and a ring (group 1), with Matrigel (group 2), or natively without Matrigel or a ring (group 3). Real-time imaging techniques, such as various ultrasound modalities, optical coherence tomography, infrared imaging, and imaging analyses with Image J for tumor growth and extension, as well as color doppler, optical coherence angiography, and fluorescein angiography for angiogenesis, were performed on ED18 as alternative monitoring instruments. The tumor samples were excised on ED18 for histological assessment. Results: There were no significant differences between the three tested experimental groups regarding the length and width of the grafts during the development period. A statistically significant increase in volume (p = 0.0007) and weight (p = 0.0216) between ED7 and ED18 was only documented for tumor specimens of group 2. A significant correlation of the results for the cross-sectional area, largest basal diameter, and volume was documented between the different imaging and measurement techniques and the excised grafts. The formation of a vascular star around the tumor and of a vascular ring on the base of the tumor was observed for the majority of the viable developing grafts as a sign of successful engraftment. Conclusion: The establishment of a CAM-PDX uveal melanoma model could elucidate the biological growth patterns and the efficacy of new therapeutic options in vivo. The methodological novelty of this study, investigating different implanting techniques and exploiting advances in real-time imaging with multiple modalities, allows precise, quantitative assessment in the field of tumor experimentation, underlying the feasibility of CAM as an in vivo PDX model

Electrochemotherapy with Bleomycin Enhances Radiosensitivity of Uveal Melanomas: First In Vitro Results in 3D Cultures of Primary Uveal Melanoma Cell Lines

Electrochemotherapy (ECT) is emerging as a complementary treatment modality for local tumor control in various cancer entities. Irradiation is an established therapeutic option for oncologic patients, which is commonly combined with chemotherapy due to its insufficient targeting ability. The efficiency of radiotherapy for tumors can be enhanced with different radiosensitizers. ECT can potentiate the radiosensitizing effect of chemotherapeutic agents such as bleomycin. The present study aims to evaluate the radiosensitizing effect of concomitant ECT with bleomycin on 3D tumor spheroids with primary and radioresistant uveal melanoma cell lines (UPMD2, UPMM3, UM92.1, Mel270) and irradiation. The changes in the spheroid growth and the cell viability as well the cytotoxic long-term effect of the combination treatment were evaluated with various combinations of electroporation settings and bleomycin concentrations as well as radiotherapy doses. A broad range of radiosensitivity was documented among the spheroids from different uveal melanoma cell lines. The primary cell lines showed a higher radiosensitivity and required lower irradiation and bleomycin doses. The maximal tumor control with a reduction of cell survival <10% was achieved with a 5 Gy irradiation only in the primary uveal melanoma cell lines and in combination with all tested ECT settings, whereas the same result could be obtained in UM92.1 spheroids only after ECT with 20 Gy irradiation. Based on the spheroid growth and the measurement of the cross-sectional area, the Mel270 spheroids, originating from a previously irradiated recurrent uveal melanoma, required higher doses of bleomycin and ECT settings after irradiation with 5 Gy in order to achieve a significant growth reduction. No significant difference could be demonstrated for the reduction of cell viability in the combination therapy with 20 Gy and 1000 V/cm between 1 and 2.5 µg/mL bleomycin even in Mel270 spheroids, underlying the importance of a drug delivery system to potentiate the radiosensitizing effect of agents in lower doses. ECT should be further assessed for its applicability in clinical settings as a therapeutic radiosensitizing option for radioresistant tumors and a sufficient local tumor control with lower chemotherapy and irradiation doses

Optimisation of the Chicken Chorioallantoic Membrane Assay in Uveal Melanoma Research

The treatment of uveal melanoma and its metastases has not evolved sufficiently over the last decades in comparison to other tumour entities, posing a great challenge in the field of ocular oncology. Despite improvements in the conventional treatment regime and new discoveries about the genetic and molecular background of the primary tumour, effective treatment strategies to either prevent tumours or treat patients with advanced or metastatic disease are still lacking. New therapeutic options are necessary in order to achieve satisfactory local tumour control, reduce the risk of metastasis development, and preserve the eyeball and possibly the visual function of the eye. The development of in vivo model systems remains crucial for the identification and investigation of potential novel treatment modalities. The aim of this study was the optimisation of the chorioallantoic membrane (CAM) model for uveal melanoma research. We analysed the established CAM assay and its modification after the implantation of three-dimensional spheroids. The chorioallantoic membrane of a chick embryo was used to implant uveal melanoma-cell-line-derived spheroids in order to study their growth rate, angiogenic potential, and metastatic capability. Using the UM 92.1, UPMD2, UPMM3, and Mel270 cell lines, we were able to improve the viability of the embryos from 20% to >80% and to achieve up to a fourfold volume increase of the transplanted spheroid masses. The results point to the value of an optimised chicken embryo assay as an in vivo model for testing novel therapies for uveal melanoma by simplifying the research conditions and by contributing to a considerable reduction in animal experiments

Influence of biological sex, age and smoking on Graves’ orbitopathy – a ten-year tertiary referral center analysis

PurposeSeverity of Graves’ orbitopathy (GO) shows wide individual differences. For optimal treatment, it is important to be able to predict the natural course of the disease as accurate as possible to counteract with anti-inflammatory and surgical treatment. Therefore, we aimed to further elucidate the impact of sex, age and smoking on GO.MethodsWe collected the clinical and demographic data of all patients of our tertiary referral center from January 2008 till December 2018 and analyzed it with descriptive statistics. Only patients with a complete data set were included in the further analysis. Odds ratio’s for moderate-to-severe and sight-threatening GO in relation to age, sex and smoking were calculated by means of multivariate logistic regression models.ResultsWe evaluated the data of 4260 patient with GO and complete data sets. Most of these were women (83%). There were no significant differences between male and female patients regarding smoking habits and thyroid treatment. Men were significantly older at initial manifestation of TED (51.8 vs. 49.9y, p&lt;0.01) and showed significant more often severe stages (61% vs. 53%, p&lt;0.0001). Therefore, they needed significantly more intense treatment with steroids, irradiation, orbital decompression and muscle surgery. In multivariate logistic regression analyses age (OR 0.97, 95% CI:0.97-0.98, p&lt;0.0001), male sex (OR 1.64, 95% CI:1.38-1.9, p&lt;0.0001), smoking (OR 1.19, 95% CI:1.04-1.36, p=0.01), Grave’s disease (OR 1.55, 95% CI:1.26-1.90, p&lt;0.0001) and history of radioiodine treatment (RAI) (OR 2.44, 95% CI:2.10-2.86, p&lt;0.0001) showed an significant association with severe stages of GO.DiscussionOur retrospective analysis showed once more that women are more often afflicted by GO. In contrast, men seem to be more severely afflicted and in need of anti-inflammatory and surgical treatments. This might be due to a different approach to the health system and resilience to GO specific symptoms, as well as previously described worse thyroid control. Estrogen mediated effects might also play a role as in other autoimmune diseases and should be subject of further trials. Besides the biological sex, smoking could again be confirmed as serious risk factor for severe GO. Of note, RAI was associated with more severe stages of GO, which should be subject to further investigation

Potential involvement of the bone marrow in experimental Graves’ disease and thyroid eye disease

IntroductionGraves’ disease is an autoimmune disorder caused by auto-antibodies against the thyroid stimulating hormone receptor (TSHR). Overstimulation of the TSHR induces hyperthyroidism and thyroid eye disease (TED) as the most common extra thyroidal manifestation of Graves’ disease. In TED, the TSHR cross talks with the insulin-like growth factor 1 receptor (IGF-1R) in orbital fibroblasts leading to inflammation, deposition of hyaluronan and adipogenesis. The bone marrow may play an important role in autoimmune diseases, but its role in Graves’ disease and TED is unknown. Here, we investigated whether induction of experimental Graves’ disease and accompanying TED involves bone marrow activation and whether interference with IGF-1R signaling prevents this activation.ResultsImmunization of mice with TSHR resulted in an increase the numbers of CD4-positive T-lymphocytes (p ≤0.0001), which was normalized by linsitinib (p = 0.0029), an increase of CD19-positive B-lymphocytes (p= 0.0018), which was unaffected by linsitinib and a decrease of GR1-positive cells (p= 0.0038), which was prevented by linsitinib (p= 0.0027). In addition, we observed an increase of Sca-1 positive hematopietic stem cells (p= 0.0007) and of stromal cell-derived factor 1 (SDF-1) (p ≤0.0001) after immunization with TSHR which was prevented by linsitinib (Sca-1: p= 0.0008, SDF-1: p ≤0.0001). TSHR-immunization also resulted in upregulation of CCL-5, IL-6 and osteopontin (all p ≤0.0001) and a concomitant decrease of the immune-inhibitory cytokines IL-10 (p= 0.0064) and PGE2 (p ≤0.0001) in the bone marrow (all p≤ 0.0001). Treatment with the IGF-1R antagonist linsitinib blocked these events (all p ≤0.0001). We further demonstrate a down-regulation of arginase-1 expression (p= 0.0005) in the bone marrow in TSHR immunized mice, with a concomitant increase of local arginine (p ≤0.0001). Linsitinib induces an upregulation of arginase-1 resulting in low arginase levels in the bone marrow. Reconstitution of arginine in bone marrow cells in vitro prevented immune-inhibition by linsitinib.ConclusionCollectively, these data indicate that the bone marrow is activated in experimental Graves’ disease and TED, which is prevented by linsitinib. Linsitinib-mediated immune-inhibition is mediated, at least in part, by arginase-1 up-regulation, consumption of arginine and thereby immune inhibition

GA4GH Phenopackets: A Practical Introduction.

The Global Alliance for Genomics and Health (GA4GH) is developing a suite of coordinated standards for genomics for healthcare. The Phenopacket is a new GA4GH standard for sharing disease and phenotype information that characterizes an individual person, linking that individual to detailed phenotypic descriptions, genetic information, diagnoses, and treatments. A detailed example is presented that illustrates how to use the schema to represent the clinical course of a patient with retinoblastoma, including demographic information, the clinical diagnosis, phenotypic features and clinical measurements, an examination of the extirpated tumor, therapies, and the results of genomic analysis. The Phenopacket Schema, together with other GA4GH data and technical standards, will enable data exchange and provide a foundation for the computational analysis of disease and phenotype information to improve our ability to diagnose and conduct research on all types of disorders, including cancer and rare diseases