Neutrophil-driven and interleukin-36γ-associated ocular surface inflammation in chronic Stevens-Johnson syndrome.

PURPOSE: This study aims to elucidate the tear proteome and understand the underlying molecular mechanisms involved in the ocular complications following Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). METHODS: Mass spectrometry (MS) was performed to quantify the tear fluid proteins from chronic SJS/TEN patients (n = 22 eyes) and age- and gender-matched controls (n = 22 eyes). The candidate proteins were validated using ELISA (n = 80 eyes) in tear samples and immunohistochemistry (IHC; n = 12) in eyelid margin specimens. These proteins were compared for significant differences based on age, gender, disease duration, and ocular severity. RESULTS: A total of 1692 tear fluid proteins were identified, of which 470 were significantly differentially regulated in chronic SJS/TEN. The top 10 significantly upregulated proteins were neutrophil secretions including neutrophil elastase (p < .0001), defensin (p < .0001), and matrix metalloproteinase 8 (p < .0001). The presence of neutrophils was confirmed by the upregulation of IL-8 (p < .001) in tears, a key cytokine known for recruiting neutrophils. Additionally, positive expression of myeloperoxidase was observed in the keratinized eyelid margins of SJS/TEN to validate the presence of neutrophils. Among 41 unique proteins identified by MS, IL-36γ (p < .01) was expressed in three SJS/TEN patients and was confirmed in SJS/TEN tears and eyelid margins by ELISA and IHC, respectively. IL-36γ was specifically expressed in the superficial layers of eyelid margin keratinized conjunctiva. The majority of the significantly downregulated proteins were lacrimal gland secretions such as lacritin (p < .0001) and opiorphin (p < .002). Neutrophil elastase (p < .02) was significantly elevated in patients with severe eyelid margin keratinization. CONCLUSION: Our observations indicate a clear correlation between eyelid margin keratinization and the expression of IL-36γ, potentially mediated by neutrophils recruited via IL-8. Future experimental studies are needed to test the role of therapies targeting IL-8 and/or IL-36γ in reducing eyelid margin keratinization and its associated ocular complications in SJS/TEN.

Development and validation of a reliable rabbit model of limbal stem cell deficiency by mechanical debridement using an ophthalmic burr.

A simple and reproducible method is necessary to generate reliable animal models of limbal stem cell deficiency (LSCD) for assessing the safety and efficacy of new therapeutic modalities. This study aimed to develop and validate a rabbit model of LSCD through mechanical injury. The corneal and limbal epithelium of New Zealand White rabbits (n = 18) were mechanically debrided using an ophthalmic burr (Algerbrush II) with a 1.0-mm rotating head after 360° conjunctival peritomy. The debrided eyes were serially evaluated for changes in corneal opacity, neo-vascularization, epithelial defect and corneal thickness using clinical photography, slit lamp imaging, fluorescein staining, and anterior segment optical coherence tomography scanning (AS-OCT). Following this, an assessment of histopathology and phenotypic marker expression of the excised corneas was conducted. The experimental eyes were grouped as mild (n = 4), moderate (n = 10), and severe (n = 4) based on the grade of LSCD. The moderate group exhibited abnormal epithelium, cellular infiltration in the stroma, and vascularization in the central, peripheral, and limbal regions of the cornea. The severe group demonstrated central epithelial edema, peripheral epithelial thinning with sparse goblet cell population, extensive cellular infiltration in the stroma, and dense vascularization in the limbal region of the cornea. A significant decrease in the expression of K12 and p63 (p < 0.0001) was observed, indicating the loss of corneal epithelium and limbal epithelial stem cells in the LSCD cornea. This study demonstrates that the Alger brush-induced mechanical debridement model provides a reliable model of LSCD with comprehensive clinic-pathological features and that is well suited for evaluating novel therapeutic and regenerative approaches.

Human cornea-derived extracellular matrix hydrogel for prevention of post-traumatic corneal scarring: A translational approach.

Corneal scarring and opacification are a significant cause of blindness affecting millions worldwide. The current standard of care for corneal blindness is corneal transplantation, which suffers from several drawbacks. One alternative approach that has shown promise is the use of xenogeneic corneal extracellular matrix (ECM), but its clinical applicability is challenging due to safety concerns. This study reports the innovative use of human cornea-derived ECM to prevent post-traumatic corneal scarring. About 30 - 40% of corneas donated to the eye banks do not meet the standards defined for clinical use and are generally discarded, although they are completely screened for their safety. In this study, human cornea-derived decellularized ECM hydrogel was prepared from the non-transplantation grade human cadaveric corneas obtained from an accredited eye-bank. The prepared hydrogel was screened for its efficacy against corneal opacification following an injury in an animal model. Our in vivo study revealed that, the control collagen-treated group developed corneal opacification, while the prophylactic application of human cornea-derived hydrogel effectively prevented corneal scarring and opacification. The human hydrogel-treated corneas were indistinguishable from healthy corneas and comparable to those treated with the xenogeneic bovine corneal hydrogel. We also demonstrated that the application of the hydrogel retained the biological milieu including cell behavior, protein components, optical properties, curvature, and nerve regeneration by remodeling the corneal wound after injury. The hydrogel application is also sutureless, resulting in faster corneal healing. We envision that this human cornea-derived ECM-based hydrogel has potential clinical application in preventing scarring from corneal wounding. STATEMENT OF SIGNIFICANCE: There are significant challenges surrounding corneal regeneration after injury due to extensive scarring. Although there is substantial research on corneal regeneration, much of it uses synthetic materials with chemical cross-linking methods or xenogeneic tissue-based material devices which have to undergo exhaustive safety analysis before clinical trials. Herein, we demonstrate the potential application of a human corneal extracellular matrix hydrogel without any additional materials for scarless corneal tissue regeneration, and a method to reduce the wasting of donated allogenic corneal tissue from eye banks. We found no difference in efficacy between the usage of human tissues compared to xenogeneic sources. This may help ease clinical translation and can be used topically without sutures as an outpatient procedure.

Expansion and characterization of human limbus-derived stromal/mesenchymal stem cells in xeno-free medium for therapeutic applications.

BACKGROUND: Mesenchymal stem cells (MSCs) have been proven to prevent and clear corneal scarring and limbal stem cell deficiency. However, using animal-derived serum in a culture medium raises the ethical and regulatory bar. This study aims to expand and characterize human limbus-derived stromal/mesenchymal stem cells (hLMSCs) for the first time in vitro in the xeno-free medium. METHODS: Limbal tissue was obtained from therapeutic grade corneoscleral rims and subjected to explant culture till tertiary passage in media with and without serum (STEM MACS XF; SM), to obtain pure hLMSCs. Population doubling time, cell proliferation, expression of phenotypic markers, tri-lineage differentiation, colony-forming potential and gene expression analysis were carried out to assess the retention of phenotypic and genotypic characteristics of hLMSCs. RESULTS: The serum-free medium supported the growth of hLMSCs, retaining similar morphology but a significantly lower doubling time of 23 h (*p < 0.01) compared to the control medium. FACS analysis demonstrated ≥ 90% hLMSCs were positive for CD90+, CD73+, CD105+, and ≤ 6% were positive for CD45-, CD34- and HLA-DR-. Immunofluorescence analysis confirmed similar expression of Pax6+, COL IV+, ABCG2+, ABCB5+, VIM+, CD90+, CD105+, CD73+, HLA-DR- and CD45-, αSMA- in both the media. Tri-lineage differentiation potential and gene expression of hLMSCs were retained similarly to that of the control medium. CONCLUSION: The findings of this study demonstrate successful isolation, characterization and culture optimization of hLMSCs for the first time in vitro in a serum-free environment. This will help in the future pre-clinical and clinical applications of MSCs in translational research.

A review of rabbit models of meibomian gland dysfunction and scope for translational research.

Dry eye disease (DED) is an emerging global health concern with meibomian gland dysfunction (MGD) being the most common subtype of DED. Despite being quite prevalent, the pathophysiological mechanisms governing MGD are poorly understood. Animal models for MGD can be a valuable resource to advance our understanding of this entity and explore novel diagnostic and therapeutic modalities. Although a lot of literature on rodent MGD models exists, a comprehensive review on rabbit animal models is lacking. Rabbits offer a great advantage over other animals as models for studying both DED and MGD. Rabbits have a widely exposed ocular surface and meibomian gland anatomy comparable with humans, which makes performing dry eye diagnostic tests possible using clinically validated imaging platforms. The existing MGD models in rabbits can broadly be classified as pharmacologically induced and surgically induced models. Most models show keratinization of the meibomian gland orifice with plugging as the final common pathway for developing MGD. Thus, understanding the advantages and disadvantages of each rabbit MGD model can help researchers choose the appropriate experimental plan based on the objective of the study. In this review, we discuss the comparative anatomy of the meibomian glands in humans and rabbits, various rabbit models of MGD, translational applications, unmet needs, and future directions in developing MGD models in rabbits.

Optimization and evaluation of tear protein elution from Schirmer's strips in dry eye disease.

PURPOSE: Extraction of tear protein from Schirmer's strip is a prerequisite for the proper identification and screening of biomarkers in dry eye disease. The study compares different methods of extraction of tear proteins from the Schirmer's strip. METHODS: Reflex tear was collected from healthy controls (HC; n = 12), Stevens-Johnson syndrome (SJS; n = 3) and dry eye disease (DED; n = 3) patients using capillary tube. This tear was used to measure the volume absorbed by Schirmer's strip per microliter. Different buffers (6) were used to compare the protein yield from the Schirmer's strip in four different conditions. The tear proteins extracted using the highest protein yield buffer were analyzed by mass spectrometry. RESULTS: A linear relationship between the tear volume and wetting length was observed (r = 0.0.997, n = 6). The highest yield was observed after incubation of the Schirmer's strip in 100 mM ammonium bicarbonate (ABC) with 0.25% Nonidet P-40(NP-40) at 4°C for an hour (P < 0.00005). The in-solution digestion of tear eluted in the above condition 100 Mm ABC + 0.25% NP-40 with one-hour incubation yielded a total of 2119 proteins in HC, SJS, and DED. The unique protein observed in SJS and DED was 0.6% and 17.9%, respectively. The significantly expressed proteins are associated with innate immune response, proteolysis, wound healing, and defense response. CONCLUSION: A method for extraction of protein from Schirmer's strip was optimized for increase in protein yield from the tear sample. SJS and DED tear samples have unique protein signature. The study will aid in better design of tear protein-based experimental study.

Identification and in silico analysis of a spectrum of SLC4A11 variations in Indian familial and sporadic cases of congenital hereditary endothelial dystrophy.

BACKGROUND: Congenital hereditary endothelial dystrophy (CHED) is a rare form of corneal dystrophy caused by SLC4A11 gene variations. This study aims to find the genetic alterations in SLC4A11, in two Indian familial CHED cases with affected members n = 3 and n = 2 respectively and five sporadic CHED cases using direct sequencing, followed by in silico analysis and characterization of the identified variants. RESULTS: All three affected members of the first CHED family were identified with a novel homozygous c.1514C > G (p.Ser489Trp) variation while second family showed presence of a compound heterozygous variation c.529A > C (p.Arg161Arg) + c.2461insT (p.Val805fs). Among five sporadic cases, two showed novel changes, homozygous c.1487G > T (p.Ser480Ile) and c.620-2A > G, while the other one had previously reported homozygous c.2653C > T (p.Arg869Cys) variation. The remaining two cases did not reveal the presence of SLC4A11-related pathogenic variations. The identified variations were excluded from the Indian control (n = 80). In silico analysis using homology-based protein modeling and pathogenicity prediction tools, which revealed these alterations as pathogenic, changing their protein stability, local flexibility, residue contact clashes, and the hydrogen bond interactions. CONCLUSIONS: This study contributed to the CHED mutational spectrum, adding four novel variations and confirming a previously reported one. It demonstrates different type of variations in CHED cases, including coding, non-coding, homozygous, synonymous, and compound heterozygous variations. The identified variations revealed different degrees of pathogenic effects in silico. Moreover, two sporadic cases could not be identified with pathogenic variation emphasizing the involvement of other genes or genetic mechanisms.

Differential expression of tear film cytokines in Stevens-Johnson syndrome patients and comparative review of literature.

To investigate the differential expression of tear cytokine levels among chronic Stevens-Johnson syndrome (SJS) patients to better understand the role of significantly altered cytokines in disease development. Tear samples were collected using Schirmer strips in 24 eyes of chronic SJS, 24 eyes of age and gender-matched controls, and 14 eyes of aqueous deficiency dry eye disease (DED) patients. The cytokine analysis was performed among 18 analytes which include pro-inflammatory, anti-inflammatory factors, and ELR-negative CXC chemokines. String analysis was performed for the significantly altered cytokines to understand their co-expression and role in the disease development. Additionally, a literature review was conducted to identify the signature cytokines present in chronic SJS tears. The differential expression of IL-6 (p ≤ 0.029), CXCL8/IL-8 (p ≤ 0.009), IL-1ß (p ≤ 0.041), IL-2 (p ≤ 0.025), IL-10 (p ≤ 0.053), and CXCL-10 (p ≤ 0.044) were observed in chronic SJS patients and healthy controls. Whereas, IL-6 (p ≤ 0.029), CXCL8/IL-8 (p ≤ 0.058), CCL4 (p ≤ 0.056), GM-CSF (p ≤ 0.0001) IL-10 (p ≤ 0.025), and CXCL-10 (p ≤ 0.010), were differentially expressed in SJS as compared to severe DED patients. String analysis of the significantly altered cytokines revealed the involvement of several biological processes including the chronic inflammatory response, nitric oxide synthesis, angiogenesis, and cellular response to drugs. Among all the cytokines evaluated, the expression of CXCL8/IL-8 and CXCL10 levels were consistently reported in the literature. There was a differential expression of tear cytokines in SJS when compared to DED and healthy controls. The differential expression of CXCL8/IL-8 and CXCL10 was in line with existing literature and their role in chronic SJS pathogenesis merits further evaluation.

Human Cadaveric Donor Cornea Derived Extra Cellular Matrix Microparticles for Minimally Invasive Healing/Regeneration of Corneal Wounds.

Biological materials derived from extracellular matrix (ECM) proteins have garnered interest as their composition is very similar to that of native tissue. Herein, we report the use of human cornea derived decellularized ECM (dECM) microparticles dispersed in human fibrin sealant as an accessible therapeutic alternative for corneal anterior stromal reconstruction. dECM microparticles had good particle size distribution (≤10 µm) and retained the majority of corneal ECM components found in native tissue. Fibrin-dECM hydrogels exhibited compressive modulus of 70.83 ± 9.17 kPa matching that of native tissue, maximum burst pressure of 34.3 ± 3.7 kPa, and demonstrated a short crosslinking time of ~17 min. The fibrin-dECM hydrogels were found to be biodegradable, cytocompatible, non-mutagenic, non-sensitive, non-irritant, and supported the growth and maintained the phenotype of encapsulated human corneal stem cells (hCSCs) in vitro. In a rabbit model of anterior lamellar keratectomy, fibrin-dECM bio-adhesives promoted corneal re-epithelialization within 14 days, induced stromal tissue repair, and displayed integration with corneal tissues in vivo. Overall, our results suggest that the incorporation of cornea tissue-derived ECM microparticles in fibrin hydrogels is non-toxic, safe, and shows tremendous promise as a minimally invasive therapeutic approach for the treatment of superficial corneal epithelial wounds and anterior stromal injuries.

Prevention of Corneal Myofibroblastic Differentiation In Vitro Using a Biomimetic ECM Hydrogel for Corneal Tissue Regeneration.

Corneal scarring is one of the major causes of blindness, affecting millions worldwide. Despite recent advancements in surgical strategies, there is an unmet need for a clinically feasible material and methods to prevent scarring following corneal injury. In this study, we report the potential utility of a hydrogel derived from cadaveric animal corneas, using a decellularized corneal matrix hydrogel (abbreviated as dCMH), which is prepared by a simple method. This hydrogel is easily injectable, biocompatible, and has the ability to maintain good shape-retention properties at 37 °C, which make it suitable for in vivo applications. Furthermore, our gene expression studies and immunofluorescence studies indicate that dCMH maintains the morphology and function of keratocytes in vitro and prevents their transdifferentiation to myofibroblasts. From the above results, it is evident that dCMH maintains the keratocytes with the ability to regenerate the corneal defect without scar. We thus suggest a simple yet effective approach for corneal tissue decellularization and that dCMH can be a promising material for prophylaxis against blinding scar formation in an injured cornea.