A semifluorinated alkane (F4H5) as novel carrier for cyclosporine A: a promising therapeutic and prophylactic option for topical treatment of dry eye

Cyclosporine A (Cs) has been used as effective topical therapy for inflammatory dry eye disease since more than a decade. However, due to its lipophilic character, Cs is formulated as emulsions or oily solutions for topical application. This experimental study aimed to test if the use of semifluorinated alkanes (SFAs) as a preservative-free, well-tolerated non-stinging or burning vehicle maintains or even improves the benefits of Cs in the topical therapy of dry-eye disease. Desiccating stress was applied to C57BL/6 mice for 14 consecutive days to induce experimental dry-eye. Cs dissolved in SFA (perfluorobutylpentane = F4H5with 0.5% Ethanol), F4H5 with 0.5% ethanol only, 0.05% Cs (RestasisA (R)), and dexamethasone (MonodexA (R)) were applied three times daily beginning either at day 4 or day 11 of desiccating stress for up to 3 weeks after end of dry-eye induction. In comparison to other groups, Cs/F4H5 demonstrated high efficacy and earlier reduction of corneal staining. In this study, Cs/F4H5 had the ability to maintain conjunctival goblet cell density once applied on day 4. Flow cytometry analysis from cervical lymphnodes demonstrated a significantly lower CD4+ and CD8+ T-cells in the Cs/F4H5 group following 3 weeks of therapy than at baseline, but no difference in regulatory T cells from regional lymphnodes were seen. Overall, compared to a commercially available Cs formulation (RestasisA (R)) and dexamethasone, Cs/F4H5 was shown to be equally effective but with a significantly faster therapeutic response in reducing signs of dry-eye disease in an experimental mouse model

Increased Anti-Inflammatory Therapeutic Potential and Progenitor Marker Expression of Corneal Mesenchymal Stem Cells Cultured in an Optimized Propagation Medium

There is a huge unmet need for new treatment modalities for ocular surface inflammatory disorders (OSIDs) such as dry eye disease and meibomian gland dysfunction. Mesenchymal stem cell therapies may hold the answer due to their potent immunomodulatory properties, low immunogenicity, and ability to modulate both the innate and adaptive immune response. MSC-like cells that can be isolated from the corneal stroma (C-MSCs) offer a potential new treatment strategy; however, an optimized culture medium needs to be developed to produce the ideal phenotype for use in a cell therapy to treat OSIDs. The effects of in vitro expansion of human C-MSC in a medium of M199 containing fetal bovine serum (FBS) was compared to a stem cell medium (SCM) containing knockout serum replacement (KSR) with basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (LIF), investigating viability, protein, and gene expression. Isolating populations expressing CD34 or using siRNA knockdown of CD34 were investigated. Finally, the potential of C-MSC as a cell therapy was assessed using co-culture with an in vitro corneal epithelial cell injury model and the angiogenic effects of C-MSC conditioned medium were evaluated with blood and lymph endothelial cells. Both media supported proliferation of C-MSC, with SCM increasing expression of CD34, ABCG2, PAX6, NANOG, REX1, SOX2, and THY1, supported by increased associated protein expression. Isolating cell populations expressing CD34 protein made little difference to gene expression, however, knockdown of the CD34 gene led to decreased expression of progenitor genes. C-MSC increased viability of injured corneal epithelial cells whilst decreasing levels of cytotoxicity and interleukins-6 and -8. No pro-angiogenic effect of C-MSC was seen. Culture medium can significantly influence C-MSC phenotype and culture in SCM produced a cell phenotype more suitable for further consideration as an anti-inflammatory cell therapy. C-MSC show considerable potential for development as therapies for OSIDs, acting through anti-inflammatory action

ABCB5+ limbal epithelial stem cells inhibit developmental but promote inflammatory (lymph) angiogenesis while preventing corneal inflammation

The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) but not in adult mice. Conversely, when using a cornea suture model, the WT animals exhibited a mild but significant increase in the number of lymphatic vessel sprouts compared to the ABCB5KO, suggesting a contextual anti-lymphangiogenic effect of ABCB5 on the limbal vasculature during development, but a pro-lymphangiogenic effect under inflammatory challenge in adulthood. In addition, conditioned media from ABCB5-positive cultured human limbal epithelial cells (ABCB5+) stimulated human blood and lymphatic endothelial cell proliferation and migration. Finally, a proteomic analysis demonstrated ABCB5+ cells have a pro(lymph)angiogenic as well as an anti-inflammatory profile. These data suggest a novel dual, context-dependent role of ABCB5+ LESCs, inhibiting developmental but promoting inflammatory (lymph)angiogenesis in adulthood and exerting anti-inflammatory effects. These findings are of high clinical relevance in relation to LESC therapy against blindness

Future directions in managing aniridia-associated keratopathy

Congenital aniridia is a panocular disorder that is typically characterized by iris hypoplasia and aniridia-associated keratopathy (AAK). AAK results in the progressive loss of corneal transparency and thereby loss of vision. Currently, there is no approved therapy to delay or prevent its progression, and clinical management is challenging because of phenotypic variability and high risk of complications after interventions; however, new insights into the molecular pathogenesis of AAK may help improve its management. Here, we review the current understanding about the pathogenesis and management of AAK. We highlight the biological mechanisms involved in AAK development with the aim to develop future treatment options, including surgical, pharmacological, cell therapies, and gene therapies

Quantitative Analysis of BTF3, HINT1, NDRG1 and ODC1 Protein Over-Expression in Human Prostate Cancer Tissue

Prostate carcinoma is the most common cancer in men with few, quantifiable, biomarkers. Prostate cancer biomarker discovery has been hampered due to subjective analysis of protein expression in tissue sections. An unbiased, quantitative immunohistochemical approach provided here, for the diagnosis and stratification of prostate cancer could overcome this problem. Antibodies against four proteins BTF3, HINT1, NDRG1 and ODC1 were used in a prostate tissue array (> 500 individual tissue cores from 82 patients, 41 case pairs matched with one patient in each pair had biochemical recurrence). Protein expression, quantified in an unbiased manner using an automated analysis protocol in ImageJ software, was increased in malignant vs non-malignant prostate (by 2-2.5 fold, p<0.0001). Operating characteristics indicate sensitivity in the range of 0.68 to 0.74; combination of markers in a logistic regression model demonstrates further improvement in diagnostic power. Triple-labeled immunofluorescence (BTF3, HINT1 and NDRG1) in tissue array showed a significant (p<0.02) change in co-localization coefficients for BTF3 and NDRG1 co-expression in biochemical relapse vs non-relapse cancer epithelium. BTF3, HINT1, NDRG1 and ODC1 could be developed as epithelial specific biomarkers for tissue based diagnosis and stratification of prostate cancer

Bevacizumab Induces Upregulation of Keratin 3 and VEGFA in Human Limbal Epithelial Cells in Vitro

Topical application of vascular endothelial growth factor A (VEGFA) inhibitors including Bevacizumab is used for antiangiogenic therapy at the ocular surface. While clinical studies have suggested that this approach is well-tolerated, the effect of the drug on limbal epithelial stem cells has not been studied. In this study, the effect of Bevacizumab on phenotype and functionality of putative limbal epithelial stem cells (SC) was investigated. The effect of Bevacizumab on human limbal epithelial cells was assessed in terms of metabolic activity and scratch wound closure. The different treatment groups featured no difference in proliferation and colony forming efficiency (CFE) of limbal epithelial cells or their putative SC marker expression. A significant delay in scratch closure of all the Bevacizumab-treated groups was detected at 4 h. RNA and protein quantification indicated a dose-responsive increase of keratin 3. VEGFA RNA expression also increased while VEGFC and D as well as VEGFR1, 2 and 3 were unchanged. This study highlights previously unknown effects of Bevacizumab on cultured putative limbal epithelial SC: a dose-related increase of keratin 3, an increase in VEGFA as well as a delay in scratch wound closure. These in vitro data should be considered when using Bevacizumab in the context of limbal epithelial SC transplantation

Characterisation and biocompatibility of novel chitosan-alginata blend membranes

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UV Protection in the Cornea: Failure and Rescue

Simple Summary The sun is a deadly laser, and its damaging rays harm exposed tissues such as our skin and eyes. The skin's protection and repair mechanisms are well understood and utilized in therapeutic approaches while the eye lacks such complete understanding of its defenses and therefore often lacks therapeutic support in most cases. The aim here was to document the similarities and differences between the two tissues as well as understand where current research stands on ocular, particularly corneal, ultraviolet protection. The objective is to identify what mechanisms may be best suited for future investigation and valuable therapeutic approaches. Ultraviolet (UV) irradiation induces DNA lesions in all directly exposed tissues. In the human body, two tissues are chronically exposed to UV: the skin and the cornea. The most frequent UV-induced DNA lesions are cyclobutane pyrimidine dimers (CPDs) that can lead to apoptosis or induce tumorigenesis. Lacking the protective pigmentation of the skin, the transparent cornea is particularly dependent on nucleotide excision repair (NER) to remove UV-induced DNA lesions. The DNA damage response also triggers intracellular autophagy mechanisms to remove damaged material in the cornea; these mechanisms are poorly understood despite their noted involvement in UV-related diseases. Therapeutic solutions involving xenogenic DNA-repair enzymes such as T4 endonuclease V or photolyases exist and are widely distributed for dermatological use. The corneal field lacks a similar set of tools to address DNA-lesions in photovulnerable patients, such as those with genetic disorders or recently transplanted tissue

Overestimation of Relative Risk and Prevalence Ratio: Misuse of Logistic Modeling

The extensive use of logistic regression models in analytical epidemiology as well as in randomized clinical trials, often creates inflated estimates of the relative risk (RR). Particularly, in cases where a binary outcome has a high or moderate incidence in the studied population (&gt;10%), the bias in assessing the relative risk may be very high. Meta-analysis studies have estimated that about 40% of the relative risk estimates in prospective investigations, through binary logistic models, lead to extensive bias of the population parameters. The problem of risk inflation also appears in cross-sectional studies with binary outcomes, where the parameter of interest is the prevalence ratio. As an alternative to the use of logistic regression models in both longitudinal and cross-sectional studies, the modified Poisson regression model is proposed