Donor Corneal Epithelium Research Articles

Factors affecting the epithelial integrity of human donor corneas.

To evaluate various factors affecting the integrity of human donor corneal epithelium. Donor corneal buttons were evaluated for epithelial defect (ED) and exposure. The slit-lamp photographs were taken on day 01, and the data such as age and gender of the donor, cause of death, refrigeration of cadavers, death-to-preservation time (DPT), experience of technician, and distance from site of collection to eye bank were collected. A total of 100 consecutive corneal buttons belonging to 56 donors were evaluated. The median age of donors was 50 years. Males constituted 45 (80.4%). The mean DPT was 9.7 ± 5.3 hours. After death, 34 donors (60.7%) were refrigerated before the collection/retrieval. Most of the corneas (80%) were recovered by technicians having an experience of 0-5 years. Thirty-one donors (55.3%) were located at 1-50 km from the eye bank. The mean area of exposure was 15 ± 4.3 mm2. The mean area of ED was 28.7 ± 5.9 mm2. ED was significantly associated with refrigeration of cadavers and longer DPT. On multivariate analysis, only DPT was found to be significantly associated (P = 0.006; odds ratio [OR] = 1.54 ± 0.24) with the presence of ED. After transplantation, only two corneas had persistent epithelial defects and were treated successfully using various interventions. Integrity of donor corneal epithelium is mainly influenced by the refrigeration of cadavers and DPT.

Preservation of donor corneal epithelium in McCarey-Kaufman medium.

To evaluate the role of McCarey-Kaufman (MK) medium in maintaining the integrity of donor corneal epithelium. Nineteen corneal buttons were harvested and stored in MK media at 2°C-8°C for four days. Serial photographs were done every day till the 3rd day, and images were then analyzed with ImageJ software (LOCI, University of Wisconsin, USA). The area of exposure and epithelial defect (ED) was calculated every day for each corneal button. The average age of the donors was 56.5 ± 22.7 years and mean time from death to preservation of the corneal buttons was 7.7 ± 3.1 hours. The average corneal area was 145.6 ± 18.8 mm2. The total mean area of exposure was 3.6 ± 4.8, 7.2 ± 9.2, and 9.0 ± 11.9 mm2, and ED was 1.7 ± 4.6, 2.8 ± 5.3, and 3.3 ± 5.9 mm2 on days 1, 2, and 3, respectively. The percentage of increase in the area of exposure and ED in MK media was 3.71% and 1.1% from day 1 to day 3, respectively. Six out of 19 corneal buttons (31.57%) were utilized for keratoplasties, of which two were utilized in house and four were distributed outside. Of the two utilized corneas, none had epithelial defect on postoperative day 1. Rest 13 corneas were either used for training and research purposes, stored in glycerol media, or discarded. Since the percentage change in area of exposure/ED is not much at the end of day 3, corneas stored in MK media can be safely used even after three days of storage. Hence, MK medium serves as an excellent medium in maintaining the integrity of donor corneal epithelium.

Interleukin-32 induced thymic stromal lymphopoietin plays a critical role in the inflammatory response in human corneal epithelium

Interleukin (IL)-32, a novel cytokine, participates in a variety of inflammatory disorders. Thymic stromal lymphopoietin (TSLP) plays important roles in mucosal epithelial cells, especially in allergy-induced inflammation, through the TSLP-TSLPR (thymic stromal lymphopoietin receptor) signalling pathway. However, the association of IL-32 with TSLP on the ocular surface remains unclear. The present work aimed to assess the functional association of IL-32 with TSLP in the control of pro-inflammatory cytokine levels in the corneal epithelium. Human corneal tissue specimens and human corneal epithelial cells (HCECs) were administered different concentrations of IL-32 in the presence or absence of various inhibitors to assess TSLP levels and localization, as well as the molecular pathways that control pro-inflammatory cytokine production. TSLP mRNA levels were determined by real time RT- PCR, while protein levels were quantitated by ELISA and immunohistochemical staining. TSLP protein expression was examined in donor corneal epithelium samples. IL-32 significantly upregulated TSLP and pro-inflammatory cytokines (TNFα and IL-6) in HCECs at the gene and protein levels. The production of pro-inflammatory molecules by IL-32 was increased by recombinant TSLP. Interestingly, both NF-κB (quinazoline) and caspase-1 (VX-765) inhibitors suppressed the IL-32-related upregulation of pro-inflammatory cytokines (TNFα and IL-6). These findings demonstrate that IL-32 and IL-32-induced-TSLP are critical cytokines that participate in inflammatory responses through the caspase-1 and NF-κB signalling pathways in the corneal epithelium, suggesting new molecular targets for inflammatory diseases of the ocular surface. The effects of IL-32 on cell proliferation and apoptosis were investigated by MTT assays and RT-PCR,respectively. The results demonstrated that IL-32 inhibits cells apoptosis in HCECs.

TGF-β and NF-κB signaling pathway crosstalk potentiates corneal epithelial senescence through an RNA stress response.

The corneal epithelium plays important roles in the maintenance of corneal transparency for good vision, and acts as a protective barrier against foreign insults. Structural and functional changes with aging in the corneal epithelium have been documented. Here we found that transforming growth factor-β (TGF-β) is highly expressed in the elderly donor corneal epithelium, as are senescence-associated genes, such as p16 and p21. In human corneal epithelial cell (HCEC) models, TGF-β induces cellular senescence, characterized by increased SA-β-gal positive cells and elevated expression of p16 and p21. Pharmacological inhibition of TGF-β signaling alleviates TGF-β-induced cellular senescence. In addition, we determined that senescence-associated inflammation was significantly aggravated in TGF-β-induced cellular senescence by detecting the expression of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNFα). Both genetic and pharmacological approaches revealed that blocking nuclear factor-κB (NF-κB) signaling not only inhibited the production of inflammatory factors, but also rescued the senescent phenotype induced by TGF-β in HCECs. Mechanistically, TGF-β induced an atypical RNA stress responses, leading to accelerated mRNA degradation of IκBα, an inhibitor of NF-κB. Together, our data indicate that TGF-β-driven NF-κB activation contributes to corneal epithelial senescence via RNA metabolism and the inflammation blockade can attenuate TGF-β-induced senescence.

A Novel Interleukin 33/ST2 Signaling Regulates Inflammatory Response in Human Corneal Epithelium

Interleukin (IL) 33, a member of IL-1 cytokine family, is well known to promote Th2 type immune responses by signaling through its receptor ST2. However, it is not clear whether ST2 is expressed by mucosal epithelium, and how it responds to IL-33 to induce inflammatory mediators. This study was to identify the presence and function of ST2 and explore the role of IL-33/ST2 signaling in regulating the inflammatory cytokine production in corneal epithelial cells. Human corneal tissues and cultured primary human corneal epithelial cells (HCECs) were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of inflammatory cytokine and chemokine. The mRNA expression was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 mRNA and protein were detected in donor corneal epithelium and cultured HCECs, and ST2 signal was enhanced by exposure to IL-33. IL-33 significantly stimulated the production of inflammatory cytokines (TNF-α, IL-1β and IL-6) and chemokine IL-8 by HCECs at both mRNA and protein levels. The stimulated production of inflammatory mediators by IL-33 was blocked by ST2 antibody or soluble ST2 protein. Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein phosphorylation and nuclear translocation, and also suppressed the production of these inflammatory cytokines and chemokine induced by IL-33. These findings demonstrate that ST2 is present in human corneal epithelial cells, and IL-33/ST2 signaling plays an important role in regulating IL-33 induced inflammatory responses in ocular surface.

Regulation of interleukin 33/ST2 signaling of human corneal epithelium in allergic diseases.

To identify the function of ST2 and explore the role of IL-33/ST2 signaling in regulating the pro-allergic cytokine production in human corneal epithelial cells (HCECs). Human corneal tissues and cultured primary HCECs were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of pro-allergic cytokine and chemokine. The expression of mRNA was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 protein was detected in donor corneal epithelium, and ST2 signal was enhanced by exposure to IL-33. IL-33 significantly stimulated production of pro-allergic cytokines thymic stromal lymphopoietin (TSLP) and chemokine (CCL2, CCL20, CCL22) in HCECs at both mRNA and protein levels. These stimulated productions of pro-allergic mediators by IL-33 were blocked by ST2 antibody or soluble ST2 protein (P<0.05). Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein nuclear translocation, and also suppressed the productions of these pro-allergic cytokines and chemokine induced by IL-33. These findings demonstrate that IL-33/ST2 signaling plays an important role in regulating IL-33 induced pro-allergic responses. IL-33 and ST2 could become novel molecular targets for the intervention of allergic diseases in ocular surface.

Cross Presentation of Antigen on MHC Class II via the Draining Lymph Node after Corneal Transplantation in Mice

We investigated Ag trafficking from the cornea and T effector cell activation in secondary lymphoid tissue after corneal transplantation. In preliminary experiments, the central cornea was shown to contain a population of CD45(+), CD11b(+), CD11c- cells, with a few MHC class II(+) cells, and F4/80(+) cells. However, MHC class II(+) passenger leukocytes in donor cornea after allografting did not traffic to the draining lymph node. Instead, Ag (plasmid) delivered to the eye via the donor cornea during allograft was detected in host CD11c(+) and F4/80(+) APC in the draining lymph nodes and spleen. The earliest detection of APC-associated Ag was at 6 h in the draining lymph node and 24 h in the spleen. After 48 h Ag was not detected in the draining lymph node but was still present in the spleen. Ag applied to the donor corneal epithelium before allografting induced Ag-specific T cell activation and expansion in the draining lymph node with a peak response at 4-6 days, indicating that cross-presentation of Ag had occurred. We conclude therefore, that Ag is transported from the donor cornea within host APC and that this event occurs within hours after grafting. Ag is cross-presented to host CD4(+) T cells on MHC class II and leads to the activation of Ag-specific effector T cells and clonal expansion in the draining lymph node.

Distinct Populations of Dendritic Cells in the Normal Human Donor Corneal Epithelium

To characterize dendritic cells (DC) in normal human corneal epithelium. Normal human donor corneal epithelium was examined by fluorescence microscopy with single and double staining for multiple markers. Morphologic studies were also performed by confocal microscopy. HLA-DRa, CD1c, and CD16 mRNA expression in the corneal epithelium was examined by RT-PCR. CD45+ cells were separated from the corneal epithelium with magnetic beads and then were stimulated with TNF-alpha and lipopolysaccharide in vitro. CD45+ cells were mainly located in the basal-cell layer of the corneal epithelium and partly in the wing/surface layers. CD45-positive cell numbers were significantly higher in the peripheral cornea (3-6 mm from the center) than in the central cornea (0-3 mm from the center). All these cells expressed HLA-DR and CD11c but not CD3, CD11b, CD14, CD19, CD56, or CD66, suggesting that these were bone marrow-derived myeloid DC. Some DR+CD11c+ DCs from the periphery expressed CD1c and CD16. HLA-DRa, CD1c, and CD16 mRNAs were detected in normal corneal epithelium. These CD11c+ DCs did not express CD123, CD1a, DC marker (CMRF56), CD40, CD80, or CD86. When CD45+ cells were isolated from the corneal epithelium by magnetic cell sorting, CD40 and CD86 expression were detected after in vitro stimulation with TNF-alpha and lipopolysaccharide. These findings demonstrate that normal human corneal epithelium contains at least three DC phenotypes, with HLA-DR+ myeloid lineage CD11c+CD16- DCs as the main population plus a small number of CD11c+CD16+ DCs and CD11c+CD1c+ DCs. These cells can be discriminated from bone marrow-derived cells in the human corneal stroma.

Distinct Populations of Dendritic Cells in the Normal Human Corneal Epithelium

To characterize dendritic cells (DC) in normal human corneal epithelium.Normal human donor corneal epithelium was examined by fluorescence microscopy with single and double staining for multiple markers. Morphologic studies were also performed by confocal microscopy. HLA-DRa, CD1c, and CD16 mRNA expression in the corneal epithelium was examined by RT-PCR. CD45+ cells were separated from the corneal epithelium with magnetic beads and then were stimulated with TNF-alpha and lipopolysaccharide in vitro.CD45+ cells were mainly located in the basal-cell layer of the corneal epithelium and partly in the wing/surface layers. CD45-positive cell numbers were significantly higher in the peripheral cornea (3-6 mm from the center) than in the central cornea (0-3 mm from the center). All these cells expressed HLA-DR and CD11c but not CD3, CD11b, CD14, CD19, CD56, or CD66, suggesting that these were bone marrow-derived myeloid DC. Some DR+CD11c+ DCs from the periphery expressed CD1c and CD16. HLA-DRa, CD1c, and CD16 mRNAs were detected in normal corneal epithelium. These CD11c+ DCs did not express CD123, CD1a, DC marker (CMRF56), CD40, CD80, or CD86. When CD45+ cells were isolated from the corneal epithelium by magnetic cell sorting, CD40 and CD86 expression were detected after in vitro stimulation with TNF-alpha and lipopolysaccharide.These findings demonstrate that normal human corneal epithelium contains at least three DC phenotypes, with HLA-DR+ myeloid lineage CD11c+CD16- DCs as the main population plus a small number of CD11c+CD16+ DCs and CD11c+CD1c+ DCs. These cells can be discriminated from bone marrow-derived cells in the human corneal stroma.

Effect of Donor Epithelium on Corneal Transplant Survival

Two hundred thirty-two penetrating keratoplasties in 228 eyes were performed with or without the removal of donor corneal epithelium in a prospective, randomized clinical trial to determine the effect of epithelial removal on graft survival. The removal of epithelium did not reduce the likelihood of graft failure, irreversible allograft reaction, or reversible allograft reaction.

A suction trephine block for marking donor corneal buttons.

We describe a technique to accurately align the circumferences of the donor button and host cornea during penetrating keratoplasty. The donor cornea is trephined on a block into which four narrow, intersecting, equally spaced radial cuts are made. Suction is applied to the donor cornea through the four cuts, resulting in four radial marks in the donor corneal epithelium. Four radial marks are also made in the host cornea before trephination, using a radial keratotomy marker. When the donor button is placed within the recipient opening, the radial marks are aligned and used as guides for the cardinal sutures. This simple technique allows for matching of the donor and host circumferences, even if there is some collapse of the peripheral host cornea and sclera at surgery, and should minimize astigmatism resulting from donor/host misalignment.

Corneal Epithelium in Penetrating Keratoplasty

We studied corneal epithelium in 66 patients with bullous keratopathy treated with penetrating keratoplasty using McCarey-Kaufman stored donor corneas. Epithelium was evaluated at times of storage, surgery, and postoperative dressing changes. Epithelium was intact in 43 of the donor corneas at storage, and 23 had 5 to 100% (median, 50%) epithelium missing. At the end of the keratoplasty procedure, 16 grafts had epithelium intact, and 50 had 5 to 100% (median, 20%) epithelium missing. Postoperative epithelial healing time ranged from one to 12 days, with a median of two days. Postoperative healing was significantly prolonged when donor corneal epithelium was missing at keratoplasty. As the amount of epithelium intact at the end of surgery decreased, the number of days to heal postoperatively increased. We found that donor corneas could be stored as long as 79 hours, with 63 hours in McCarey-Kaufman medium, and still have epithelium intact at the end of the keratoplasty procedure.

Immunological activity to different corneal antigens in patients with corneal diseases.

Patients suffering from various corneal diseases and waiting for keratoplasty have been immunologically investigated in order to establish sensitisation to corneal antigens. The presence of lymphocytes sensitised to the soluble from human corneas, bovine corneal epithelium, and bovine corneal stroma, which all possess common antigenicity, could be demonstrated in 30%, 50%, and 23%, respectively, of all patients. In none of these patients could a positive plasma antibody titre to human corneal antigens be detected. The results suggest the dominance of T-lymphocyte activity. No correlation was found between the degree of corneal vascularisation and the presence of sensitised lymphocytes to human corneal antigens. Arrangement of the patients according to diagnosis showed that especially those suffering from herpes simplex virus keratitis reacted positively to human corneal antigens. A possible explanation is given. Lymphocytes of controls showed no or only very low stimulation with the soluble fractions of human corneas or bovine corneal stromas. The soluble fraction of bovine corneal epithelium stimulated the lymphocytes of 6 out of 19 controls. The elimination of the donor corneal epithelium before transplantation may be beneficial in view of the involvement of histocompatibility antigens.