Atsuyoshi Dota

Amniotic Membrane as a Substrate for Cultivating Limbal Corneal Epithelial Cells for Autologous Transplantation in Rabbits.

Purpose. To examine the viability of using human amniotic membrane as substrate for culturing corneal epithelial cells and transplanting them onto severely injured rabbit eyes. Methods. An ocular-surface injury was created in the right eye of eight rabbits by a lamellar keratectomy extending 5 mm outside the limbus. Next, from the limbal region of the uninjured left eyes of five of these animals, a small biopsy of corneal epithelial cells was taken and cultured on acellular human amniotic membrane. One month later, the invading conjunctiva that covered the corneal surface of all eight injured eyes was surgically removed. Five of the eyes then received grafts of amniotic membrane containing autologous cultured epithelial cells, whereas the other three received grafts of acellular amniotic membrane alone. Results. A confluent primary culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 14 days. Cells were partially stratified and fairly well attached to the underlying amniotic membrane, although a fully formed basement membrane was not evident. The three rabbits that received amniotic membrane transplantation alone all had total epithelial defects on the graft in the early postoperative period. Eyes that were grafted with amniotic membrane that contained cultivated epithelial cells, however, were all successfully epithelialized up to 5 days after surgery. Conclusion. Autologous transplantation of cultivated corneal epithelium is feasible by using acellular amniotic membrane as a carrier.

Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

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.

Isolation and Chromosomal Localization of a Cornea-Specific Human Keratin 12 Gene and Detection of Four Mutations in Meesmann Corneal Epithelial Dystrophy

Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span approximately 6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved alpha-helix-initiation motif of rod domain 1A or in the alpha-helix-termination motif of rod domain 2B. These sites are essential for keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the alpha-helix-termination motif in type I keratin.

Amyloid and Pro501Thr-mutated βig-h3 gene product colocalize in lattice corneal dystrophy type IIIA

PURPOSE To assess the relative distribution in the cornea of amyloid and (beta)ig-h3 gene product in lattice corneal dystrophy type IIIA (LCD-IIIA). METHODS Serial sections from the cornea of a patient with LCD-IIIA were subjected to either Congo red staining or immunohistochemistry employing an antibody to (beta)ig-h3. Also, genomic DNA was isolated from peripheral blood and used as a template for polymerase chain reaction to amplify all exons of (beta)ig-h3. RESULTS Exon 11 of (beta)ig-h3 was mutated (Pro501Thr). Subepithelial and intrastromal congophilic deposits exhibited a birefringency characteristic of amyloid. These regions of the tissue were also highly immunoreactive with the antibody to the (beta)ig-h3 gene product. CONCLUSION Amyloid and Pro501Thr-mutated (beta)ig-h3 protein accumulate and colocalize in LCD-IIIA.

Apolipoproteins J and E co-localise with amyloid in gelatinous drop-like and lattice type I corneal dystrophies

AIMS Apolipoprotein J (apoJ) and apolipoprotein E (apoE) are thought to contribute to amyloid formation in patients with Alzheimer’s disease. The aim of this investigation was to discover whether or not these apolipoproteins associate with corneal amyloid in gelatinous drop-like corneal dystrophy (GDCD) and lattice corneal dystrophy type I (LCD-I). METHODS Corneas from three eyes of three patients with GDCD and one eye of one patient with LCD-I were examined immunohistochemically using antibodies against apoJ and apoE. Two normal corneas were similarly examined. Tissue sections of brain from a patient with Alzheimer’s disease were used as positive controls for the antibodies. For all negative controls, mouse IgG was used instead of the primary antibody. RESULTS Intense apoJ and apoE immunoreactivities were found in congophilic amyloid deposits in GDCD and LCD-I. These deposits were located subepithelially in GDCD, and subepithelially and intrastromally in LCD-I. In GDCD, immunostaining of subepithelial amyloid with anti-apoJ was noticeably stronger than with anti-apoE. CONCLUSIONS As in senile plaques in brain from a patient with Alzheimer’s disease, apoJ and apoE co-localise with amyloid in corneas with GDCD and LCD-I.

Epithelial hyperproliferation and transglutaminase 1 gene expression in Stevens-Johnson syndrome conjunctiva

In Stevens-Johnson syndrome, pathological keratinization of the ordinarily nonkeratinized corneal and conjunctival mucosal epithelia results in severe visual loss. We examined conjunctiva covering cornea in five eyes in the chronic cicatricial phase of Stevens-Johnson syndrome. Normal conjunctiva from five age-matched individuals was studied also. The number of epithelial cells in Stevens-Johnson syndrome conjunctiva that were immunoreactive with a monoclonal antibody, Ki-67, to a nuclear antigen found only in proliferating cells was greater than normal (93.8+/-19.8 cells above 100 basal cells versus 12.8+/-0.5 cells above 100 basal cells; P = 0.009). In addition, although clinical inflammation was mild, massive lymphocytic infiltration was seen in the substantia propria of conjunctiva covering cornea. In situ hybridization documented transglutaminase 1 (keratinocyte transglutaminase) mRNA in suprabasal cells of the abnormally thickened conjunctival epithelium in all Stevens-Johnson syndrome patients. In contrast, no message was detected in normal conjunctival or corneal epithelia. Transglutaminase 1 is expressed during the terminal differentiation of keratinocytes where it helps synthesize cornified cell envelopes. We speculate that in Stevens-Johnson syndrome, epithelial hyperproliferation, and transglutaminase 1 gene expression lead to the pathological keratinization of ocular surface mucosal epithelia.

Epithelial Barrier Function and Ultrastructure of Gelatinous Drop-like Corneal Dystrophy.

Purpose. Recently, mutations in the M1S1 gene have been identified as responsible for gelatinous drop-like corneal dystrophy (GDLD). How the abnormal M1S1 gene product causes GDLD is not known, although evidence suggests that it may compromise corneal epithelial function. This investigation attempted to determine the effect of the abnormal M1S1 gene product by assessing epithelial barrier function and epithelial ultrastructure in GDLD corneas. Methods. Epithelial barrier function was assessed on the basis of fluorescein uptake. The method used a modified slit-lamp fluorophotometer. High-resolution scanning electron and atomic force microscopy was used to investigate the amyloid deposits and epithelial cell structure. Results. Epithelial permeability was orders of magnitude higher in GDLD corneas than normal. The structure of the amyloid deposits was characterized, and clear abnormalities in epithelial morphology and cell junctions were observed. Conclusions. The high epithelial permeability observed in GDLD corneas was directly correlated with abnormalities in epithelial structure, including irregular cell junctions. This suggests that the abnormal M1S1 gene product may affect epithelial cell junctions resulting in increased cell permeability in GDLD corneas.

Detection of herpes simplex virus DNA in atypical epithelial keratitis using polymerase chain reaction

AIM To study herpes simplex virus (HSV) DNA in tears from patients with atypical epithelial keratitis of unknown aetiology. METHODS Tear samples were collected from 17 affected eyes of 17 consecutive patients suffering from epithelial keratitis in whom HSV keratitis was suspected but whose diagnosis was difficult on the basis of clinical manifestations alone. Using reduced sensitivity polymerase chain reaction (PCR), tear samples were tested for HSV DNA. Tears from the unaffected eyes of the 17 patients were also examined, along with 38 tear samples from 19 normal volunteers. Southern blot analysis was performed to confirm that amplified DNA bands were specific for HSV. Clinical correlation with photographs of corneal lesions was also investigated. RESULTS HSV DNA was detected in tears from the affected eyes of eight of the 17 patients with suspected HSV keratitis. Tears from the affected eyes of the other patients were PCR negative, as were tears from the unaffected eyes of all 17 patients, and from the 38 normal eyes. There was no correlation between PCR results and clinical manifestation of keratitis. CONCLUSIONS Based on the sensitivity of the PCR system, eight of 17 suspected HSV keratitis patients were confirmed as suffering from HSV keratitis. HSV keratitis should therefore be considered as a possible diagnosis in atypical epithelial keratitis.