Diabetic Retinopathy Screening in Patients with Diabetes Using a Handheld Fundus Camera: The Experience from the South-Eastern Region in Hungary.

PURPOSE: Diabetic retinopathy (DR) is the leading cause of vision loss among active adults in industrialized countries. We aimed to investigate the prevalence of diabetes mellitus (DM), DR and its different grades, in patients with DM in the Csongrád County, South-Eastern region, Hungary. Furthermore, we aimed to detect the risk factors for developing DR and the diabetology/ophthalmology screening patterns and frequencies, as well as the effect of socioeconomic status- (SES-) related factors on the health and behavior of DM patients. METHODS: A cross-sectional study was conducted on adults (>18 years) involving handheld fundus camera screening (Smartscope Pro Optomed, Finland) and image assessment using the Spectra DR software (Health Intelligence, England). Self-completed questionnaires on self-perceived health status (SPHS) and health behavior, as well as visual acuity, HbA1c level, type of DM, and attendance at healthcare services were also recorded. RESULTS: 787 participants with fundus camera images and full self-administered questionnaires were included in the study; 46.2% of the images were unassessable. T1D and T2D were present in 13.5% and 86.5% of the participants, respectively. Among the T1D and T2D patients, 25.0% and 33.5% had DR, respectively. The SES showed significant proportion differences in the T1D group. Lower education was associated with a lower DR rate compared to non-DR (7.7% vs. 40.5%), while bad/very bad perceived financial status was associated with significantly higher DR proportion compared to non-DR (63.6% vs. 22.2%). Neither the SPHS nor the health behavior showed a significant relationship with the disease for both DM groups. Mild nonproliferative retinopathy without maculopathy (R1M0) was detected in 6% and 23% of the T1D and T2D patients having DR, respectively; R1 with maculopathy (R1M1) was present in 82% and 66% of the T1D and T2D groups, respectively. Both moderate nonproliferative retinopathy with maculopathy (R2M1) and active proliferative retinopathy with maculopathy (R3M1) were detected in 6% and 7% of the T1D and T2D patients having DR, respectively. The level of HbA1c affected the attendance at the diabetology screening (HbA1c > 7% associated with >50% of all quarter-yearly attendance in DM patients, and with 10% of the diabetology screening nonattendance). CONCLUSION: The prevalence of DM and DR in the studied population in Hungary followed the country trend, with a slightly higher sight-threatening DR than the previously reported national average. SES appears to affect the DR rate, in particular, for T1D. Although DR screening using handheld cameras seems to be simple and dynamic, much training and experience, as well as overcoming the issue of decreased optic clarity is needed to achieve a proper level of image assessability, and in particular, for use in future telemedicine or artificial intelligence screening programs.

Ex vivo 3D human corneal stroma model for Schnyder corneal dystrophy - role of autophagy in its pathogenesis and resolution.

INTRODUCTION: Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. METHODS: Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50 nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours. RESULTS: CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. CONCLUSIONS: MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.

Cultivation and characterization of pterygium as an ex vivo study model for disease and therapy.

PURPOSE: Development of ex vivo model to study pathogenesis, inflammation and treatment modalities for pterygium. METHODS: Pterygium obtained from surgery was cultivated (3 months). Gravitational attachment method using viscoelastic facilitated adherence of graft and outgrowing cells. Medium contained serum as the only growth supplement with no use of scaffolds. Surface profiling of the multi-layered cells for hematopoietic- and mesenchymal stem cell markers was performed. Examination of cells by immunohistochemistry using pluripotency, oxidative stress, stemness, migration and proliferation, epithelial and secretory markers was performed. The effect of anti-proliferative agent Mitomycin C upon secretion of pro-inflammatory cytokines IL-6 and IL-8 was assessed. RESULTS: Cells showed high expression of migration- (CXCR4), secretory- (MUC1, MUC4) and oxidative damage- (8-OHdG) markers, and low expression of hypoxia- (HIF-1α) and proliferation- (Ki-67) markers. Moderate and low expression of the pluripotency markers (Vimentin and ΔNp63) was present, respectively, while the putative markers of stemness (Sox2, Oct4, ABCG-2) and epithelial cell markers- (CK19, CK8-18) were weak. The surface marker profile of the outgrowing cells revealed high expression of the hematopoietic marker CD47, mesenchymal markers CD90 and CD73, minor or less positivity for the hematopoietic marker CD34, mesenchymal marker CD105, progenitor marker CD117 and attachment protein markers while low levels of IL-6 and IL-8 secretion ex vivo, were inhibited upon Mitomycin C treatment. CONCLUSION: Ex vivo tissue engineered pterygium consists of a mixture of cells of different lineage origin, suitable for use as a disease model for studying pathogenesis ex vivo, while opening possibilities for new treatment and prevention modalities.

Osteogenic differentiation of human lens epithelial cells might contribute to lens calcification.

Calcification of the human lens has been described in senile cataracts and in young patients with congenital cataract or chronic uveitis. Lens calcification is also a major complication of cataract surgery and plays a role in the opacification of intraocular lenses. A cell-mediated process has been suggested in the background of lens calcification, but so far the exact mechanism remained unexplored. Lens calcification shares remarkable similarities with vascular calcification; in both pathological processes hydroxyapatite accumulates in the soft tissue. Vascular calcification is a regulated, cell-mediated process in which vascular cells undergo osteogenic differentiation. Our objective was to investigate whether human lens epithelial cells (HuLECs) can undergo osteogenic transition in vitro, and whether this process contributes to lens calcification. We used inorganic phosphate (Pi) and Ca to stimulate osteogenic differentiation of HuLECs. Osteogenic stimuli (2.5mmol/L Pi and 1.2mmol/L Ca) induced extracellular matrix mineralization and Ca deposition in HuLECs with the critical involvement of active Pi uptake. Osteogenic stimuli almost doubled mRNA expressions of osteo-/chondrogenic transcription factors Runx2 and Sox9, which was accompanied by a 1.9-fold increase in Runx2 and a 5.5-fold increase in Sox9 protein expressions. Osteogenic stimuli induced mRNA and protein expressions of alkaline phosphatase and osteocalcin in HuLEC. Ca content was higher in human cataractous lenses, compared to non-cataractous controls (n=10). Osteocalcin, an osteoblast-specific protein, was expressed in 2 out of 10 cataractous lenses. We conclude that osteogenic stimuli induce osteogenic differentiation of HuLECs and propose that this mechanism might play a role in lens calcification.

Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications.

Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.