Evaluation of the Possible Influence of Povidone Iodine (PVI) Solution and Polyhexanide (PHMB) on Wound Healing in Corneal Epithelial Regeneration.

This study investigates the possible toxic effects of the preoperative antiseptic substances povidone iodine (PVI) and polyhexanide (PHMB; Serasept® 2) on wound healing in ophthalmology. To assess this impact, human telomerase-immortalized corneal epithelial (hTCEpi) cells and human telomerase-immortalized conjunctival epithelial (hCjE) cells were exposed to 1% and 5% PVI or 0.04% PHMB for different periods to evaluate the cytotoxicity of these two antiseptics. Furthermore, the toxicity of these antiseptics was investigated in a human tissue-specific corneal epithelial construct and porcine eye culture model. The results reveal the high cytotoxicity of PVI and PHMB in the hTCEpi and hCjE in monolayer cell culture models, independent of the incubation time and concentration of these substances. However, after hTCEpi cell differentiation into a tissue-specific corneal epithelial construct, contact with these antiseptics for the relevant preoperative time did not alter cPARP1 or Ki67 expression. Furthermore, the wound-healing process in the porcine cornea was not significantly influenced after incubation with these antiseptics. In summary, corneal and conjunctival epithelial cell lines are very sensitive to PVI and PHMB, whereas no significant alterations were found in intact tissue-specific corneal epithelial constructs or porcine corneas. Therefore, we could not identify PVI and PHMB as reasons for postoperative eye irritation.

Allogeneic Limbal Transplants Integrate into the Corneal Surface and Lead to an Improved Visual Acuity.

Limbal stem cell deficiency (LSCD) severely impairs vision and can lead to blindness. LSCD causes include chemical burns, infections, multiple previous operations and congenital malformations. Allogeneic limbal transplantation is a procedure for treating LSCD where prepared limbal tissue is attached using a double running suture during allogeneic penetrating keratoplasty (PKP). A total of 22 patients underwent ALT surgery between February 2019 and June 2022 at the University Hospital Halle (Saale). Regular follow-up was performed postoperatively every three months and included visual acuity testing, pressure measurement, slit lamp microscopic examination, fundoscopy, corneal topography and anterior segment optical coherence tomography (AS-OCT). The mean patient age was 69.5 years, and the mean follow-up was 19 months. All included patients had LSCD and multiple previous surgeries. Patient LSCD etiology was 59% infectious and 41% traumatic. ALTs integrated into corneal surfaces in all patients, demonstrated on AS-OCT. Since most patients initially received allogeneic limbal transplants, none of the operated eyes had surgical complications. Overall, visual acuity improved postoperatively from an initial 2.06 to 1.44 logarithm of the minimum angle of resolution (logMAR). Allogeneic limbal transplantation can be used to treat LSCD and its integration into the surrounding corneal tissue can be observed on AS-OCT.

ALT (allogeneic limbal transplantation): a new surgical technique for limbal stem cell deficiency.

PURPOSE: Limbal stem cell deficiency (LSCD) is a rare but extremely relevant disease of the eye. LSCD patients often require a variety of surgical procedures, including keratoplasty in some cases. However, the outcome of these surgeries, including opacification and revascularization, is often frustrating due to LSCD relapse. METHODS: We developed a new surgical technique for the treatment of LSCD in which partial allogenic limbal transplantation (ALT) is carried out as part of penetrating keratoplasty (PK). After the PK, 1-8 slices from the limbal tissue of the donor graft are prepared and placed under the double running sutures attaching the corneal graft. This procedure was performed on 14 patients with LSCD, caused by severe ocular burn in 5 cases and by infection in 9. Between one and eight limbal transplants were used depending on the extension of the LSCD. RESULTS: All 14 patients showed stable or increased visual acuity after the ALT surgery compared to their preoperative visual acuity. All of the grafts were integrated into the superficial corneal layers without progression of corneal vascularization beyond the limbal grafts. The median follow-up period was 12 months on average. CONCLUSION: The ALT method seems to be a promising surgical procedure for the treatment of patients with LSCD. It can be properly carried out in the context of keratoplasty and does not require a separate donor tissue. The ALT grafts may offer the possibility of constructing a new limbal region, resulting in stable or even increased visual acuity and the absence of corneal vascularization.

CTG18.1 repeat expansion may reduce TCF4 gene expression in corneal endothelial cells of German patients with Fuchs' dystrophy.

PURPOSE: It was the aim of this investigation to elucidate the functional effects of CTG18.1 trinucleotide repeat expansion and the polymorphism rs613872 in the transcription factor 4 (TCF4) in corneas of patients affected by Fuchs' endothelial corneal dystrophy (FECD). METHODS: Sixty-one unrelated German patients with FECD and 113 unaffected controls were investigated and genotyped for the CTG18.1 locus by triplet primed PCR (TP-PCR) and the rs613872 polymorphism via Sanger sequencing and by employing genomic DNA from peripheral blood leucocytes. DNA and RNA retrieved from human corneal endothelial explants were examined for alterations in the gene expression of TCF4, ZEB1, E-cadherin, N-cadherin, as well as the CTG18.1 locus. RESULTS: The CTG18.1 trinucleotide repeat expansion (>50 repeats) was detected in the peripheral blood in 77% of affected FECD patients and 11.5% of the healthy volunteers. Applying the TP-PCR method, the length of CTG18.1 repeat expansions correlates in the blood and corneal cells. We noted that the CTG18.1 trinucleotide repeat expansion was associated with reduced TCF4 and ZEB1 gene expression, especially in the explanted corneal endothelial cells. While E-cadherin gene expression was not detected in any corneal endothelial cells, expression of CDH2 (N-cadherin) was detected in FECD-affected endothelium and in our controls. CONCLUSIONS: The CTG18.1 repeat expansion may reduce gene expression of TCF4 and ZEB1, suggesting that a mechanism triggering a loss of function may contribute to FECD. The correlation of CTG18.1 repeat expansion from blood and the cornea may represent the first step toward investigating the potential relevance of testing the blood of cornea donors to minimize the risk of transplanting grafts potentially affected with FECD.

Identification of two novel mutations in the cornea-specific TGFBI gene causing unique phenotypes in patients with corneal dystrophies.

The purpose of this study was to report on two novel missense mutations of the cornea-specific TGFBI gene in one single patient and in two generations of a family diagnosed with unique corneal dystrophy (CD) phenotypes. Ophthalmologic examination, in several cases ocular coherence tomography of the anterior segment (AS-OCT), was performed in 21 affected patients and in two unaffected members of one affected family. Coding regions of the TGFBI gene were direct sequenced in all 23 individuals. The two novel mutations were verified by RFLP analysis. A novel mutation c.1640T > G (p.Phe574Cys) in exon 12 of the TGFBI gene was detected in one single patient with recurrent granular intrastromal deposits comparable to a type of granular dystrophy. In AS-OCT, the deposit pattern reached up to the Descemet's layer. A further novel mutation c.393G > T(p.Glu131Asp) in exon 4 of the TGFBI gene was detected in all three affected members of one family with superficial cloud- and honeycomb-like opacifications, comparable to a Schnyder corneal dystrophy. Two unaffected members did not carry this alteration. The two identified novel mutations add other two phenotypes in patients suffering from TGFBI-linked CD to those reported so far. In one case, clinical finding indicates a Schnyder corneal dystrophy-like phenotype due to its superficial crystalline shape, and in the second one, granular deposits who reach Descemet's layer indicate a granular CD subtype. Molecular genetic analysis may help to distinguish those subtypes and to decide for specific treatment in time of a wide variation of corneal surgical techniques.

Hypoxia supports reprogramming of mesenchymal stromal cells via induction of embryonic stem cell-specific microRNA-302 cluster and pluripotency-associated genes.

Pluripotency is characterized by specific transcription factors such as OCT4, NANOG, and SOX2, but also by pluripotency-associated microRNAs (miRs). Somatic cells can be reprogrammed by forced expression of these factors leading to induced pluripotent stem cells (iPSCs) with characteristics similar to embryonic stem cells (ESCs). However, current reprogramming strategies are commonly based on viral delivery of the pluripotency-associated factors, which affects the integrity of the genome and impedes the use of such cells in any clinical application. In an effort to establish nonviral, nonintegrating reprogramming strategies, we examined the influence of hypoxia on the expression of pluripotency-associated factors and the ESC-specific miR-302 cluster in primary and immortalized mesenchymal stromal cells (MSCs). The combination of hypoxia and fibroblast growth factor 2 (FGF2) treatments led to the induction of OCT4 and NANOG in an immortalized cell line L87 and primary MSCs, accompanied with increased doubling rates and decreased senescence. Most importantly, the endogenous ECS-specific cluster miR-302 was induced upon hypoxic culture and FGF2 supplementation. Hypoxia also improved reprogramming of MSCs via episomal expression of pluripotency factors. Thus, our data illustrate that hypoxia in combination with FGF2 supplementation efficiently facilitates reprogramming of MSCs.

DNA microarrays reveal relationship of Ewing family tumors to both endothelial and fetal neural crest-derived cells and define novel targets.

Ewing family tumors (EFTs) are small round blue cell tumors that show features of neuroectodermal differentiation. However, the histogenetic origin of EFTs is still a matter of debate. We used high-density DNA microarrays for the identification of EFT-specific gene expression profiles in comparison with normal tissues of diverse origin. We identified 37 genes that are up-regulated in EFTs compared with normal tissues and validated expression of these genes in EFTs by both conventional and quantitative reverse transcription-polymerase chain reaction. The expression pattern of EFT-associated genes in normal tissues indicated a high similarity between EFTs and fetal and neuronal as well as endothelial tissues and supports the concept that a primitive neural crest-derived progenitor at the transition to mesenchymal and endothelial differentiation is transformed in EFTs. EFT-associated genes could be used for molecular discrimination between EFTs and other small round blue cell tumors and clearly identified a cell line (SK-N-MC) that was initially established as neuroblastoma as being an EFT. Ectopic expression of the EFT-specific EWS-FLI1 fusion protein in human embryonic kidney (HEK293) cells was not sufficient to induce the complete EFT-specific gene expression signature, suggesting that the EFT-specific gene expression profile is not just a consequence of EWS-FLI1 expression but depends on the histogenetic background of the EFT stem cell.