The aim of this study was to find out whether protease inhibitor 9 (PI-9) and granzyme B (GrB) molecules that contribute to immune response and the immunological privilege of various tissues are expressed in healthy and pathological human corneas. Using cryosections, cell imprints of control corneoscleral discs, we showed that PI-9 was expressed particularly in the endothelium, the superficial and suprabasal epithelium of healthy corneas, limbus, and conjunctiva. GrB was localized in healthy corneal and conjunctival epithelium, while the endothelium showed weak immunostaining. The expression of PI-6 and GrB was confirmed by qRT-PCR. Increased expression levels of the PI-9 and GrB genes were determined when the corneas were cultured with proinflammatory cytokines. Fluorescent and enzymatic immunohistochemistry of pathological corneal explants (corneal melting and herpes virus keratitis) showed pronounced PI-9, GrB, human leucocyte antigen (HLA)-DR, and leukocyte-common antigen (CD45) signals localized in multicellular stromal infiltrates and inflammatory cells scattered in the corneal stroma. We conclude that increased expression of the PI-9 and GrB proteins under pathological conditions and their upregulation in an inflammatory environment indicate their participation in immune response of the cornea during the inflammatory process.

• Keywords
• cornea, corneal endothelium, corneal epithelium, granzyme B, protease inhibitor 9,
• Publication type
• Journal Article MeSH

The corneal endothelium plays a key role in maintaining corneal transparency. Its dysfunction is currently treated with penetrating or lamellar keratoplasty. Advanced cell therapy methods seek to address the persistent global deficiency of donor corneas by enabling the renewal of the endothelial monolayer with tissue-engineered grafts. This review provides an overview of recently published literature on the preparation of endothelial grafts for transplantation derived from cadaveric corneas that have developed over the last decade (2010-2021). Factors such as the most suitable donor parameters, culture substrates and media, endothelial graft storage conditions, and transplantation methods are discussed. Despite efforts to utilize alternative cellular sources, such as induced pluripotent cells, cadaveric corneas appear to be the best source of cells for graft preparation to date. However, native endothelial cells have a limited natural proliferative capacity, and they often undergo rapid phenotype changes in ex vivo culture. This is the main reason why no culture protocol for a clinical-grade endothelial graft prepared from cadaveric corneas has been standardized so far. Currently, the most established ex vivo culture protocol involves the peel-and-digest method of cell isolation and cell culture by the dual media method, including the repeated alternation of high and low mitogenic conditions. Culture media are enriched by additional substances, such as signaling pathway (Rho-associated protein kinase, TGF-β, etc.) inhibitors, to stimulate proliferation and inhibit unwanted morphological changes, particularly the endothelial-to-mesenchymal transition. To date, this promising approach has led to the development of endothelial grafts for the first in-human clinical trial in Japan. In addition to the lack of a standard culture protocol, endothelial-specific markers are still missing to confirm the endothelial phenotype in a graft ready for clinical use. Because the corneal endothelium appears to comprise phenotypically heterogeneous populations of cells, the genomic and proteomic expression of recently proposed endothelial-specific markers, such as Cadherin-2, CD166, or SLC4A11, must be confirmed by additional studies. The preparation of endothelial grafts is still challenging today, but advances in tissue engineering and surgery over the past decade hold promise for the successful treatment of endothelial dysfunctions in more patients worldwide.

• Keywords
• Cell culture, Corneal endothelium, Endothelial phenotypic markers, Storage, Tissue engineering, Transplantation,
• MeSH
• Antiporters metabolism   MeSH
• Endothelial Cells transplantation   MeSH
• Humans MeSH
• Anion Transport Proteins metabolism   MeSH
• Proteomics MeSH
• Cornea MeSH
• Endothelium, Corneal * metabolism   transplantation   MeSH
• Corneal Transplantation * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Antiporters MeSH
• Anion Transport Proteins MeSH
• SLC4A11 protein, human MeSH Browser

PURPOSE: Posterior polymorphous corneal dystrophy (PPCD) is characterized by abnormal proliferation of corneal endothelial cells. It was shown that TGF-β2 present in aqueous humor (AH) could help maintaining the corneal endothelium in a G1-phase-arrest state. We wanted to determine whether the levels of this protein are changed in AH of PPCD patients. METHODS: We determined the concentrations of active TGF-β2 in the AH of 29 PPCD patients (42 samples) and 40 cadaver controls (44 samples) by ELISA. For data analysis the PPCD patients were divided based on either the molecular genetic cause of their disease as PPCD1 (37 samples), PPCD3 (1 sample) and PPCDx (not linked to a known PPCD loci, 4 samples) or on the presence (17 samples) or absence (25 samples) of secondary glaucoma or on whether they had undergone penetrating keratoplasty (PK, 32 samples) or repeated PK (rePK, 7 samples). RESULTS: The level of active TGF-β2 in the AH of all PPCD patients (mean ± SD; 386.98 ± 114.88 pg/ml) in comparison to the control group (260.95 ± 112.43 pg/ml) was significantly higher (P = 0.0001). Compared to the control group, a significantly higher level of active TGF-β2 was found in the PPCD1 (P = 0.0005) and PPCDx (P = 0.0022) groups. Among patients the levels of active TGF-β2 were not significantly affected by gender, age, secondary glaucoma or by the progression of dystrophy when one or repeated PK were performed. CONCLUSION: The levels of active TGF-β2 in the AH of PPCD patients are significantly higher than control values, and thus the increased levels of TGF-β2 could be a consequence of the PPCD phenotype and can be considered as another feature characterizing this disease.

• MeSH
• Corneal Dystrophies, Hereditary metabolism   MeSH
• Glaucoma metabolism   MeSH
• Keratoplasty, Penetrating methods   MeSH
• Aqueous Humor metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Cornea metabolism   MeSH
• Endothelium, Corneal metabolism   MeSH
• Transforming Growth Factor beta2 metabolism   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Transforming Growth Factor beta2 MeSH

PURPOSE: To present cytokeratin (CK)7 (OV-TL 12/30 clone) as a newly identified, reliable marker for distinguishing between the conjunctival and corneal surface epithelia, which will contribute to the precise diagnosis of limbal stem cell deficiency (LSCD). METHODS: Corneal and conjunctival epithelial imprints from 12 cadaveric bulbi and from 9 patients with clinically diagnosed LSCD were used for CK7 and CK19 immunocytochemistry. Specimens on nitroacetate cellulose filter papers obtained from the patients were stained with a combination of periodic acid-Schiff (PAS) and Gill's modified Papanicolaou stains, to assess the presence of goblet cells (GCs). RESULTS: CK7 was present in almost all superficial conjunctival epithelial cells from the cadaveric specimens. No immunostaining was observed on the corneal surface. A prominent sharp border of stain was found between the positive conjunctiva and the completely negative epithelium of the central cornea. A more gradual centrifugal decrease in the number of positive cells between the conjunctiva and cornea was observed for CK19. Several CK19-positive cells were detected in the central corneal epithelium. All corneal specimens from affected eyes (unilateral as well as bilateral LSCD patients) revealed strong positivity for CK7, and GCs were present in only 78% of patients. CONCLUSIONS: In cases in which GCs are severely decreased or are absent from the conjunctival surface, the detection of CK7 (OV-TL 12/30 clone) clearly confirms the overgrowth of the conjunctival epithelium over the cornea. Moreover, CK7 is a more reliable marker for distinguishing between the corneal and conjunctival epithelia compared with CK19.

• MeSH
• Biomarkers MeSH
• Adult Stem Cells pathology   MeSH
• Adult MeSH
• Keratin-19 immunology   metabolism   MeSH
• Keratin-7 genetics   immunology   metabolism   MeSH
• Conjunctiva metabolism   pathology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Limbus Corneae metabolism   pathology   MeSH
• Cadaver MeSH
• Corneal Diseases metabolism   pathology   MeSH
• Antibodies immunology   MeSH
• Epithelium, Corneal metabolism   pathology   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Antibody Specificity MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers MeSH
• Keratin-19 MeSH
• Keratin-7 MeSH
• Antibodies MeSH