Mina Nakatsukasa

Cultivated human conjunctival epithelial transplantation for total limbal stem cell deficiency.

PURPOSE To determine the feasibility of cultivated conjunctiva as a viable epithelial sheet for transplantation and corneal resurfacing in eyes with limbal stem cell deficiency (LSCD). METHODS Human corneal epithelial (HCE) and human conjunctival epithelial (HCjE) cells were cultivated on human amniotic membrane (AM) to confluence and then air lifted to allow further stratification and differentiation. Denuded AM and cultivated HCE and cultivated HCjE cells were then transplanted into 18 eyes of rabbits with induced LSCD. The cultivated and engrafted epithelia were examined by transmission electron microscopy (TEM) and immunohistochemistry. Two weeks after transplantation, the eyes were examined by slit lamp biomicroscopy and scored on epithelial integrity, corneal haze, and corneal neovascularization. RESULTS Both cultivated and engrafted HCjE sheets demonstrated confluent epithelial sheets with five to six layers of well-stratified epithelium. TEM examination of engrafted HCjE revealed numerous microvilli, desmosomes, and hemidesmosomes, identical with in vivo corneal epithelium. Immunohistochemical analysis of both HCjE and HCE cells showed the presence of CK3, CK4, and CK12, with absence of Muc5AC. Clinical outcomes for eyes receiving HCjE transplants and HCE transplants were comparable, with most having transparent, smooth corneas, free of epithelial defects. CONCLUSIONS The study showed that microscopically, HCjE cells have features similar to HCE cells, with clinically equivalent outcomes. The ex vivo cultivation of conjunctiva to form transplantable epithelial sheets for corneal replacement is a promising new treatment modality in patients with LSCD.

Amino Acid Profiles in Human Tear Fluids Analyzed by High-Performance Liquid Chromatography and Electrospray Ionization Tandem Mass Spectrometry

PURPOSE To identify the 23 amino acid profiles in human tear fluids, and to evaluate whether the ocular disease conditions reflect the amino acid profiles. DESIGN Laboratory investigation. METHODS We evaluated the concentrations and relative composition of 23 amino acids in tear fluids obtained from 31 healthy volunteers using reversed-phase high-performance liquid chromatography and electrospray ionization tandem mass spectrometry, and compared them with those in plasma and aqueous humor. We also evaluated the tear-fluid amino acid profiles from 33 affected subjects. RESULTS The amino acid profiles of the basal tear and reflex tear were found to be similar, and 4 distinct groups of healthy volunteers (male, female, young, and elderly) showed similar profiles. Absolute concentrations of taurine (Tau) and L-glutamine were significantly dominant in these tear fluids. The relative compositions of Tau, L-glutamic acid, L-arginine (Arg), and citrulline in the tear fluid were significantly higher than those in the plasma and aqueous humor. Analysis of the hierarchical clustering of the amino acid profiles clearly distinguished severe ocular surface diseases from non-ocular surface diseases. The relative compositions of Tau, L-methionine, and Arg decreased in severe ocular surface disease subjects compared with non-ocular surface disease subjects. CONCLUSIONS Tear-fluid amino acid profiles differ from those in plasma and aqueous humor. Steady-state tear-fluid amino acid profiles might reflect ocular-surface homeostasis and the observed changes of amino acids might have a close relation with the disease conditions on the ocular surface.

Tumor-Associated Calcium Signal Transducer 2 Is Required for the Proper Subcellular Localization of Claudin 1 and 7 : Implications in the Pathogenesis of Gelatinous Drop-Like Corneal Dystrophy

Gelatinous drop-like dystrophy (GDLD) is a rare autosomal recessive form of corneal dystrophy characterized by subepithelial amyloid depositions on the cornea. Previous clinical and laboratory observations have strongly suggested that epithelial barrier function is significantly decreased in GDLD. Despite the decade-old identification of the tumor-associated calcium signal transducer 2 (TACSTD2) gene as a causative gene for GDLD, the mechanism by which the loss of function of this causative gene leads to the pathological consequence of this disease remains unknown. In this study, we investigated the functional relationship between the TACSTD2 gene and epithelial barrier function. Through the use of immunoprecipitation and a proximity ligation assay, we obtained evidence that the TACSTD2 protein directly binds to claudin 1 and 7 proteins. In addition, the loss of function of the TACSTD2 gene leads to decreased expression and change in the subcellular localization of tight junction-related proteins, including claudin 1, 4, 7, and ZO1 and occludin, both in diseased cornea and cultured corneal epithelial cells. These results indicate that loss of function of the TACSTD2 gene impairs epithelial barrier function through decreased expression and altered subcellular localization of tight junction-related proteins in GDLD corneas.

Genomic Aberrations and Cellular Heterogeneity in SV40-Immortalized Human Corneal Epithelial Cells

PURPOSE Simian virus (SV)40-immortalized human corneal epithelial (HCE-T) cells have been widely used as an in vitro model of human corneal epithelial cells. The nature of this cell line was assessed for genomic aberrations and cellular heterogeneity. METHODS For the quantitative measurement of genomic aberrations, array-based comparative genomic hybridization (CGH) analysis was performed. For identification of cellular heterogeneity, cell morphology, growth kinetics, transepithelial electrical resistance, and transfection/transcriptional efficiency were analyzed. Real-time PCR and chromosomal fluorescent in situ hybridization (cFISH) against some gained or lost loci were performed, to assess genomic heterogeneity. Expressed sequence tags (ESTs) for this cell line were collected to assess differences in the gene expression profiles between HCE-T cells and normal corneal epithelial cells. Southern blot analysis and inverse PCR analyses were used to determine the genomic integration site of the SV40 large T antigen gene (LTAG). RESULTS Array CGH analysis demonstrated that the genomic content of HCE-T cells is different from the normal healthy genome. The results from cellular functional assays, real-time PCR, and cFISH strongly indicated that HCE-T cells consist of a significant number of heterogeneous cell populations. The genomic integration site of the SV40 large T antigen was at p22.1 of chromosome 9. CONCLUSIONS The results indicate that HCE-T cells have an altered genomic content and that they are composed of heterogeneous cell populations. This should be considered when conducting experiments or interpreting the results of studies that use this cell line.

Diagnosis of multiple myeloma in a patient with atypical corneal findings.

Purpose: To report the case of a 67-year-old woman with a diagnosis of multiple myeloma (MM) and atypical corneal opacity as the first appearance. Methods: The patient consulted an ophthalmologist regarding blurred vision that progressed gradually without any systemic symptoms. The corneal epithelium and anterior stroma of both eyes harbored diffuse gray-white deposits of unknown etiology. She underwent a general checkup that included molecular genetic analysis. At cataract surgery, performed 5 months after the diagnosis, the corneal epithelium was removed with an epikeratome. The epithelial sheet was analyzed histologically. Results: Her serum immunoglobulin G (IgG) level was increased; serum immunoelectrophoresis showed a monoclonal γ-globulin spike of the IgG κ-type. The presence of 30.4% pleomorphic plasma cells in the bone marrow confirmed the diagnosis of MM. Her visual acuity improved after combined cataract surgery and superficial keratectomy. The surgically removed epithelial sheet showed IgG deposits in the corneal epithelium; electron-dense intracellular deposits were observed in corneal epithelial cells. Conclusions: In some individuals, unusual corneal deposits may constitute the first sign of MM. Superficial keratectomy with an epikeratome is a minimally invasive treatment of MM with corneal opacification.

Establishment of a Human Corneal Epithelial Cell Line Lacking the Functional TACSTD2 Gene as an In Vitro Model for Gelatinous Drop-Like Dystrophy

PURPOSE Gelatinous drop-like corneal dystrophy (GDLD) is characterized by subepithelial amyloid deposition that engenders severe vision loss. The exact mechanism of this disease has yet to be elucidated. No fundamental treatment exists. This study was conducted to establish an immortalized corneal epithelial cell line to be used as a GDLD disease model. METHODS A corneal tissue specimen was obtained from a GDLD patient during surgery. Corneal epithelial cells were enzymatically separated from the cornea and were dissociated further into single cells. The epithelial cells were immortalized by the lentiviral transduction of the simian virus 40 (SV40) large T antigen and human telomerase reverse transcriptase (hTERT) genes. For the immortalized cells, proliferative kinetics, gene expressions, and functional analyses were performed. RESULTS The immortalized corneal epithelial cells continued to proliferate despite cumulative population doubling that exceeded 100. The cells showed almost no sign of senescence and displayed strong colony-forming activity. The cells exhibited a low epithelial barrier function as well as decreased expression of tight-junction-related proteins claudin 1 and 7. Using the immortalized corneal epithelial cells derived from a GDLD patient, we tested the possibility of gene therapy. CONCLUSIONS We established an immortalized corneal epithelial cell line from a GDLD patient. The immortalized cells exhibited cellular phenotypes similar to those of in vivo GDLD. The immortalized cells are thought to be useful for the development of new therapies for treating GDLD corneas and for elucidation of the pathophysiology of GDLD.