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Importance: Exfoliation syndrome is a systemic disorder characterized by progressive accumulation of abnormal fibrillar protein aggregates manifesting clinically in the anterior chamber of the eye. This disorder is the most commonly known cause of glaucoma and a major cause of irreversible blindness. Objective: To determine if exfoliation syndrome is associated with rare, protein-changing variants predicted to impair protein function. Design, Setting, and Participants: A 2-stage, case-control, whole-exome sequencing association study with a discovery cohort and 2 independently ascertained validation cohorts. Study participants from 14 countries were enrolled between February 1999 and December 2019. The date of last clinical follow-up was December 2019. Affected individuals had exfoliation material on anterior segment structures of at least 1 eye as visualized by slit lamp examination. Unaffected individuals had no signs of exfoliation syndrome. Exposures: Rare, coding-sequence genetic variants predicted to be damaging by bioinformatic algorithms trained to recognize alterations that impair protein function. Main Outcomes and Measures: The primary outcome was the presence of exfoliation syndrome. Exome-wide significance for detected variants was defined as P < 2.5 × 10-6. The secondary outcomes included biochemical enzymatic assays and gene expression analyses. Results: The discovery cohort included 4028 participants with exfoliation syndrome (median age, 78 years [interquartile range, 73-83 years]; 2377 [59.0%] women) and 5638 participants without exfoliation syndrome (median age, 72 years [interquartile range, 65-78 years]; 3159 [56.0%] women). In the discovery cohort, persons with exfoliation syndrome, compared with those without exfoliation syndrome, were significantly more likely to carry damaging CYP39A1 variants (1.3% vs 0.30%, respectively; odds ratio, 3.55 [95% CI, 2.07-6.10]; P = 6.1 × 10-7). This outcome was validated in 2 independent cohorts. The fi

Hironobu Jimura,1 Yuuki Mishima,1 Chie Sotozono,2 Akihide Watanabe,2 Jun Asai,3 Chiho Ohbayashi,4 Nahoko Ogata1 1Department of Ophthalmology, Nara Medical University, Nara, Japan; 2Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; 3Department of Dermatology, Kyoto Prefectural University of Medicine, Kyoto, Japan; 4Department of Diagnostic Pathology, Nara Medical University, Nara, JapanCorrespondence: Nahoko OgataDepartment of Ophthalmology, Nara Medical University, Nara, JapanTel +81-744-29-8884 (ext.3432)Fax +81-744-23-8032Email ogata@naramed-u.ac.jpAbstract: Primary acquired melanosis (PAM) of the conjunctiva is a potentially serious melanocytic lesion that can lead to the development of a melanoma. A 60-year-old woman noticed pigmentation of the conjunctiva of her left eye for more than 10 years. She underwent excisional biopsy combined with cryotherapy and was diagnosed with PAM without atypia by intraoperative consultation. She was followed for 7 years, and no changes were observed. Fourteen years after the initial biopsy, she noted a growing conjunctival tumor, and a melanoma was suspected. She underwent orbital exenteration and skin grafting procedures. Histopathological examination of the specimen led to a diagnosis of conjunctival malignant melanoma. Re-examination of the initial biopsy specimen revealed that there was a proliferation of melanocytes that partially expanded over the basal layer of the conjunctiva which had been diagnosed as PAM with moderate atypia. We conclude that this case of conjunctival PAM had progressed to a conjunctival malignant melanoma after 14 years. Pathological evaluation of intraepithelial lesions has its limitations; thus, cases of PAM, even in the absence of obvious atypia, require careful follow-up.Keywords: primary acquired melanosis, conjunctival malignant melanoma, malignant melanoma, conjunctival melanoma, conjunctival tumor

Norihiko Yokoi,1 Aoi Komuro,1 Chie Sotozono,1 Shigeru Kinoshita2 1Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; 2Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan Purpose: Surgical punctal occlusion is indispensable for the treatment of severe dry eye in cases where punctal-plug insertion is not applicable due to an enlarged or deformed punctum. However, permanent punctal occlusion is difficult in some cases. In our aim to establish a more reliable punctal occlusion, we have devised a new surgical approach for punctal occlusion.Patients and methods: This study involved 20 puncta of 12 eyes of 12 patients (1 male and 11 females; mean age: 65.2 years) with severe aqueous-tear-deficient dry eye. A new surgical procedure for punctal occlusion using fibrous tissue from under the lacrimal caruncle into the diathermy-induced deepithelialized canaliculus as supporting tissue for punctal closure was performed. In all patients, the assessment of eye symptoms, as well as the condition of punctal occlusion by slit-lamp biomicroscopy, tear volume (tear-meniscus radius [TMR] measurement by meniscometry), the condition of precorneal tear film (graded by interferometry [IG]), measurement of fluorescein breakup time (FBUT), and scoring of ocular surface staining (fluorescein score of area [FSA] and density [FSD], and lissamine green score [LGS]) were performed, and the preoperative and 6-month-postoperative values were compared.Results: In regard to the postoperative improvement of symptoms, 11 patients showed remarkable improvement, 1 patient showed improvement, and no reopening of the closed punctum was found in any patient. Test values were all significantly improved post surgery (all: P<0.05) as compared to those prior to surgery (respective values [mean ± SD], and the pre- and postoperative P-values were: TMR (mm) [0.18±0.08; 0.56&amp

Exfoliation syndrome (XFS) is the most common known risk factor for secondary glaucoma and a major cause of blindness worldwide. Variants in two genes, LOXL1 and CACNA1A, have previously been associated with XFS. To further elucidate the genetic basis of XFS, we collected a global sample of XFS cases to refine the association at LOXL1, which previously showed inconsistent results across populations, and to identify new variants associated with XFS. We identified a rare protective allele at LOXL1 (p.Phe407, odds ratio (OR) = 25, P = 2.9 × 10−14) through deep resequencing of XFS cases and controls from nine countries. A genome-wide association study (GWAS) of XFS cases and controls from 24 countries followed by replication in 18 countries identified seven genome-wide significant loci (P < 5 × 10−8). We identified association signals at 13q12 (POMP), 11q23.3 (TMEM136), 6p21 (AGPAT1), 3p24 (RBMS3) and 5q23 (near SEMA6A). These findings provide biological insights into the pathology of XFS and highlight a potential role for naturally occurring rare LOXL1 variants in disease biology.

Osamu Hieda, Norihiko Yokoi, Chie Sotozono Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan Purpose: To report a case of filamentary keratitis occurring in the cornea hidden under the eyelids by squint surgery.Methods: A 69-year-old female patient with a history of amblyopia was referred for intractable filamentary keratitis in the left eye. The strabismus angle was 35Δ hypertrophic, and ocular motility was within the normal range. Slit-lamp examination of her left eye revealed filamentary keratitis in more than one-third of the upper cornea behind the upper eyelid. Her right eye was diagnosed as supranuclear double elevator palsy. We performed strabismus surgery on her right eye, including inferior rectus muscle recession (5 mm) in combination with superior rectus muscle resection (5 mm) under local anesthesia. Following surgery, the left eye squint angle was improved. The filamentary keratitis of the left eye disappeared, and there was no recurrence over the following 5 years.Conclusion: The squint surgery of paralyzed right eye decreased the strabismus angle, subsequently resulting in the disappearance of the filamentary keratitis in the left eye via the resolution of the relative blepharoptosis. Although the squint operation performed was not for the purpose of improving binocular function, we want to conclude that it can treat the filamentary keratitis behind the eyelid. Keywords: filamentary keratitis, squint surgery, double elevator palsy, amblyopia

Ocular surface reconstruction (OSR) using tissue-engineered cultivated oral mucosal epithelial cell sheets (COMECS) is a promising newly developed treatment for patients with severe ocular surface disease. Until now, this technique has used exogenic and undefined components such as mouse-derived 3T3 feeder cells and fetal bovine serum. To minimize associated risks of zoonotic infection or transmission of unknown pathogens and so establish a safe and effective protocol for the next generation of treatment modality, we developed a novel technique for the COMECS protocol, using a feeder-free and serum-free (FFSF) culture system. Following this new protocol, COMECS exhibited 4-5 layers of well-stratified and differentiated cells, and we successfully produced functional COMECS that included holoclone-type stem cells. Immunohistochemistry confirmed the presence of markers for cell junction (ZO1, Desmoplakin), basement membrane assembly (Collagen 7, Laminin 5), differentiation (K13, K3), proliferation (Ki67) and stem/progenitor cells (p75) in the FFSF COMECS. When transplanted to the ocular surfaces of rabbits, the tissue survived for up to 2 weeks. This study represents a first step toward assessing the development of functional FFSF COMECS for safe and ideal OSR.

Ocular surface reconstruction (OSR) using tissue-engineered cultivated oral mucosal epithelial cell sheets (COMECS) is a promising newly developed treatment for patients with severe ocular surface disease. Until now, this technique has used exogenic and undefined components such as mouse-derived 3T3 feeder cells and fetal bovine serum. To minimize associated risks of zoonotic infection or transmission of unknown pathogens and so establish a safe and effective protocol for the next generation of treatment modality, we developed a novel technique for the COMECS protocol, using a feeder-free and serum-free (FFSF) culture system. Following this new protocol, COMECS exhibited 4-5 layers of well-stratified and differentiated cells, and we successfully produced functional COMECS that included holoclone-type stem cells. Immunohistochemistry confirmed the presence of markers for cell junction (ZO1, Desmoplakin), basement membrane assembly (Collagen 7, Laminin 5), differentiation (K13, K3), proliferation (Ki67) and stem/progenitor cells (p75) in the FFSF COMECS. When transplanted to the ocular surfaces of rabbits, the tissue survived for up to 2 weeks. This study represents a first step toward assessing the development of functional FFSF COMECS for safe and ideal OSR.

Yoko Ikeda,1,2,* Kazuhiko Mori,1,* Kaori Tada,3 Morio Ueno,1 Shigeru Kinoshita,4 Chie Sotozono1 1Department of Ophthalmology, Kyoto Prefectural University of Medicine, 2Oike-Ganka Ikeda Clinic, 3Department of Ophthalmology, Japanese Red Cross Society Kyoto Daini Hospital, 4Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan *These authors contributed equally to this work Purpose: To evaluate and compare the intraocular pressure (IOP) reduction efficacy and safety between the ophthalmic solutions 0.005% latanoprost (Lat) and 0.0015% tafluprost (Taf) in Japanese patients with normal-tension glaucoma (NTG).Methods: In this randomized nonmasked study, we prospectively enrolled 30 Japanese NTG patients who had used Lat monotherapy for more than 4 weeks, and randomly divided them into the following two groups: 1) Lat-to-Taf group (LT group) and 2) Taf-to-Lat group (TL group). At the beginning of the study, both groups were switched from initial Lat to Lat or Taf for 12 weeks, and then switched over to the other drug (crossover) for 12 additional weeks. At 0, 4, 12, 16, and 24 weeks, we evaluated each patient’s IOP, conjunctival injection, and corneal epitheliopathy score, and at 0, 12, and 24 weeks, we evaluated their eyelash changes and pigmentation of the eyelids and irises.Results: The mean IOP of the LT group (15 eyes) was 10.5, 10.6, and 11.1 mmHg, at 0, 12, and 24 weeks, respectively, whereas that of the TL group (15 eyes) was 11.7, 11.1, and 10.5 mmHg at 0, 12, and 24 weeks, respectively. No significant differences were found between the two groups and in the intragroup comparisons. Moreover, no significant differences were found between Lat and Taf in regard to the conjunctival injection score and corneal epitheliopathy score. Eyelash changes and eyelid and iris pigmentation were similar in both groups.Conclusion: The findings of this study show that Lat and Taf

Purpose: To determine the long-term epithelial lineage of origin of surgically removed grafts after allogeneic cultivated corneal limbal epithelial transplantation (CLET). Design: Interventional case reports. Participants: We studied 2 eyes from 2 patients with total corneal stem cell destruction; 1 eye was from a patient with Stevens-Johnson syndrome and 1 eye had sustained chemical injury. Methods: Allogeneic cultivated corneal limbal epithelial sheets on human amniotic membrane (AM) were transplanted onto the ocular surface. Regrafting (1 eye, 42 months later) or penetrating keratoplasty (1 eye, 75 months later) were performed after the initial transplantation procedure for further visual rehabilitation. Main Outcome Measures: The excised grafts were subjected to clinical evaluation and to light-and transmission electron microscopy (TEM) examination and to immunohistochemical analysis. Results: In clinically conjunctival grafts, TEM and immunohistochemical analysis disclosed only small areas where the original cultivated corneal epithelial cells persisted. Neighboring conjunctival epithelial cells had apparently invaded a large portion of the corneal surface (keratin 3/12(-), Muc5ac(+)). In clinically corneal grafts, transplanted allogeneic cultivated corneal epithelial cells clearly survived for a long period of time (keratin 3/12(+), Muc5ac(-)); there was no infiltration by inflammatory cells, nor was there dissolution of the AM substrate. Conclusions: We theorize that the process of graft opacification after allogeneic CLET is responsible for the loss of transplanted cultivated corneal epithelial cells and that this is followed by conjunctival cell invasion onto the corneal surface. The results of this study confirmed that in the clinically evaluated corneal graft, transplanted cultivated corneal epithelial cells indeed survived for a long period of time on the corneal surface and maintained ocular surface integrity, even though the transplanted cells were all

PURPOSE. To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS). METHODS. AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry. RESULTS. BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy. CONCLUSIONS. AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

PURPOSE. To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS). METHODS. AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry. RESULTS. BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy. CONCLUSIONS. AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

PURPOSE. To examine the feasibility of using sterilized, freeze-dried amniotic membrane (FD-AM) as a substrate for cultivating autologous corneal epithelial cells for ocular surface reconstruction. METHODS. Human AM deprived of amniotic epithelial cells by incubation with EDTA was freeze dried, vacuum packed, and sterilized with -irradiation. The resultant FD-AM was characterized for its physical, biological, and morphologic properties by stretch stress tests, immunohistochemistry, electron microscopy, and cell culture. In addition, 3 weeks after an ocular surface injury, the conjunctivalized corneal surfaces of eyes in eight rabbits were surgically reconstructed by transplantation of autologous cultivated corneal epithelial cells on FD-AM. RESULTS. A stretch stress test revealed no significant differences between sterilized FD-AM and cryopreserved AM. Immunohistochemistry for several extracellular matrix molecules and electron microscopic analysis of FD-AM revealed that the process of drying and irradiation did not affect its biological and morphologic properties. The corneal epithelial cells cultivated on FD-AM had four to five stratified, well-differentiated cell layers. Corneas that were grafted with the cultivated corneal epithelial cells on FD-AM were clear and were all epithelialized at 10 days after surgery. CONCLUSIONS. The sterilized, freeze-dried AM retained most of the physical, biological, and morphologic characteristics of cryopreserved AM; consequently, it is a useful biomaterial for ocular surface reconstruction.

PURPOSE. To examine the feasibility of using sterilized, freeze-dried amniotic membrane (FD-AM) as a substrate for cultivating autologous corneal epithelial cells for ocular surface reconstruction. METHODS. Human AM deprived of amniotic epithelial cells by incubation with EDTA was freeze dried, vacuum packed, and sterilized with -irradiation. The resultant FD-AM was characterized for its physical, biological, and morphologic properties by stretch stress tests, immunohistochemistry, electron microscopy, and cell culture. In addition, 3 weeks after an ocular surface injury, the conjunctivalized corneal surfaces of eyes in eight rabbits were surgically reconstructed by transplantation of autologous cultivated corneal epithelial cells on FD-AM. RESULTS. A stretch stress test revealed no significant differences between sterilized FD-AM and cryopreserved AM. Immunohistochemistry for several extracellular matrix molecules and electron microscopic analysis of FD-AM revealed that the process of drying and irradiation did not affect its biological and morphologic properties. The corneal epithelial cells cultivated on FD-AM had four to five stratified, well-differentiated cell layers. Corneas that were grafted with the cultivated corneal epithelial cells on FD-AM were clear and were all epithelialized at 10 days after surgery. CONCLUSIONS. The sterilized, freeze-dried AM retained most of the physical, biological, and morphologic characteristics of cryopreserved AM; consequently, it is a useful biomaterial for ocular surface reconstruction.

Purpose. To investigate the expression of growth factor mRNA and the level of growth factor protein in preserved human amniotic membrane (AM). Methods. RT-PCR was used to examine the expression of mRNA for eight growth factors (EGF, TGF-a, KGF, HGF, bFGF, TGF-1, -2, -3) and two growth factor receptors (KGFR and HGFR) in human AM preserved at -80°C for one month. In addition, ELISAs were used to measure the protein concentrations of seven growth factors (EGF, TGF-a, KGF, HGF, bFGF, TGF-1, -2) in preserved human corneas and in AM both with and without amniotic epithelium. Results. RT-PCR revealed that human AM expresses mRNA for EGF, TGF-a, KGF, HGF, bFGF, TGF-1, -2, -3, KGFR and HGFR, while ELISAs showed that it contains EGF, TGF-a, KGF, HGF, bFGF, TGF-1, -2. AM without amniotic epithelium also contains all seven growth factors examined, however, in this tissue the protein levels of EGF, KGF, HGF and bFGF were found to be significantly lower than in native AM. Conclusions. Preserved human AM expresses mRNAs for a number of growth factors and contains several growth factor proteins that might benefit epithelialization after AM transplantation. High levels of EGF, KGF, HGF and bFGF in AM with amniotic epithelium as compared to AM without amniotic epithelium suggest an epithelial origin for these growth factors. We feel that EGF, KGF and HGF in particular might play important roles in ocular surface wound healing after AM transplantation.