Assessment of the corneal biomechanical features of granular corneal dystrophy type 2 using dynamic ultra-high-speed Scheimpflug imaging.

PURPOSE: To evaluate the corneal biomechanical features of eyes with granular corneal dystrophy type 2 (GCD2) by analyzing corneal biomechanical indices obtained using a Corvis ST (CST) dynamic ultra-high-speed Scheimpflug imaging device. METHODS: In this retrospective case-control study, 35 CST parameters were compared in normal eyes (control) and eyes of patients with GCD2 treated at Osaka University Hospital, Osaka, Japan. The parameters included the Corvis Biomechanical Index (CBI), which is important in differentiating eyes with keratoconus from normal eyes. We measured the deposition rates of lesions in the central 7-mm region of the eye and assessed the correlation between the deposition rate and the CBI. RESULTS: Twenty-one eyes with GCD2 and 23 control eyes were analyzed. Eyes with GCD2 showed significantly less corneal stiffness in 15 CST parameters than did control eyes. In particular, the CBI was remarkably higher in eyes with GCD2 than in control eyes (P = 0.000006). Additionally, the deposition rate and the CBI were positively correlated. CONCLUSIONS: GCD2 eyes had softer corneas than did control eyes in most biomechanical CST parameters, and one of the parameters (the CBI) was linked to the rate of deposited lesions. Since IOP may be underestimated in GCD2 eyes, management should be especially careful in GCD2 cases complicated by glaucoma.

An outbreak of serotype 19A pneumococcal pneumonia in a relief facility in Japan.

An outbreak of serotype 19A Streptococcus pneumoniae occurred among the residents of a relief facility. Pneumonia developed in 5 of 99 residents (attack rate, 5.1%). We obtained pharyngeal specimens from non-onset residents, and S. pneumoniae was isolated from 6 individuals (6.4%), 5 of whom had serotype 19A.

Fusobacterium necrophorum Endocarditis with Liver Abscesses: A Case Report and Review of the Literature.

Fusobacterium necrophorum is a very rare cause of endocarditis. We herein report a case of F. necrophorum endocarditis with liver abscesses in a 51-year-old woman. This is the first reported case of monomicrobial F. necrophorum endocarditis to present in a patient over 50 years old. We also reviewed 10 reported cases, including the present case. Our review indicated that anaerobic bacteria, including Gram-negative anaerobic bacilli such as F. necrophorum, should be considered in the differential diagnosis of infective endocarditis, especially in patients without preexisting organic heart disease.

Rubicon regulates A2E-induced autophagy impairment in the retinal pigment epithelium implicated in the pathology of age-related macular degeneration.

Waste product deposition and light stress in the retinal pigment epithelium (RPE) are crucial factors in the pathogenesis of various retinal degenerative diseases, including age-related macular degeneration (AMD), a leading cause of vision loss in elderly individuals worldwide. Given that autophagy in the RPE suppresses waste accumulation, determining the molecular mechanism by which autophagy is compromised in degeneration is necessary. Using polarized human RPE sheets, we found that bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E), a major toxic fluorophore of lipofuscin, causes significant impairment of autophagy and the simultaneous upregulation of Rubicon, a negative regulator of autophagy. Importantly, this impairment was reversed in Rubicon-specific siRNA-treated RPE sheets. In a retinal functional analysis using electroretinograms (ERGs), mice with the RPE-specific deletion of Rubicon showed no significant differences from control cre-expressing mice but presented partially but significantly enhanced amplitudes compared with Atg7 knockout mice. We also found that an inflammatory reaction in the retina in response to chronic blue light irradiation was alleviated in mice with the RPE-specific deletion of Rubicon. In summary, we propose that upregulating basal autophagy by targeting Rubicon is beneficial for protecting the RPE from functional damage with ageing and the inflammatory reaction caused by light-induced cellular stress.

Role of oncostatin M in the pathogenesis of vernal keratoconjunctivitis: focus on tissue remodeling.

PURPOSE: Vernal keratoconjunctivitis (VKC) is a severe and recurrent allergic conjunctivitis, the mechanism of which is not well understood. In this study, we investigated the role of oncostatin M (OSM) in the pathogenesis of VKC, with a focus on tissue remodeling. STUDY DESIGN: Clinical and experimental. PATIENTS AND METHODS: The OSM concentrations in tear fluid samples obtained from VKC patients and healthy controls were measured using ELISA, and the expression of OSM mRNA and protein in giant papillae resected from VKC patients was investigated using RT-PCR and immunohistochemistry, respectively. In cultured human conjunctival epithelial cells (HconEpiCs), expression of OSM receptor ß (OSMRß) was detected using immunocytochemical and FACS analyses. Finally, we investigated whether recombinant OSM activated STAT1 and STAT3 to induce the expression of various genes related to tissue remodeling in HconEpiCs, by using Western blot analysis, microarray analysis, and RT-PCR. RESULTS: The OSM concentration was higher in the tear fluid of VKC patients than in that of the healthy controls, and strong expression of OSM mRNA was found in the giant papillae. We also detected T cells expressing OSM in the giant papillae. In addition, HconEpiCs showed surface expression of OSMRß. Recombinant human OSM strongly activated both STAT1 and STAT3 in HconEpiCs and induced various tissue remodeling-related genes, including MMP-1, MMP-3, IL-24, IL-20, serpinB3, S100A7, tenascin C, and SOCS3. CONCLUSION: Our results suggest that OSM is one of the key molecules involved in remodeling of giant papillae in VKC.

Corneal Opacity Induced by Light in a Mouse Model of Gelatinous Drop-Like Corneal Dystrophy.

Gelatinous drop-like corneal dystrophy (GDLD) is a severe inherited corneal dystrophy characterized by subepithelial corneal amyloid deposition. We had previously succeeded in identifying the responsible gene, TACSTD2, and subsequently found that the epithelial barrier function is significantly decreased. As with GDLD patients, the knockout mice showed severe loss of tight junction, progressive opacity, and neovascularization in the cornea. We devised an easy method to confirm the loss of the corneal barrier function even before corneal opacity is observed. Furthermore, by using knockout mice, we were able to verify clinical findings, such as the wound healing delay and light-induced acceleration of the disease. This mouse model should prove to be a highly useful tool for investigating the pathology of GDLD and for developing new therapies.

Comparison of ocular surface squamous neoplasia and pterygium using anterior segment optical coherence tomography angiography.

PURPOSE: To compare ocular surface squamous neoplasia (OSSN) and pterygium using anterior segment optical coherence tomography angiography (AS-OCTA). OBSERVATIONS: Flow patterns of conjunctival vessels in patients with OSSN and pterygium were investigated using AS-OCTA. In case 1, slit-lamp examination of a 72-year-old woman revealed an elevated lesion with increased permeability of fluorescein in the inferior nasal conjunctiva of her left eye. AS-OCTA showed markedly meandering large blood vessels in both the superficial and deep layers. Histopathological evaluation of the conjunctival biopsy indicated conjunctival intraepithelial neoplasia. Case 2 was that of a 79-year-old man with a history of three conjunctival tumor excisions. Slit-lamp examination showed an elevated lesion with hyperpermeability of fluorescein in the nasal conjunctiva of his left eye. AS-OCTA revealed increased meandering vasculature in both the superficial and deep layers. Histopathological investigation concluded that the diagnosis was squamous cell carcinoma. Case 3 involved a 61-year-old man with a pterygium. Slit-lamp examination showed typical findings of an elevated nasal lesion accompanied by a head that appeared triangular with a blunt apex. AS-OCTA revealed increased straight vasculature in the superficial layer and an avascular area in the deep layer of the pterygium head. CONCLUSIONS AND IMPORTANCE: AS-OCTA revealed abnormal "zigzag vessel patterns" in both the superficial and deep layers denoting meandering vessels in the patients with OSSN. In the patient with the pterygium, it showed "straight vessel patterns" signifying unbending stretched vessels in the superficial layer and an avascular zone in the deep layer of the pterygium head. These findings may be useful for the differential diagnosis of OSSN and pterygium.

Quantitative Analysis of the Association Between Follow-Up Duration and Severity of Limbal Stem Cell Deficiency or Visual Acuity in Aniridia.

Purpose: This study aimed to quantitatively analyze the association between follow-up duration and the severity of limbal stem cell deficiency (LSCD) or visual acuity in patients with aniridia. Methods: A total of 52 eyes of 27 patients with aniridia were enrolled at Osaka University Hospital. Medical records were retrospectively reviewed to obtain information on the severity of LSCD and corrected distance visual acuity (CDVA). LSCD severity was based on a modified severity grading scale. We used an ordered logistic regression model to examine the association between follow-up duration and LSCD severity, and a linear regression model with a generalized linear mixed model for the association between follow-up duration and visual acuity. Results: The mean follow-up duration was 5.2 ± 6.3 years. The mean age at the last follow-up visit was 40.5 ± 18.9 years. The mean CDVA was 1.52 ± 1.09 logMAR. At the last follow-up, 1 examined eye (1.9%) was categorized as stage 0, 7 (13.5%) as Ia, 9 (17.3%) as Ib, 5 (9.6%) as Ic, 2 (3.8%) as IIb, 12 (23.1%) as IIc, and 11 (21.2%) as III. Five eyes (9.6%) were unclassifiable. There was a significant association between follow-up duration and LSCD severity (odds ratio per +1 year, 1.41; P < 0.001). CDVA significantly decreased as follow-up duration increased. Each increase of 1 year in the follow-up duration was associated with a mean difference of +0.021 logMAR (95% confidence interval [CI] 0.01-0.03; P < 0.001). Conclusion: We quantitatively demonstrate that LSCD severity and visual impairment significantly progress as follow-up duration increases.

Generation and validation of a PITX2-EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells.

PITX2 (Paired-like homeodomain transcription factor 2) plays important roles in asymmetric development of the internal organs and symmetric development of eye tissues. During eye development, cranial neural crest cells migrate from the neural tube and form the periocular mesenchyme (POM). POM cells differentiate into several ocular cell types, such as corneal endothelial cells, keratocytes, and some ocular mesenchymal cells. In this study, we used transcription activator-like effector nuclease technology to establish a human induced pluripotent stem cell (hiPSC) line expressing a fluorescent reporter gene from the PITX2 promoter. Using homologous recombination, we heterozygously inserted a PITX2-IRES2-EGFP sequence downstream of the stop codon in exon 8 of PITX2 Cellular pluripotency was monitored with alkaline phosphatase and immunofluorescence staining of pluripotency markers, and the hiPSC line formed normal self-formed ectodermal autonomous multizones. Using a combination of previously reported methods, we induced PITX2 in the hiPSC line and observed simultaneous EGFP and PITX2 expression, as indicated by immunoblotting and immunofluorescence staining. PITX2 mRNA levels were increased in EGFP-positive cells, which were collected by cell sorting, and marker gene expression analysis of EGFP-positive cells induced in self-formed ectodermal autonomous multizones revealed that they were genuine POM cells. Moreover, after 2 days of culture, EGFP-positive cells expressed the PITX2 protein, which co-localized with forkhead box C1 (FOXC1) protein in the nucleus. We anticipate that the PITX2-EGFP hiPSC reporter cell line established and validated here can be utilized to isolate POM cells and to analyze PITX2 expression during POM cell induction.

Efficacy of therapeutic soft contact lens in the management of gelatinous drop-like corneal dystrophy.

BACKGROUND/AIMS: To investigate the efficacy of therapeutic soft contact lenses (SCLs) in gelatinous drop-like corneal dystrophy (GDLD) management. METHODS: This was a retrospective, consecutive, observational case series, including 20 patients (40 eyes) with GDLD treated in Osaka University Hospital within the last 15 years. We tested the effects of therapeutic SCL on clinical features, visual acuity and surgical interventions. Examinations for clinical features and visual acuity were done on patients who had no surgical intervention for 3 years. Scoring and evaluation of changes in three main clinical GDLD features and visual acuity (logMAR units) were performed using Fisher's exact test and Mann-Whitney U test. Surgery-free survival time was compared by Kaplan-Meier analyses in all patients. RESULTS: We found a significantly lower rate of progression in GDLD nodular lesions in patients wearing SCLs compared with those who did not (p=0.0179). No suppressant effects were observed regarding opacity and neovascularisation, and no significant improvements were found in visual acuity (in logMAR values, SCL-on: mean=- 0.036, median=0; SCL-off: mean=0.149, median=+ 0.088; p=0.14). The surgery-free survival time for all 16 SCL-on eyes was 2770 ± 1918 days, significantly longer than that for 22 SCL-off eyes, 1342 ± 1323 days (Kaplan-Meier analysis, p=0.0007), suggesting that therapeutic SCL extends the period until surgical intervention and reduces their necessity in patients with GDLD. CONCLUSION: Wearing therapeutic SCLs in GDLD slows the progression of nodular lesions and decreases the need for surgical interventions.

Correlation Between Angle Parameters and Central Corneal Thickness in Fuchs Endothelial Corneal Dystrophy.

PURPOSE: To evaluate the correlation between anterior chamber parameters and central corneal thickness (CCT) or peripheral corneal thickness (PCT) in patients with Fuchs endothelial corneal dystrophy (FECD) using anterior segment optical coherence tomography. METHODS: This case-control study included 20 eyes from 20 patients with FECD and 31 eyes from 31 patients with healthy corneas. CCT was measured as an indicator of FECD severity. Anterior chamber angle parameters, including trabecular-iris angle (TIA500) and angle opening distance (AOD500), were measured as an indicator of peripheral anterior chamber morphology. We also analyzed PCT and lens vault (LV). The relationships between CCT or PCT and anterior chamber parameters were also analyzed in patients with FECD. RESULTS: Patients with FECD had a larger CCT (593.9 ± 54.6 µm vs. 533.0 ± 25.4 µm, P < 0.001), smaller TIA500 (21.8 ± 9.9 vs. 32.5 ± 11.2 degrees, P = 0.002), smaller AOD500 (0.21 ± 0.11 vs. 0.34 ± 0.18 mm, P = 0.002), and greater LV (0.60 ± 0.27 vs. 0.40 ± 0.29 mm, P = 0.02) than control subjects. In patients with FECD, CCT was negatively correlated with the angle parameters TIA500 (R = 0.29, P = 0.009) and AOD500 (R = 0.19, P = 0.03). There were no significant correlations between PCT and TIA500 (R = 0.008, P = 0.29) or AOD500 (R = 0.007, P = 0.29). There were also no significant correlations between CCT and LV (R = 0.02, P = 0.55). CONCLUSIONS: Larger CCT was significantly associated with narrower anterior chamber angle width, but not with LV. We showed that the severity of FECD is associated with angle chamber morphology.

A novel mutation in gelatinous drop-like corneal dystrophy and functional analysis.

We identified a novel mutation of the tumor-associated calcium signal transducer 2 (TACSTD2) gene in a Japanese patient with gelatinous drop-like corneal dystrophy (GDLD). Genetic analysis revealed a novel homozygous mutation (c.798delG, which may result in frameshift mutation p.Lys267SerfsTer4) in the TACSTD2 gene. This mutated gene was devoid of its original function in helping the claudin (CLDN) 1 and 7 proteins transfer from the cytoplasm to the plasma membrane.

Homeostasis of SLC4A11 protein is mediated by endoplasmic reticulum-associated degradation.

The cornea is an important tissue that refracts light, and the corneal endothelium prevents edema of the corneal stroma by acting as a barrier and a pump for the transport of essential molecules/ions. Sodium bicarbonate transporter-like protein 11 (SLC4A11) is a transporter present in the corneal endothelium, and its mutation causes corneal endothelial disease. Here, we aimed to investigate the degradation pathway of SLC4A11. Quantitative PCR analysis revealed that two variants of SLC4A11 transcripts, variant 2 (SLC4A11-B) and variant 3 (SLC4A11-C), were expressed in human corneal endothelial tissues. Transient overexpression of these variants in HEK293T cells revealed that SLC4A11-B abundantly localized to the cell membrane. Furthermore, SLC4A11-B-transfected HEK293T cells expressed the mature glycosylated forms and immature non-glycosylated forms of SLC4A11. Cycloheximide chase experiments revealed that mature SLC4A11 showed high degradation stability; however, degradation of immature SLC4A11-B was significantly faster than that of immature SLC4A11-C. Therefore, we performed further degradation analysis of the SLC4A11 mutants, which are classified into ER-retained and cell surface-associated mutants similar to the wild type. Compared to the wild type, ER-retained mutants S213P and W240P showed delayed degradation but the cell surface-associated mutants showed minimal degradation. Further analysis using proteasome inhibitors revealed that degradation of immature SLC4A11 was delayed after treatment with the proteasome inhibitors, MG-132 and bortezomib, and was mediated by poly-ubiquitination. Moreover, the degradation of immature SLC4A11 protein was suppressed by Eeyarestatin I, an ER-associated protein degradation (ERAD) inhibitor. Collectively, these data suggest that SLC4A11 protein is degraded via ERAD.

Clinical outcomes and time to recurrence of phototherapeutic keratectomy in Japan.

To assess the indications, outcomes and time to recurrence of phototherapeutic keratectomy (PTK) for anterior corneal pathology.This study involved 714 eyes of 477 consecutive patients (mean age: 66.0 ±â€Š15.2 years; range: 6-101 years) who underwent PTK as the initial surgical intervention for an anterior corneal pathology. In case of each patient, the cornea treated by PTK, followed up by slit-lamp examination and corrected distance visual acuity (CDVA) testing. Main outcome measures included Slit-lamp findings (1), CDVA (2), patients' complaints (3).The mean follow-up period was 44.0 ±â€Š38.8 months (range: 1-156 months).We treated 376 granular corneal dystrophy (GCD) eyes, 238 band keratopathy (BK) eyes, 23 epithelium attachment disorder eyes, 16 gelatinous drop-like corneal dystrophy (GDLD) eyes, 13 lattice corneal dystrophy (LCD) eyes, and 48 eyes with other corneal diseases. The CDVA significantly improved from LogMAR 0.65 ±â€Š0.61 pre PTK to LogMAR 0.26 ±â€Š0.39 post PTK. A 2 or more lines increase of CDVA was observed in GCD eyes (67.8%), BK eyes (49.2%), epithelium attachment disorder eyes (57.1%), GDLD eyes (87.5%), LCD eyes (76.9%), and other corneal disease eyes (60.4%). The recurrence of BK was rare. GCD recurred slowly. Epithelium attachment disorder eyes remitted simultaneously, and recurred comparatively faster.PTK was proved to be a successful therapy for all 6 corneal disease categories. Disease recurrence after PTK differed among the diseases, and surgeons should recognize the different rates of disease recurrence after PTK surgery.

Conjunctival dysbiosis in mucosa-associated lymphoid tissue lymphoma.

To investigate the conjunctival microbiota and the association between the development of conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma and dysbiosis, DNA samples were collected from 25 conjunctival MALT lymphoma patients and 25 healthy controls. To compare the microbiota, samples were collected from the following four body locations: conjunctiva, meibomian gland, periocular skin and hand. Extracted DNA was analyzed by 16S rRNA sequences, and libraries were sequenced on an Illumina MiSeq sequencer. The differences in bacteria were characterized by using principal coordinate analysis of metagenomics data, and the differences in bacterial compositions were evaluated by linear discriminant analysis effect size. The conjunctival microbiota of MALT lymphoma patients was compositionally different from that of healthy controls. For the conjunctival MALT lymphoma patients, alterations in the microbial composition were detected, and a remarkable change was detected at the conjunctiva. Detailed analysis showed that a specific population of the microbiota, the genus Delftia, was significantly more abundant in conjunctival MALT lymphoma patients, and the genera Bacteroides and Clostridium were less abundant in the MALT lymphoma patients. A specific microbiota on the ocular surface in conjunctival MALT lymphoma patients was detected, and dysbiosis may play an important role in the pathophysiology of conjunctival MALT lymphoma.

Clinical evaluation of a newly developed graft inserter (NS Endo-Inserter) for Descemet stripping automated endothelial keratoplasty.

PURPOSE: The aim of this study was to describe the postoperative outcomes of Descemet stripping automated endothelial keratoplasty (DSAEK) performed using our newly developed graft inserter (NS Endo-Inserter) and compare the findings with those for DSAEK performed using the Busin glide. PATIENTS AND METHODS: In this retrospective, case-control, institutional study, we studied the clinical outcomes of DSAEK performed using the NS Endo-Inserter (NS group, n=13) or the Busin glide (Busin group, n=10) for patients with corneal endothelial dysfunction. Clinical parameters, including the distance-corrected visual acuity (DCVA), endothelial cell (EC) loss, and intraoperative/postoperative complications, were assessed over a 6-month follow-up period. RESULTS: At 6 months after surgery, the mean DCVA showed no significant difference between the two groups. EC loss at 3 and 6 months after DSAEK was 9.1%±20.7% and 18.2%±22.6%, respectively, in the NS group and 44.0%±25.5% and 46.5%±23.3%, respectively, in the Busin group; differences between groups were statistically significant at both 3 and 6 months (P=0.024 and P=0.016, respectively). Anterior chamber hemorrhage was observed in one patient in the Busin group. Rebubbling after surgery was required for one eye in the Busin group. No complications were observed in the NS group. CONCLUSION: Our newly developed graft inserter for DSAEK may cause significantly less EC damage than the conventional pull-through technique using the Busin glide. Our inserter permits safe endothelial graft delivery without anterior chamber collapse and can result in successful graft attachment without complications at 6 months after surgery.

Transcription factor TFAP2B up-regulates human corneal endothelial cell-specific genes during corneal development and maintenance.

The corneal endothelium, which originates from the neural crest via the periocular mesenchyme (PM), is crucial for maintaining corneal transparency. The development of corneal endothelial cells (CECs) from the neural crest is accompanied by the expression of several transcription factors, but the contribution of some of these transcriptional regulators to CEC development is incompletely understood. Here, we focused on activating enhancer-binding protein 2 (TFAP2, AP-2), a neural crest-expressed transcription factor. Using semiquantitative/quantitative RT-PCR and reporter gene and biochemical assays, we found that, within the AP-2 family, the TFAP2B gene is the only one expressed in human CECs in vivo and that its expression is strongly localized to the peripheral region of the corneal endothelium. Furthermore, the TFAP2B protein was expressed both in vivo and in cultured CECs. During mouse development, TFAP2B expression began in the PM at embryonic day 11.5 and then in CECs during adulthood. siRNA-mediated knockdown of TFAP2B in CECs decreased the expression of the corneal endothelium-specific proteins type VIII collagen α2 (COL8A2) and zona pellucida glycoprotein 4 (ZP4) and suppressed cell proliferation. Of note, we also found that TFAP2B binds to the promoter of the COL8A2 and ZP4 genes. Furthermore, CECs that highly expressed ZP4 also highly expressed both TFAP2B and COL8A2 and showed high cell proliferation. These findings suggest that TFAP2B transcriptionally regulates CEC-specific genes and therefore may be an important transcriptional regulator of corneal endothelial development and homeostasis.

Sustained and Widespread Gene Delivery to the Corneal Epithelium via In Situ Transduction of Limbal Epithelial Stem Cells, Using Lentiviral and Adeno-Associated Viral Vectors.

Corneal epithelial dystrophies are typically characterized by symptoms such as pain, light sensitivity, and corneal opacification leading to impaired vision. The development of gene therapy for such conditions has been hindered by an inability to achieve sustained and extensive gene transfer, as the epithelium is highly replicative and has evolved to exclude foreign material. We undertook a comprehensive study in mice aiming to overcome these impediments. Direct injection of lentiviral vector within the stem cell niche resulted in centripetal streaks of epithelial transgene expression sustained for >1 year, indicating limbal epithelial stem cell transduction in situ. The extent of transgene expression varied markedly but at maximum covered 26% of the corneal surface. After intrastromal injection, adeno-associated viral (AAV) vectors were found to penetrate Bowman's membrane and mediate widespread, but transient (12-16 days), epithelial transgene expression. This was sufficient, when applied within a Cre/lox system, to result in recombined epithelium covering up to approximately 80% of the corneal surface. Lastly, systemic delivery of AAV2/9 in neonatal mice resulted in extensive corneal transduction, despite the relative avascularity of the tissue. These findings provide the foundations of a gene therapy toolkit for the corneal epithelium, which might be applied to correction of inherited epithelial dystrophies.

A New in Vitro Model of GDLD by Knocking Out TACSTD2 and Its Paralogous Gene EpCAM in Human Corneal Epithelial Cells.

PURPOSE: Gelatinous drop-like corneal dystrophy (GDLD) is a rare autosomal recessive corneal dystrophy that causes severe vision loss. Because of its poor prognosis, there is a demand for novel treatments for GDLD. Here, we establish a new in vitro disease model of GDLD based on immortalized human corneal epithelial (HCE-T) cells. METHODS: By using transcription activator-like effector nuclease plasmids, tumor-associated calcium signal transducer 2 (TACSTD2) and its paralogous gene, epithelial cell adhesion molecule (EpCAM), were knocked out in HCE-T cells. Fluorescence-activated cell sorting was performed to obtain cells in which both TACSTD2 and EpCAM were knocked out (DKO cells). In DKO cells, the expression levels and subcellular localizations of claudin (CLDN) 1, 4, and 7, and ZO-1 were investigated, along with epithelial barrier function. By using DKO cells, the feasibility of gene therapy for GDLD was also investigated. RESULTS: DKO cells exhibited decreased expression and aberrant subcellular localization of CLDN1 and CLDN7 proteins, as well as decreased epithelial barrier function. Transduction of the TACSTD2 gene into DKO cells nearly normalized expression levels and subcellular localization of CLDN1 and CLDN7 proteins, while significantly increasing epithelial barrier function. CONCLUSIONS: We established an in vitro disease model of GDLD by knocking out TACSTD2 and its paralogous gene, EpCAM, in HCE-T cells. This cell line accurately reflected pathological aspects of GDLD. TRANSLATIONAL RELEVANCE: We expect that the cell line will be useful to elucidate the pathogenesis of GDLD and develop novel treatments for GDLD.

Novel PAX6 mutation reported in an aniridia patient.

An aniridia patient was found to have a novel PAX6 mutation. A genetic duplication within PAX6, which caused a frameshift mutation, ultimately created a nonsense stop codon and premature truncation of the protein. Consequently, the patient presented with a clouded cornea as a result of partial limbal stem cell deficiency, foveal hypoplasia, nystagmus and a pale, cupped optic disc caused by glaucoma.