Koji Kitazawa

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.

Measurement of Corneal Endothelial Surface Area Using Anterior Segment Optical Coherence Tomography in Normal Subjects.

Purpose: To calculate the number of corneal endothelial cells (CEC) on the posterior surface of the normal human cornea by measuring specific surface areas through anterior segment optical coherence tomography. The effects of age, sex, and variations between the right and left eyes were also investigated. Methods: This study involved 60 eyes of 30 normal subjects with no history of corneal disease. Subjects were divided into the following groups according to age: group A (20–39 years old), group B (40–59 years old), and group C (60–79 years old). Set areas of the corneal posterior surface were imaged and analyzed using anterior segment optical coherence tomography, and the number of CECs was calculated based on the area measured and a CEC density. Results: The posterior corneal surface area measured within the central circular diameters of 10.2, 8.5, and 7.5 mm was 86.8, 63.8, and 48.4 mm2 in group A, 86.0, 63.8, and 48.4 mm2 in group B, and 87.0, 64.2, and 48.6 mm2 in group C, respectively. The total number of CECs on the posterior surface in the diameters of 10.2, 8.5, and 7.5 mm was 2.3 × 105, 1.7 × 105, and 1.3 × 105, respectively. No clinically relevant differences in the posterior surface were found in relation to age, sex, or variation between right and left eyes. Conclusions: Calculation of the CEC number on the corneal posterior surface is essential for determining the number of cells applied during penetrating keratoplasty, endothelial keratoplasty, or cultivated CEC injection therapy when available.

Nasal and conjunctival screening prior to refractive surgery: an observational and cross-sectional study

Objectives To investigate bacterial flora of clinically healthy conjunctiva and nasal cavity among patients prior to refractive surgery, as well as the characteristics of patients with methicillin-resistant Staphylococcus aureus (MRSA) colonisation. Design Observational and cross-sectional study. Setting A single-centre study in Japan. Participants 120 consecutive patients pre-refractive surgery. Primary and secondary outcome measures methods Samples were obtained from the right conjunctival sac and the nasal cavity of 120 consecutive patients prior to refractive surgery and were then measured for the levels of the minimum inhibitory concentration (MIC) of antibiotics. Patients were interviewed regarding their occupation, family living situation and any personal history of atopic dermatitis, asthma, smoking or contact lens wear. Results Propionibacterium acnes (P. acnes) (32.5%) and Staphylococcus epidermidis (4.2%) were detected from the conjunctival sac. S. epidermidis was the most commonly isolated (68.3%) in the nasal cavity. Of the 30 patients (25.0%) with colonisation by S. aureus, 2 patients, both of whom were healthcare workers with atopic dermatitis, were found to be positive for MRSA in the nasal cavity. A history of contact lens wear, asthma or smoking, as well as patient gender and age, was not associated with MRSA colonisation. Conclusions There were only 2 patients who were colonised with MRSA, both of whom were healthcare workers with atopic dermatitis. P. acnes was predominantly found in the conjunctival sac. Further study is needed to investigate the involvement between nasal and conjunctival flora, and risk factors for infectious complications.

Safety of anterior chamber paracentesis using a 30-gauge needle integrated with a specially designed disposable pipette

Aims To investigate the safety of anterior chamber (AC) paracentesis using a 30-gauge needle integrated with a specially designed disposable pipette. Methods In this retrospective observational case-series study, AC paracentesis was performed on 301 eyes of 301 patients between September 2009 and August 2016 at the Department of Ophthalmology, Kyoto Prefectural University of Medicine and the Baptist Eye Institute, Kyoto, Japan. AC paracentesis was performed with the patient placed in the supine position using a 30-gauge needle integrated with a disposable pipette with one hand, and the safety post procedure was then evaluated. Results The indications for AC paracentesis were virus detection (ie, corneal endotheliitis, anterior infectious uveitis, cytomegalovirus retinitis and acute retinal necrosis) in 264 eyes, bacterial detection (ie, endophthalmitis) in 8 eyes and malignancy (ie, primary intraocular lymphoma, leukaemia and retinoblastoma) in 29 eyes. No serious complications such as infection, hyphema, lens trauma or severe inflammation including hypopyon and AC fibrin formation were observed. Conclusions Our findings show that AC paracentesis with a disposable pipette is safe with no severe complications.

The existence of dead cells in donor corneal endothelium preserved with storage media

Aim To investigate the viability of donor corneal endothelial cells (CECs) preserved in storage media by histological examination. Methods Twenty-eight donor corneas were obtained from SightLife Eye Bank (Seattle, Washington), and redundant peripheral portions of those corneas were used for histological examination after removal of the centre corneal graft for transplantation. To assess cell viability in the corneal endothelium, biostaining experiments were performed using propidium iodide, calcein-AM, Hoechst 33 342, annexin V, anti-vimentin antibody and toluidine blue. Results Histological analysis of the endothelium showed that the cytoplasm of dead cells had low-intensity fluorescence and that their nuclei stained red, while almost all living cells had green cytoplasm and blue-stained nuclei. The mean dead cell rate in the 28 donor corneas was 4.9%±3.3% (mean ±SD) (range: 0.6%–10.5%). The propidium iodide-positive cells stained positive for annexin V, negative for vimentin and pale for toluidine blue. After the specimens were incubated in a culture medium, the red nucleus dead cells dropped off from the level of the blue nucleus living cells. Conclusion Our findings showed the existence of dead cells in storage-media-preserved donor corneal endothelium and that they dropped off after incubation, thus suggesting that the decrease of CECs following keratoplasty may be related to the presence of dead cells.

Diffuse Anterior Retinoblastoma with Sarcoidosis-Like Nodule.

Background: Retinoblastomas account for 4% of malignancies in children, 1-2% of which are diffuse infiltrating retinoblastomas. Diffuse anterior retinoblastoma is rare and does not involve the retina. Here, we report on a diffuse anterior retinoblastoma with large sarcoidosis-like nodules on the iris that were responsive to anti-inflammatory therapy. Case: We present a 6-year-old girl who had anterior uveitis with white nodules on the iris and posterior surface of the cornea in her right eye. The nodules initially responded well to anti-inflammatory treatment. However, anterior segment optical coherence tomography (AS-OCT) showed that the nodules gradually grew, shrinking the iris. We then collected the aqueous humor for diagnosis. A biopsy revealed clusters of small cells with a high nuclear-to-cytoplasm ratio with partial rosette formation. Therefore, we diagnosed diffuse anterior retinoblastoma without retinal involvement and performed enucleation of the right eye. The histopathology demonstrated undifferentiated cells similar to those seen on the biopsy, and tumor cells invaded the iris stroma, posterior surface of the cornea, ciliary body, and sclera. After the enucleation, she underwent chemotherapy and remains alive. Conclusion: A differential diagnosis of retinoblastoma should be considered when white nodules refractory to anti-inflammatory therapy occur in the eye, even in the absence of obvious retinal masses. AS-OCT findings are useful in assessing retinoblastoma.

Predictive clinical factors of cystoid macular edema in patients with Descemet’s stripping automated endothelial keratoplasty

The purpose of this present study was to investigate predictive clinical factors associated with cystoid macular edema (CME) post Descemet’s stripping automated endothelial keratoplasty (DSAEK) in a large case series. Of 393 consecutive patients who underwent DSAEK at Baptist Eye Institute, Kyoto, Japan between July 2011 and November 2016, 241 patients without CME at the pre- or early-postoperative periods were enrolled. The occurrence of anatomic CME was prospectively examined via optical coherence tomography (OCT). Possible predictive clinical factors for CME were analyzed by multivariate logistic regression analysis. At 1-month post DSAEK, CME occurred in 27 (11.2%) of the 241 patients. Multivariate analysis revealed that primary angle closure glaucoma (PACG) was significantly associated with postoperative CME (odds ratio = 6.4, P = 0.04). The findings of this study revealed that DSAEK in patients with PACG showed a high incidence of CME, thus indicating that they should undergo a careful postoperative observation of the macula via OCT.

A case of fungal keratitis and endophthalmitis post penetrating keratoplasty resulting from fungal contamination of the donor cornea

Purpose Fungal infections post keratoplasty due to contamination of the donor corneal graft have become important issues that need to be addressed. Here we report a case of fungal keratitis and endophthalmitis post penetrating keratoplasty (PKP) due to fungal contamination of the donor corneal graft. Observations We present a 52-year-old male who underwent PKP with a donor corneal graft that was later found to be contaminated with fungus. At 4-weeks postoperative, infectious infiltrates suddenly appeared at the border between the host and donor corneal graft, and endophthalmitis concomitantly occurred. A culture of the remnant donor corneoscleral rims and the vitreous fluid obtained during vitreous surgery was found to be positive for Candida albicans. At 6-months post vitreous surgery and intensive anti-fungal medical treatment, both corneal infiltrates and vitreous opacity completely disappeared, and the patients best-corrected visual acuity recovered to 20/40, with a transparent cornea. Conclusions and importance The findings of this case show that prompt intensive medical treatment and surgical intervention effectively saved the vision in a patient with fungal keratitis and endophthalmitis due to contamination of the donor corneal graft.

Distinct Aqueous Humour Cytokine Profiles of Patients with Pachychoroid Neovasculopathy and Neovascular Age-related Macular Degeneration

This study investigated the pathophysiological features of pachychoroid neovasculopathy (PNV) and neovascular age-related macular degeneration (nAMD) by analysing and comparing cytokine profiles in aqueous humour (AH) collected from 18 PNV, 18 nAMD and 11 control patients. Responses to intravitreal injection of aflibercept were also analysed in the PNV and nAMD groups. In the PNV group, vascular endothelial growth factor (VEGF)-A was significantly lower than in the nAMD group (p = 0.03) but was almost identical to that in the control group (p = 0.86). The nAMD group showed positive correlations between interleukin (IL)-6 and IL-8 (r = 0.78, p < 0.001), IL-6 and monocyte chemoattractant protein (MCP)-1 (r = 0.68, p = 0.002) and IL-8 and MCP-1 (r = 0.68, p = 0.002). In the nAMD group, eyes with dry maculae one month after the first aflibercept injection showed significantly lower VEGF-A and placental growth factor (PlGF) at baseline than those with wet maculae (p = 0.02 for both). However, there was no significant difference between dry and wet maculae in the PNV group. The results suggest that angiogenic factors and proinflammatory cytokines may play the distinct roles in the pathogenesis of PNV and nAMD.

Involvement of anterior and posterior corneal surface area imbalance in the pathological change of keratoconus

Keratoconus (KC) is an ectatic disorder with a high prevalence rate. However, the exact cause of the disease and possible underlying mechanisms of development remain unclear. In this present study, we aimed to investigate the anterior and the posterior corneal surface area in normal, forme fruste keratoconus (FFKC), and keratoconic eyes (as a reference group) using anterior segment optical coherence tomography (AS-OCT) in order to assess the pathological change of KC. The surface areas of the anterior or posterior cornea, and the anterior-posterior (As/Ps) ratio of corneal surface area, were measured at the central 5.0 mm-, 6.0 mm-, and 7.0 mm-diameter areas via AS-OCT, and a comparison between the normal eyes and FFKC eyes was then performed using the Mann-Whitney U test. The posterior surface area at the central 5.0 mm areas in the FFKC eyes (20.430 mm2) and KC eyes (20.917 mm2) seemed to become larger than that of normal eyes (20.389 mm2) (normal vs FFKC; P = 0.06). Moreover, the As/Ps of the corneal surface area in the FFKC eyes (0.986) and the KC eyes (0.976) was significantly smaller than that of the normal eyes (0.988) (normal vs FFKC; P < 0.01). Anterior and posterior corneal surface area imbalance may reflect keratoconic eyes at the early stage of the disease.