Aberrant HLA-DR expression in the conjunctival epithelium after autologous serum treatment in patients with graft-versus-host disease or Sjögren's syndrome.

The aim of this study was to determine the effect of autologous serum (AS) eye drops on the density of human leucocyte antigen (HLA)-DR-positive epithelial cells and Langerhans cells on the ocular surface of patients with bilateral severe dry eye disease (DED) due to graft-versus-host disease (GvHD) or Sjögren's syndrome (SS). The study was conducted on 24 patients (48 eyes). AS was applied 6-10 times daily for 3 months together with regular artificial tear therapy. HLA-DR-positive cells were detected by direct immunocytochemistry on upper bulbar conjunctiva imprints obtained before and after treatment. The application of AS drops led to a statistically significant increase in the mean density of aberrant HLA-DR-positive conjunctival epithelial cells (p < 0.05) and HLA-DR-positive Langerhans cells (p < 0.05) in the GvHD group. Aberrant HLA-DR-positive epithelial cells in the SS group were decreased non-significantly. All patients reported a significant decrease in the Ocular Surface Disease Index (p < 0.01), which indicates improvement of the patient's subjective feelings after therapy. There was an expected but non-significant decrease of aberrant HLA-DR-positive conjunctival epithelial cells in the SS group only. However, the increased density of HLA-DR-positive cells, indicating slight subclinical inflammation, does not outweigh the positive effect of AS in patients with DED from GvHD.

The effect of culture medium and carrier on explant culture of human limbal epithelium: A comparison of ultrastructure, keratin profile and gene expression.

Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).

Changes in lysyl oxidase (LOX) distribution and its decreased activity in keratoconus corneas.

Inadequate cross-linking between collagen lamellae is a characteristic feature of keratoconus corneas. The formation of covalent bonds between collagen and elastin fibrils, which maintain the biomechanical properties of the cornea, is mediated by the cuproenzyme lysyl oxidase and four lysyl oxidase-like enzymes. The aim of this study was to determine the distribution of lysyl oxidase and the total lysyl oxidase activity (lysyl oxidase and the four lysyl oxidase-like enzymes) in control and keratoconic corneas. Seven control and eight keratoconic corneas were used for the imunohistochemical detection of lysyl oxidase in corneal cryosections using two different antibodies. The total lysyl oxidase activity in the culture medium of corneal fibroblasts from six explanted keratoconic and four control corneas was measured using a fluorometric assay in the presence and absence of the lysyl oxidase inhibitor beta-aminopropionitrile and determined as the production of H(2)O(2) in nM per µg of total protein. In the control tissue, the most intense signal for lysyl oxidase was present in the corneal epithelium, in which perinuclear dots brightly projecting from more or less homogenous cytoplasmic staining may represent the lysyl oxidase propeptide. Less intense staining was present in keratocytes, the extracellular matrix and in the corneal endothelium. The epithelium of the limbus and the perilimbal conjunctiva showed intense to very intense staining. The distribution of lysyl oxidase was clearly decreased in at least five of the eight keratoconic specimens. The most marked signal reduction was observed in the stromal matrix and in keratocytes. Moreover, the signal in pathological specimens revealed a more irregular pattern, including the presence of intra- and extracellular clumps in the epithelium. Interestingly, endothelial cells showed no or very weak staining in areas just beneath negative stromal tissue. The mean activity of total lysyl oxidase in the keratoconic samples (2.60 ± 2.23 nM H(2)O(2)/µg of total protein) was more than 2.5-fold lower than in control tissue (6.83 ± 2.53 nM H(2)O(2)/µg of total protein), and the decrease was statistically significant (p = 0.0178). The location of lysyl oxidase in the healthy cornea, limbus and perilimbal conjunctiva was described. We hypothesize that the restricted lysyl oxidase distribution in keratoconic corneas, and particularly the decrease of total lysyl oxidase activity in cultured keratoconic fibroblasts, is one potential reason for the inadequate collagen cross-linking that is a hallmark of this disease.

The OV-TL 12/30 clone of anti-cytokeratin 7 antibody as a new marker of corneal conjunctivalization in patients with limbal stem cell deficiency.

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.

Mesothelial proteins are expressed in the human cornea.

The goal of our study was to determine whether proteins typical of the human mesothelial cell phenotype, such as mesothelin, HBME-1 (Hector Battifora mesothelial cell-1) protein and calbindin 2, are expressed in the human cornea, especially in endothelial cells. Cryosections and endothelial and epithelial imprints of sixteen human cadaverous corneoscleral discs were used. The presence of proteins was examined using immunohistochemistry and Western blotting, while mRNA levels were determined by qRT-PCR. A strong signal for mesothelin was present in the corneal epithelium, while less intense staining was visible in the endothelium. Similarly, higher and lower mRNA levels were detected using qRT-PCR in the corneal epithelium and endothelium, respectively. HBME-1 antibody strongly stained the corneal endothelium and stromal keratocytes. Marked positivity was present in the corneal stromal extracellular matrix, while no staining was present in the sclera. Calbindin 2 was detected using immunohistochemistry and Western blotting in the corneal epithelium, endothelium and stroma. qRT-PCR confirmed its expression in epithelial and endothelial cells. Three proteins expressed constitutively in mesothelial cells were detected in the human cornea. The possible function of mesothelin in cell-cell contact on the ocular surface is discussed. The presence of HBME-1 protein in the endothelial layer may indicate a still unknown function that could be shared with mesothelial cells of the pleura and peritoneum. The much more pronounced occurrence of calbindin 2 in the corneal epithelium compared to fewer positive endothelial cells explains the higher turnover of epithelial cells compared to the proliferatively inactive endothelium.