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1.  Comprehensive polymerase chain reaction assay for detection of pathogenic DNA in lymphoproliferative disorders of the ocular adnexa 
Scientific Reports  2016;6:36621.
Infectious agents have been identified as a major cause of specific types of human cancers worldwide. Several microorganisms have been identified as potential aggravators of ocular adnexal neoplasms; however, given the rarity of these neoplasms, large epidemiological studies are difficult to coordinate. This study aimed to conduct an exhaustive search for pathogenic DNA in lymphoproliferative disorders (LPD) of the ocular adnexa in a total of 70 patients who were diagnosed with LPD of the ocular adnexa between 2008 and 2013. Specimens were screened for bacterial, viral, fungal, and parasitic DNA by multiplex polymerase chain reaction (PCR) and quantitative real-time PCR. Among cases of conjunctival mucosa-associated lymphoid tissue lymphoma, human herpes virus (HHV)-6, HHV-7, chlamydia, Epstein-Barr virus (EBV) and bacterial 16S ribosomal DNA were detected. In cases of IgG4-related ocular disease, similar pathogens were detected but in a larger number of patients. Our PCR assays detected DNAs of various infectious agents in tumor specimens, especially HHV6, HHV7, and EBV, with different positive rates in various types of LPD. Chronic inflammatory stimulation or activation of oncogenes from these infectious agents might be involved in the pathogenesis of LPD of the ocular adnexa.
PMCID: PMC5103257  PMID: 27830722
2.  High‐dose methotrexate following intravitreal methotrexate administration in preventing central nervous system involvement of primary intraocular lymphoma 
Cancer Science  2016;107(10):1458-1464.
In order to prevent central nervous system (CNS) involvement and improve the prognosis of primary intraocular lymphoma (PIOL), we prospectively evaluated the efficacy of combined therapy using intravitreal methotrexate (MTX) and systemic high‐dose MTX on treatment‐naïve PIOL. Patients with newly diagnosed PIOL whose lymphoma was limited to the eyes were enrolled. The patients were treated with weekly intravitreal MTX until the ocular lesions were resolved, followed by five cycles of systemic high‐dose MTX (3.5 g/m2) every other week. Ten patients were enrolled in this study and completed the treatment. All patients achieved complete response for their ocular lesions with rapid decrease of intravitreal interleukin‐10 concentration. Adverse events of intravitreal and systemic high‐dose MTX were mild and tolerable. With a median follow‐up of 29.5 months, four patients (40%) experienced the CNS disease development and the mean CNS lymphoma‐free survival (CLFS) time was 51.1 months. Two‐year CLFS, which was the primary end‐point of the study, was 58.3% (95% confidence interval, 23.0–82.1%). In contrast, eight patients were treated with intravitreal MTX alone in our institute, and their 2‐year CLFS was 37.5% (95% confidence interval, 8.7–67.4%). In conclusion, systemic high‐dose MTX following intravitreal MTX is feasible and might be effective in preventing CNS involvement of PIOL. Further arrangements are worth considering in order to improve the effects. This study was registered with UMIN Clinical Trials Registry (UMIN000003921).
PMCID: PMC5084671  PMID: 27412324
Central nervous system disease; interleukin‐10; intraocular lymphoma; methotrexate; uveitis
3.  Differentiation/Purification Protocol for Retinal Pigment Epithelium from Mouse Induced Pluripotent Stem Cells as a Research Tool 
PLoS ONE  2016;11(7):e0158282.
To establish a novel protocol for differentiation of retinal pigment epithelium (RPE) with high purity from mouse induced pluripotent stem cells (iPSC).
Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated.
We obtained iPS-RPE at high purity (approximately 98%). The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation.
We successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE.
PMCID: PMC4934919  PMID: 27385038
4.  Standardization of Quantitative PCR for Human T-Cell Leukemia Virus Type 1 in Japan: a Collaborative Study 
Journal of Clinical Microbiology  2015;53(11):3485-3491.
Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory.
PMCID: PMC4609719  PMID: 26292315
5.  First case of primary intraocular natural killer t-cell lymphoma 
BMC Ophthalmology  2015;15:169.
Natural killer cell tumors can be broadly divided by origin into mature-cell and progenitor-cell types. The invasion of nasal-origin natural killer cells into the ophthalmologic field is sometimes observed in patients, but primary ocular natural killer cell tumors are a rare occurrence.
Case presentation
A 66 year-old woman without any systemic disease presented with blurred vision due to a severe vitreous opacity in the right eye. Flow cytometric analysis of the vitreous fluid suggested a natural killer cell tumor. Moreover, cytologic examination of vitreal and retinal specimens revealed the infiltration of a natural killer cell tumor, while PCR and immunocytochemistry revealed Epstein-Barr virus infection. The results of a gene rearrangement analysis were positive for IGH, while TCR beta chains were all negative. We examined the patient with whole-body magnetic resonance imaging and positron emission tomography, and performed a bone marrow examination. These examinations returned no abnormal results.
Thorough analysis of vitreal samples is essential when performing vitrectomies for vitreous opacities of unknown cause. Flow cytometric, cytologic, and PCR analysis of vitreal and retinal samples may reveal the presence and cause of severe illness.
PMCID: PMC4653874  PMID: 26585973
Natural killer cell; Vitreous sample; Flow cytometry; Cytology; Epstein-Barr virus
6.  Corneal Regeneration by Deep Anterior Lamellar Keratoplasty (DALK) Using Decellularized Corneal Matrix 
PLoS ONE  2015;10(7):e0131989.
The purpose of this study is to demonstrate the feasibility of DALK using a decellularized corneal matrix obtained by HHP methodology. Porcine corneas were hydrostatically pressurized at 980 MPa at 10°C for 10 minutes to destroy the cells, followed by washing with EGM-2 medium to remove the cell debris. The HHP-treated corneas were stained with H-E to assess the efficacy of decellularization. The decellularized corneal matrix of 300 μm thickness and 6.0 mm diameter was transplanted onto a 6.0 mm diameter keratectomy wound. The time course of regeneration on the decellularized corneal matrix was evaluated by haze grading score, fluorescein staining, and immunohistochemistry. H-E staining revealed that no cell nuclei were observed in the decellularized corneal matrix. The decellularized corneal matrices were opaque immediately after transplantation, but became completely transparent after 4 months. Fluorescein staining revealed that initial migration of epithelial cells over the grafts was slow, taking 3 months to completely cover the implant. Histological sections revealed that the implanted decellularized corneal matrix was completely integrated with the receptive rabbit cornea, and keratocytes infiltrated into the decellularized corneal matrix 6 months after transplantation. No inflammatory cells such as macrophages, or neovascularization, were observed during the implantation period. The decellularized corneal matrix improved corneal transparency, and remodelled the graft after being transplanted, demonstrating that the matrix obtained by HHP was a useful graft for corneal tissue regeneration.
PMCID: PMC4498638  PMID: 26161854
7.  Lymphangiosarcoma with systemic metastases in a Japanese domestic cat 
A 4-year-2-month-old female Japanese domestic cat was diagnosed with lymphangiosarcoma through tissue biopsy of an amputated leg. Two months later, the cat was euthanized, and postmortem findings revealed edema, and bruising at the caudal region of the trunk, pulmonary hemorrhage, pulmonary nodules and mediastinal lymphadenopathy. Microscopically, neoplastic tissues were observed in the dermis and subcutis of the trunk, lung, mediastinal lymph nodes, diaphragm, omentum and mesentery. The tumor cells were spindle to polygonal-shaped with nuclear pleomorphism aligning along pre-existing collagen bundles and forming irregular vascular channels in which the erythrocytes were rarely observed. These cells were immunopositive for vimentin, von Willebrand factor and CD31. Based on the histopathological and immunohistochemical features, the neoplasia was diagnosed as lymphangiosarcoma with systemic metastases.
PMCID: PMC4383788  PMID: 25482607
feline; lymphangiosarcoma; metastases
8.  The Relationship between Clinicopathological Features and Expression of Epithelial and Mesenchymal Markers in Spontaneous Canine Mammary Gland Tumors 
It is known that epithelial mesenchymal transition (EMT) contributes to the acquisition of malignant property in human cancers. However, the role of EMT in canine tumors remains to be elucidated. To evaluate the correlation between expression levels of protein markers involved in EMT and clinicopathological characteristics in canine mammary gland tumors, immunohistochemistry using antibodies against ZO-1, E-cadherin, vimentin, N-cadherin and fibronectin was performed on 119 clinical tissue samples. Consequently, loss of ZO-1 and E-cadherin, and gain of vimentin and N-cadherin were more frequently observed in malignant tumors than in benign tumors. However, there was no correlation among expression of these molecules. Univariate and multivariate analysis identified that loss of E-cadherin independently had a low one-year survival rate (adjusted odds ratio: 2.3, P=0.02). These results suggested that EMT might relate to acquisition of malignancy, and additionally, E-cadherin was strongly correlated with malignant behavior in canine mammary gland tumors.
PMCID: PMC4221164  PMID: 24931646
canine mammary gland tumors; epithelial mesenchymal transition; immunohistochemistry; prognosis
9.  Bone Regeneration by the Combined Use of Tetrapod-Shaped Calcium Phosphate Granules with Basic Fibroblast Growth Factor-Binding Ion Complex Gel in Canine Segmental Radial Defects 
The effect of tetrapod-shaped alpha tricalcium phosphate granules (Tetrabones® [TB]) in combination with basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) on bone defect repair was examined. Bilateral segmental defects 20-mm long were created in the radius of 5 dogs, stabilized with a plate and screws and implanted with 1 of the following: TB (TB group), TB and bFGF solution (TB/f group), and TB and f-IC gel (TB/f-IC group). Dogs were euthanized 4 weeks after surgery. Radiographs showed well-placed TB granules in the defects and equal osseous callus formation in all the groups. Histomorphometry revealed that the number of vessels and volume of new bone in the TB/f-IC group were significantly higher than those in the other groups. However, no significant differences in neovascularization and new bone formation were observed between the TB/f and TB groups. Furthermore, no significant difference in the lamellar bone volume or rate of mineral apposition was observed among groups. These results suggest that increased bone formation might have been because of the promotion of neovascularization by the f-IC gel. Therefore, the combinatorial method may provide a suitable scaffold for bone regeneration in large segmental long bone defects.
PMCID: PMC4143656  PMID: 24670963
bone regeneration; calcium phosphate; collagen; fibroblast growth factor; histomorphometry
10.  High-resolution genomic copy number profiling of primary intraocular lymphoma by single nucleotide polymorphism microarrays 
Cancer Science  2014;105(5):592-599.
Primary intraocular lymphoma (PIOL) is a rare lymphoma. Because of difficulties in obtaining tissue samples, little is known about the disease's genetic features. In order to clarify these features, we carried out single nucleotide polymorphism array karyotyping of IOL using genomic DNA extracted from vitreous fluid. We analyzed 33 samples of IOLs consisting of 16 PIOLs, 12 IOLs with a central nervous system (CNS) lesion at diagnosis (IOCNSL), and five secondary IOLs following systemic lymphoma. All were B-cell type. We identified recurrent copy number (CN) gain regions in PIOLs, most frequently on chromosome 1q followed by 18q and 19q. Chromosome 6q was the most frequent loss region. Although these CN gain regions of PIOL were in common with those of IOCNSL, loss of 6q22.33 containing PTPRK and 9p21.3 containing CDKN2A were more frequently deleted in IOCNSL. Large CN loss in 6q was detected in three of four PIOL patients who had early CNS development and short survival periods, whereas long-term survivors did not have such deletions. There was a correlation between gain of the IL-10 gene located on 1q and intravitreal interleukin-10 concentration, which was higher in IOL than in benign uveitis. The results suggest that IOCNSL is a highly malignant form of PIOL that infiltrates into the CNS at an early stage. They also indicate that genetic differences between PIOL and primary CNS lymphoma need to be clarified.
PMCID: PMC4317829  PMID: 24612100
Central nervous system; IL-10; intraocular lymphoma; single nucleotide polymorphism; vitreous body
11.  Presumed toxoplasmic central retinal artery occlusion and multifocal retinitis with perivascular sheathing 
Central retinal artery occlusion (CRAO) and multifocal retinitis with perivascular sheathing are rare in ocular toxoplasmosis. We report a case of toxoplasmic CRAO and multifocal retinitis with perivascular sheathing. A healthy 83-year-old male developed left panuveitis. Funduscopic examination of the left eye showed a swollen optic disc and sheathing of the retinal artery with a dense vitreous haze and a white retinal lesion. Serum anti-toxoplasma antibodies were positive in a latex agglutination assay. Vitrectomy was performed to improve visualization of the retinal lesions and for examination of causative microorganisms. A postoperative fundus examination revealed CRAO with optic disc involvement and multifocal retinitis with perivascular sheathing. Qualitative multiplex polymerase chain reaction detected the Toxoplasma gondii B1 gene in ocular fluid from both the aqueous and vitreous humor. The presumed diagnosis of ocular toxoplasmosis was made and treatment was started with prednisone and acetylspiramycin with subsequent improvement. Two months later, the patient developed active retinochoroiditis in the left eye. After 6 weeks of anti-toxoplasma therapy, the disease involuted. Retinal vascular occlusions and multifocal retinitis with perivascular sheathing are rare in toxoplasmosis. This is the first case report of toxoplasmic CRAO and multifocal retinitis with perivascular sheathing. The diagnosis of ocular toxoplasmosis should be considered in patients with retinal artery occlusions and multifocal retinitis with perivascular sheathing associated with inflammation.
PMCID: PMC4003269  PMID: 24790408
ocular toxoplasmosis; toxoplasma retinochoroiditis; retinal vascular occlusion; polymerase chain reaction; vitrectomy
12.  Implantation of Tetrapod-Shaped Granular Artificial Bones or β-Tricalcium Phosphate Granules in a Canine Large Bone-Defect Model 
We investigated biodegradability and new bone formation after implantation of tetrapod-shaped granular artificial bone (Tetrabone®) or β-tricalcium phosphate granules (β-TCP) in experimental critical-size defects in dogs, which were created through medial and lateral femoral condyles. The defect was packed with Tetrabone® (Tetrabone group) or β-TCP (β-TCP group) or received no implant (control group). Computed tomography (CT) was performed at 0, 4 and 8 weeks after implantation. Micro-CT and histological analysis were conducted to measure the non-osseous tissue rate and the area and distribution of new bone tissue in the defect at 8 weeks after implantation. On CT, β-TCP was gradually resorbed, while Tetrabone® showed minimal resorption at 8 weeks after implantation. On micro-CT, non-osseous tissue rate of the control group was significantly higher compared with the β-TCP and Tetrabone groups (P<0.01), and that of the β-TCP group was significantly higher compared with the Tetrabone group (P<0.05). On histology, area of new bone tissue of the β-TCP group was significantly greater than those of the Tetrabone and control groups (P<0.05), and new bone distribution of the Tetrabone group was significantly greater than those of the β-TCP and control groups (P<0.05). These results indicate differences in biodegradability and connectivity of intergranule pore structure between study samples. In conclusion, Tetrabone® may be superior for the repair of large bone defects in dogs.
PMCID: PMC3982820  PMID: 24161964
artificial bones; β-TCP granules; canine; critical-size defect; Tetrabone
13.  Acute anterior uveitis after discontinuation of tocilizumab in a patient with rheumatoid arthritis 
Tocilizumab is a humanized monoclonal anti-interleukin-6 (IL-6) receptor antibody and has been approved in Japan for the treatment of Castleman’s disease, rheumatoid arthritis (RA), and systemic juvenile idiopathic arthritis. Conjunctivitis and dry eye are known ocular adverse effects, but uveitis has not been reported.
Case report
A 72-year-old woman had undergone bilateral cataract surgery without complications. Six months after the surgery, she was diagnosed with RA and treated with tocilizumab infusion every 4 weeks. However, severe malaise and dizziness occurred after the third tocilizumab infusion, and the treatment was suspended. Since the symptoms associated with RA had resolved, she was followed without any medication thereafter. At 5 weeks after the third tocilizumab infusion, she developed severe anterior inflammation with hypopyon in her left eye, and her visual acuity dropped to less than 2/200. Considering her age and history of cataract surgery, endophthalmitis was suspected and a vitrectomy was performed, but no pathogens were detected from the intraocular fluid samples collected during surgery. The ocular inflammation was gradually resolved with systemic antibiotics and corticosteroids. However, severe anterior uveitis recurred in the same eye during the tapering of the systemic corticosteroids, when the aqueous humor IL-6 level was 46,100 pg/mL. The recurrent ocular inflammation was resolved with increased doses of topical and systemic corticosteroids, and the patient has since remained relapse-free. No symptom of inflammation was observed in the right eye during the follow-up period.
This case indicates a possibility that acute anterior uveitis may have been an adverse effect after the discontinuation of anti-IL-6 receptor antibody therapy in a patient with RA.
PMCID: PMC3891663  PMID: 24531503
14.  Dermal Cell Damage Induced by Topical Application of Non-Steroidal Anti-Inflammatory Drugs is Suppressed by Trehalose Co-Lyophilization in Ex Vivo Analysis 
Topical administration of non-steroidal anti-inflammatory drugs (NSAIDs) is generally considered safer than oral administration, although the former can occasionally induce cutaneous irritation. We hypothesized that the cutaneous irritation by topical NSAIDs might be suppressed by trehalose, which has protective effects on biological membranes. Using the three-dimensional cultured human skin model, Living Skin Equivalent-high, we found that cutaneous damage due to NSAIDs was reduced by concomitant use of trehalose and that this effect of trehalose was reinforced by co-lyophilization of NSAIDs with trehalose. The anti-inflammatory effect of co-lyophilized NSAIDs with trehalose was comparable to that seen with NSAIDs alone in a rat model. Our results suggest that co-lyophilization of NSAIDs with trehalose might be a novel procedure that can help prevent NSAIDs-induced skin irritation.
PMCID: PMC3942957  PMID: 23884023
cutaneous damage; NSAIDs; topical application; trehalose
15.  Intraocular Invasion of Adult T-Cell Leukemia Cells without Systemic Symptoms after Cataract Surgery 
Case Reports in Ophthalmology  2013;4(3):252-256.
Adult T-cell leukemia (ATL) is an aggressive lymphoid proliferation associated with the human T-lymphotropic virus type I (HTLV-I). The intraocular invasion of ATL is a rare event. A 75-year-old man without any systemic disease underwent uneventful cataract surgery of the right eye. On postoperative day 6, the patient presented with blurred vision due to severe vitreous opacity in the right eye. Analysis of the vitreous fluid revealed a suspected ATL infection based on the flow cytometric analysis. Moreover, cytological examination of the vitreous specimen revealed flower cell infiltration, and HTLV-1 DNA was detected by PCR analysis of the vitreous sample. Monoclonal T-cell receptor chain rearrangement was also detected by PCR. Thorough analysis of a vitreous sample is essential for vitrectomy in vitreous opacity of unknown cause. Flow cytometric, cytological, and PCR analysis of vitreous samples is beneficial for determining the cause of this kind of severe illness.
PMCID: PMC3861856  PMID: 24348412
Adult T-cell leukemia; Intraocular invasion; Flow cytometry
16.  CD200R signaling inhibits pro-angiogenic gene expression by macrophages and suppresses choroidal neovascularization 
Scientific Reports  2013;3:3072.
Macrophages are rapidly conditioned by cognate and soluble signals to acquire phenotypes that deliver specific functions during inflammation, wound healing and angiogenesis. Whether inhibitory CD200R signaling regulates pro-angiogenic macrophage phenotypes with the potential to suppress ocular neovascularization is unknown. CD200R-deficient bone marrow derived macrophages (BMMΦ) were used to demonstrate that macrophages lacking this inhibitory receptor exhibit enhanced levels of Vegfa, Arg-1 and Il-1β when stimulated with PGE2 or RPE-conditioned (PGE2-enriched) media. Endothelial tube formation in HUVECs was increased when co-cultured with PGE2-conditioned CD200R−/− BMMΦ, and laser-induced choroidal neovascularization was enhanced in CD200R-deficient mice. In corroboration, signaling through CD200R results in the down-regulation of BMMΦ angiogenic and pro-inflammatory phenotypes. Translational potential of this pathway was investigated in the laser-induced model of choroidal neovascularization. Local delivery of a CD200R agonist mAb to target myeloid infiltrate alters macrophage phenotype and inhibits pro-angiogenic gene expression, which suppresses pathological angiogenesis and CNV development.
PMCID: PMC3812658  PMID: 24170042
17.  Role of IL-22– and TNF-α–Producing Th22 Cells in Uveitis Patients with Behçet’s Disease 
The Journal of Immunology Author Choice  2013;190(11):5799-5808.
Behçet’s disease is a systemic inflammatory disorder with recurrent episodes of oral ulceration, skin lesions, genital ulceration, and intraocular inflammation (uveitis). The intraocular inflammation is strictly associated with Th effector cells. IL-22 is a member of the IL-10 cytokine family that is involved in inflammatory processes. Recently, Th22 cells were identified as a Th cell population that produces IL-22 and TNF-α and are distinct from Th1, Th2, and Th17 cells. In this study, we established Th22-type T cell clones from ocular samples taken from Behçet’s disease patients with active uveitis. These clones produced large amounts of IL-22 and TNF-α but not the Th1 cytokine IFN-γ and the Th17 cytokine IL-17. CD4+ T cells from the peripheral blood of Behçet’s disease patients differentiated into Th22 cells in the presence of IL-6 and TNF-α in vitro. The polarized Th22 cell lines produced large amounts of IL-22, and the polarized Th1 and Th17 cells also produced IL-22. In the presence of anti–TNF-α– and anti–IL-6–blocking Abs, Behçet’s disease Th22-type T cells failed to produce IL-22. In addition, infliximab-pretreated Th22 cells and Th22-type ocular T cells produced less IL-22 and TNF-α. Moreover, IL-22–producing T cells were isolated from mice with experimental autoimmune uveitis, an animal model of Behçet’s disease, and the intraocular T cells from uveitis models produced large amounts of IL-22 in the presence of retinal Ags. Our results suggest that inflammatory cytokines IL-22 and TNF-α may play a key role in the ocular immune response in Behçet’s disease.
PMCID: PMC3659956  PMID: 23630362
18.  Insulin-like growth factor 1 is not associated with high myopia in a large Japanese cohort 
Molecular Vision  2013;19:1074-1081.
To investigate whether genetic variations in the insulin-like growth factor 1 (IGF-1) gene are associated with high myopia in Japanese.
A total of 1,339 unrelated Japanese patients with high myopia (axial length ≥26 mm in both eyes) and two independent control groups were evaluated (334 cataract patients without high myopia and 1,194 healthy Japanese individuals). The mean axial length (mm±SD) in the case group was 29.18±1.85 mm, and the mean spherical equivalent (D±SD) of the phakic eyes was −12.69±4.54 D. We genotyped five tagging single nucleotide polymorphisms (SNPs) in IGF-1: rs6214, rs978458, rs5742632, rs12423791, and rs2162679. Chi-square tests for trend, multivariable logistic regression, and haplotype regression analysis were conducted.
We found no significant association between the IGF-1 SNPs and high or extreme myopia (axial length ≥28 mm in both eyes, 837 subjects) in the additive model, even when compared with the cataract and general population controls, with or without adjustments for age and sex. The evaluation using dominant and recessive models also did not reveal any significant associations. The haplotype analysis with a variable-sized sliding-window strategy also showed a lack of association of IGF-1 SNPs with high or extreme myopia.
The results of the present study using a Japanese subset do not support the proposal that the IGF-1 gene determines susceptibility to high or extreme myopia in Caucasians and Chinese. Further studies are needed to confirm our reports in other populations and to identify the underlying genetic determinants of these ocular pathological conditions.
PMCID: PMC3668686  PMID: 23734076
19.  Cancer-associated retinopathy caused by benign thymoma 
PMCID: PMC3487380  PMID: 20424218
20.  Inhibition of Th17 differentiation by anti-TNF-alpha therapy in uveitis patients with Behçet's disease 
The purpose of this study was to determine whether anti-tumour necrosis factor alpha (anti-TNF-α) antibody, infliximab, can inhibit T helper 17 (Th17) differentiation in uveitis patients who have Behçet's disease (BD).
To measure inflammatory cytokines, ocular fluid samples from BD patients being treated with infliximab were collected. Cluster of differentiation 4 (CD4)+ T cells from BD patients with active uveitis were co-cultured with anti-cluster of differentiation 3/cluster of differentiation 28 (CD3/CD28) antibodies in the presence of infliximab. For the induction of Th17 cells, CD4+ T cells from BD patients were co-cultured with anti-CD3/CD28, anti-interferon-gamma (anti-IFN-γ), anti-interleukin-4 (anti-IL-4), and recombinant proteins such as interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-23 (IL-23), and TNF-α. The BD T cells were co-cultured with infliximab, and the production of interleukin-17 (IL-17) was evaluated by ELISA and flow cytometry, and the expression of retinoid-acid receptor-related orphan receptor gamma t (RORγt) was also evaluated by flow cytometry. In addition, intraocular cells collected from mice with experimental autoimmune uveitis (EAU) were used for the assay with anti-TNF-α blocking antibody.
Ocular fluids from active uveitis patients who have BD contained significant amounts of inflammatory cytokines such as IFN-γ, IL-2, TNF-α, IL-6, and IL-17, while ocular fluids from infliximab patients did not contain any inflammatory cytokines. Activated CD4+ T cells from BD patients produced large amounts of TNF-α and IL-17, whereas T cells in the presence of infliximab failed to produce these cytokines. Polarized Th17 cell lines from BD patients produced large amounts of IL-17, and Th17 cells exposed to infliximab had significantly reduced IL-17 production. Polarized BD Th17 cells expressed large amounts of transcription factor RORγt. In contrast, in vitro-treated infliximab Th17 cells expressed less RORγt. Moreover, intraocular T cells from EAU mice had a high population of IL-17+ cells, and retinal antigen-specific T cells from EAU mice produced large amounts of IL-17 in the presence of retinal peptide. However, the EAU T cells produced less IL-17 if the T cells were treated with anti-TNF-α antibody.
These results indicate that anti-TNF-α therapy suppresses effector T-cell differentiation in BD patients with uveitis. Thus, suppression of effector T-cell differentiation by anti-TNF-α therapy may provide protection from severe ocular inflammation in BD.
PMCID: PMC3446476  PMID: 22546542
21.  Trehalose treatment suppresses inflammation, oxidative stress, and vasospasm induced by experimental subarachnoid hemorrhage 
Subarachnoid hemorrhage (SAH) frequently results in several complications, including cerebral vasospasm, associated with high mortality. Although cerebral vasospasm is a major cause of brain damages after SAH, other factors such as inflammatory responses and oxidative stress also contribute to high mortality after SAH. Trehalose is a non-reducing disaccharide in which two glucose units are linked by α,α-1,1-glycosidic bond, and has been shown to induce tolerance to a variety of stressors in numerous organisms. In the present study, we investigated the effect of trehalose on cerebral vasospasm, inflammatory responses, and oxidative stress induced by blood in vitro and in vivo.
Enzyme immunoassay for eicosanoids, pro-inflammatory cytokines, and endothelin-1, and western blotting analysis for cyclooxygenase-2, inducible nitric oxide synthase, and inhibitor of NF-κB were examined in macrophage-like cells treated with hemolysate. After treatment with hemolysate and hydrogen peroxide, the levels of lipid peroxide and amounts of arachidonic acid release were also analyzed. Three hours after the onset of experimental SAH, 18 Japanese White rabbits received an injection of saline, trehalose, or maltose into the cisterna magna. Angiographic and histological analyses of the basilar arteries were performed. In a separate study, the femoral arteries from 60 rats were exposed to fresh autologous blood. At 1, 3, 5, 7, 10, and 20 days after treatment, cryosections prepared from the femoral arteries were histologically analyzed.
When cells were treated with hemolysate, trehalose inhibited the production of several inflammatory mediators and degradation of the inhibitor of NF-κB and also suppressed the lipid peroxidation, the reactive oxygen species-induced arachidonic acid release in vitro. In the rabbit model, trehalose produced an inhibitory effect on vasospasm after the onset of experimental SAH, while maltose had only a moderate effect. When the rat femoral arteries exposed to blood were investigated for 20 days, histological analysis revealed that trehalose suppressed vasospasm, inflammatory response, and lipid peroxidation.
These data suggest that trehalose has suppressive effects on several pathological events after SAH, including vasospasm, inflammatory responses, and lipid peroxidation. Trehalose may be a new therapeutic approach for treatment of complications after SAH.
PMCID: PMC3422174  PMID: 22546323
Trehalose; Subarachnoid hemorrhage; Cerebral vasospasm; Inflammatory response; Oxidative stress
22.  Diagnosis of systemic metastatic retinal lymphoma 
Acta ophthalmologica  2011;89(2):e149-e154.
Systemic metastatic retinal lymphoma (SMRL) is exceptionally rare, as systemic lymphomas most often metastasize to the uvea. We have evaluated a series of SMRL cases to elucidate the clinical and pathological features of SMRL.
The pathologic specimens of intraocular lymphomas (IOLs) at the National Eye Institute from 1991–2009 were retrospectively reviewed. These cases were diagnosed by cytology, cytokine measurement (ELISA for interleukin (IL)-10 and IL-6 levels), and Immunoglobulin-Heavy (IgH) and T-cell-receptor (TCR) gene analyses.
There were 9 B-SMRLs among 96 B-cell retina lymphomas (9.4%) and 3 T-SMRLs among 5 T-cell retinal lymphomas (60%) from a total of 116 IOLs. The original sites were nasopharynx (3), testis (2), skin (2), breast (1), blood (1), retroperitoneum (1), ileo-cecum (1) and stomach (1). Cytology of vitreous samples illustrated atypical lymphoma cells with either B- or T- monoclonality. More B-SMRLs had a high ratio of vitreal IL-10 to IL-6 than T-SMRLs. Molecular pathology demonstrated lymphoma cells with gene rearrangements of IgH in all B-SMRLs and TCR in all T-SMRLs.
SMRL and primary retinal lymphoma present with similar clinical manifestations. Systemic T-cell lymphoma invades the retina and vitreous more aggressively than systemic B-cell lymphoma. A diagnosis of SMRL is made when there is a clinical history of systemic lymphoma (particularly from nasopharynx, testis, and skin) and lymphoma cells are identified in the vitreous or retina. Molecular analysis is more useful than vitreal cytokine measurement for SMRL diagnosis.
PMCID: PMC2891914  PMID: 19958292
Systemic Metastatic Retinal Lymphoma; Intraocular Lymphoma; Cytokine; IgH gene rearrangement; TCR gene rearrangement
23.  Frequency of Distinguishing Clinical Features in Vogt-Koyanagi-Harada disease 
Ophthalmology  2009;117(3):591-599.e1.
To determine the frequency of occurrence of limited clinical features which distinguish patients with Vogt-Koyanagi-Harada (VKH) disease from those with non-VKH uveitis.
Comparative case series.
1147 total patients.
All patients with bilateral ocular inflammatory disease presenting to any of ten uveitis centers in the three month period between 2006-January-01 and 2006-March-31 (inclusive) were asked to participate. The clinical and historical features of disease were obtained from the participants via direct interview and chart review. Patients were stratified based on whether they were diagnosed with VKH disease or non-VKH uveitis for statistical analysis.
Main Outcome Measures
Presence or absence of various clinical features in the two populations.
Of 1147 patients, 180 were diagnosed with VKH disease and 967 with non-VKH uveitis. Hispanics and Asians were more likely to be diagnosed with VKH than non-VKH disease compared to other ethnicities. In acute disease, the finding of exudative retinal detachment was most likely to be found in VKH disease with a positive predictive value (PPV) of 100 and negative predictive value (NPV) of 88.4, while in chronic disease, sunset glow fundus was most likely to be found, with a PPV of 94.5 and NPV of 89.2.
Numerous clinical findings have been described in the past as important in the diagnosis of VKH. The current study reveals that of these, two are highly specific to this entity in an ethnically and geographically diverse group of patients with non-traumatic bilateral uveitis. These clinical findings are 1. exudative retinal detachment during acute disease and 2. sunset glow fundus during the chronic phase of the disease.
PMCID: PMC2830365  PMID: 20036008
24.  Positive Correlation between Severity of Blepharospasm and Thalamic Glucose Metabolism 
Case Reports in Ophthalmology  2011;2(1):50-54.
A 43-year-old woman with drug-related blepharospasm was followed up for 22 months. She had undergone etizolam treatment for 19 years for indefinite complaints. We examined her cerebral glucose metabolism 5 times (between days 149 and 688 since presentation), using positron emission tomography, and identified regions of interest in the thalamus, caudate nucleus, putamen, and primary somatosensory area on both sides. The severity of the blepharospasm was evaluated by PET scanning using the Wakakura classification. Sixteen women (mean age 42.4 ± 11.7 years) were examined as normal controls. The thalamic glucose metabolism in our patient was significantly increased on days 149, 212, and 688. The severity of the blepharospasm was positively correlated with the thalamic glucose metabolism, suggesting that the severity of blepharospasms reflects thalamic activity.
PMCID: PMC3219445  PMID: 22110436
Blepharospasm; Cerebral glucose metabolism; Dystonia; Positron-emission tomography; Thalamus
25.  A Genome-Wide Association Analysis Identified a Novel Susceptible Locus for Pathological Myopia at 11q24.1 
PLoS Genetics  2009;5(9):e1000660.
Myopia is one of the most common ocular disorders worldwide. Pathological myopia, also called high myopia, comprises 1% to 5% of the general population and is one of the leading causes of legal blindness in developed countries. To identify genetic determinants associated with pathological myopia in Japanese, we conducted a genome-wide association study, analyzing 411,777 SNPs with 830 cases and 1,911 general population controls in a two-stage design (297 cases and 934 controls in the first stage and 533 cases and 977 controls in the second stage). We selected 22 SNPs that showed P-values smaller than 10−4 in the first stage and tested them for association in the second stage. The meta-analysis combining the first and second stages identified an SNP, rs577948, at chromosome 11q24.1, which was associated with the disease (P = 2.22×10−7 and OR of 1.37 with 95% confidence interval: 1.21–1.54). Two genes, BLID and LOC399959, were identified within a 200-kb DNA encompassing rs577948. RT–PCR analysis demonstrated that both genes were expressed in human retinal tissue. Our results strongly suggest that the region at 11q24.1 is a novel susceptibility locus for pathological myopia in Japanese.
Author Summary
Myopia is one of the most common ocular disorders with elongation of axis of the eyeball. Pathological myopia or high myopia, a subset of myopia which is characterized with excessive axial elongation and degenerative changes of the eye, is a leading cause of visual impairment. Since genetic factors play significant roles in its development, identification of genetic determinants is an urgent and important issue. Although family-based linkage analyses have isolated at least 16 susceptible chromosomal loci for pathological or common myopia, no gene responsible for the disease has been identified. We conducted the first genome-wide case/control association study of pathological myopia in a two-stage design using 411,777 markers with 830 Japanese patients and 1,911 Japanese controls. We identified a region strongly suggestive for the disease susceptibility at chromosome 11q24.1 containing BLID and LOC399959. Their expression was confirmed in human retina with RT–PCR. BLID encodes an inducer of apoptotic cell death, and apoptosis is known to play an important functional role in pathological myopia. We believe that our study contributes to further dissect the molecular events underlying the development and progression of pathological myopia.
PMCID: PMC2735651  PMID: 19779542

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