Yuriko Ban

Tight junction-related protein expression and distribution in human corneal epithelium.

PURPOSE To investigate the expression and cellular distribution of the tight junction-related proteins occludin, claudin and ZO-1 in human corneal epithelium. METHODS Light and electron immunohistochemistry was used to determine tissue distribution of occludin, claudin-1 and ZO-1 in the human corneal epithelium. Reverse transcription-polymerase chain reaction was used to reveal claudin mRNA expression in human corneal epithelium. RESULTS In transverse sections, occludin and ZO-1 were localized at the apical cell-cell junctions between superficial cells in stratified corneal epithelium. Both basal and basolateral membranes of superficial cells were stained by the claudin-1 antibody, but no apical membrane staining was observed. In en face sections, claudin-1 and ZO-1 antibodies showed as bands that corresponded to the junctional complex. Claudin-1 staining of superficial cell cytoplasm was also observed. Occludin staining was seen at the junctional complex, where it was not continuous, but dotted along the cell junctions. The transcripts for claudin-1 and several other claudin isotypes, such as -2, -3, -4, -7, -9 and -14 were identified. CONCLUSION Not only occludin, but also some claudins act as integral transmembrane proteins in the corneal epithelium. ZO-1 is a component of the corneal epithelial tight junction, as it is in most epithelial cells.

Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting

PURPOSE To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions (TJ) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins. METHODS Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and mRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction. RESULTS The cultures of both groups formed 4-5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3-4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium. CONCLUSIONS The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction.

Cultivated corneal endothelial transplantation in a primate: possible future clinical application in corneal endothelial regenerative medicine.

Purpose: To review our attempt to devise a method of cultivated corneal endothelial transplantation using primates in which corneal endothelium, like that of humans, has low proliferative ability. Methods: Monkey corneal endothelial cells (MCECs) were cultivated, with subcultures grown on collagen type I carriers. The corneal endothelia of 6 eyes of 6 monkeys were scraped intensively, after which cultivated MCEC sheets were inserted into the anterior chamber of 4 eyes. As controls, a collagen sheet without MCECs was transplanted in 1 eye of a monkey, and a suspension of cultivated MCECs was injected into the anterior chamber of 1 eye of another monkey. Results: MCECs produced a confluent monolayer of closely attached hexagonal cells, which expressed both ZO-1 and Na+-K+ adenosine triphosphatase. Early postoperative period MCEC sheets were attached to Descemet membrane, and corneal clarity was recovered. Six months after transplantation, MCEC-transplanted corneas remained clear, and closely attached hexagonal cells were observed. In 1 animal with longer observation, polygonal cells were observed by in vivo specular microscopy at a density of >2000 cells/mm2 and remained >1600 cells/mm2 for ≤2 years. Control eyes showed irreversible bullous keratopathy throughout the observation period. Conclusions: Cultivated MCECs become attached to the transplanted eye and maintain a clear cornea ≤2 years postoperatively, suggesting that corneal endothelial cells of primates might have proliferative ability in vivo once they have been cultured and proliferated in vitro. Our monkey model constitutes an important step forward for regenerative medicine with possible future application in patients with corneal endothelial dysfunction.

Tight junction transmembrane protein claudin subtype expression and distribution in human corneal and conjunctival epithelium.

PURPOSE The combination of the tight junction transmembrane protein claudin subtypes is one of the most important determinants of variations in the tightness of individual paired tight junction strands. The barrier function of corneal epithelium is much stronger than that of conjunctival epithelium. In this study, the expression and cellular distribution of claudin species in in vivo human corneal and conjunctival epithelium were investigated. METHODS Reverse transcription-polymerase chain reaction was used to reveal the claudin mRNA. Immunohistochemistry was used to determine the tissue distribution of tight junction-related proteins and MUC5AC. RESULTS Transcripts for claudin-1, -2, -3, -4, -7, -9, and -14 were identified in human corneal epithelium. Transcripts for claudin-1, -2, -4, -7, -9, -10, and -14 were identified in human conjunctival epithelium. By immunohistochemistry, claudin-1, -4, and -7 were found to be localized at the membrane of human corneal and conjunctival epithelial cells. In human conjunctival epithelium, claudin-10 staining was observed at several, but not all, apical epithelial cell-to-goblet cell junctions. CONCLUSIONS Claudin-1, -4, and -7 are expressed in corneal and conjunctival epithelia. Claudin-10 is prominent at several junctions between apical epithelial cells and goblet cells in conjunctival epithelium. Except for claudin-10 expression in conjunctival epithelium, the claudin subtype expressions of corneal and conjunctival epithelia are similar. Therefore, there must be a difference between these two epithelial types with regard to the specific ratio of claudin subtypes expressed or their phosphorylation status. The distribution of goblet cells in conjunctival epithelium also influences the difference in barrier function.

A novel mutation of the OPA1 gene in a Japanese patient with autosomal dominant optic atrophy.

Dear Editor: Autosomal dominant optic atrophy (ADOA; MIM#165500) is a hereditary optic neuropathy characterized by: (1) symmetrical bilateral optic atrophy associated with reduced corrected visual acuity beginning in the first 2 decades of life, (2) color vision disturbances, and (3) paracentral or caencocentral scotoma. ADOA is genetically heterogeneous, and the OPA1 gene located on chromosome 3q28–q29 has been determined to be one of the causative genes [1–3]. We report a novel mutation of the OPA1 gene in a Japanese patient with ADOA. We performed complete ocular examinations on a Japanese patient (22-year-old male) with ADOA and his two non-affected relatives (Fig. 1a). Genomic DNA samples were isolated from the whole blood of the patient and his relatives after informed consent. Each exon of the OPA1 gene and its immediate flanking sequence were amplified by polymerase chain reaction [4]. Purified amplified fragments were subjected to genetic sequencing using an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The single nucleotide polymorphism found by direct sequencing was confirmed by the single nucleotide primer extension method. The present study had the approval of the Nantan General Hospital ethics committee and was conducted in accordance with the World Medical Association Declaration of Helsinki. Clinically, the patient had reduced visual acuity during his elementary school days, with his best corrected visual acuity being 0.2 OD and 0.1 OS. The patient had bilateral temporal atrophy of the optic disc (Fig. 2a), and there were enlargement of the blind spot and temporal lower decrease of Ι/2c isopter in his right visual field (Fig. 2b). Color vision was tested using a 15-hue Farnsworth’s panel. The patient made a disorganized classification, with a specific confusion in the blue-yellow axis, suggesting tritanopia (Fig. 2c). By optical coherence tomography, the retinal nerve fiber layer thickness was very thin (Fig. 2d). No abnormality was observed by electroretinogram. His mother and grandfather were normal by all ocular examination. Sequence analyses revealed a heterozygous mutation of the OPA1 gene in exon 2 as C/T at the 154th position (Fig. 1b) which resulted in an instant termination of translation (R52X). By the reverse sequence by the SNaPshot method, the patient represents double peaks indicating heterozygous mutation at the base position of 154, which is consistent with sequencing results (data not shown). No mutation was found in his mother (Fig. 1b) and grandfather (data not shown), which is consistent with the fact that they showed no abnormalities during clinical examination. According to information obtained by interview, his deceased father had moderate visual disturbance. Unfortunately, we could not examine any other family members. Therefore, it is difficult to determine if this case was sporadic or hereditary. The inability of the wild-type allele to produce sufficient levels of protein to confer the normal phenotype—the Graefe’s Arch Clin Exp Ophthalmol (2007) 245:1581–1583 DOI 10.1007/s00417-007-0598-1