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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  Cancer-associated retinopathy caused by benign thymoma 
PMCID: PMC3487380  PMID: 20424218
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  Autofluorescence of metastatic choroidal tumor 
International Ophthalmology  2008;29(4):309-313.
PMCID: PMC2714453  PMID: 18528641
19.  In vivo evaluation of a novel scaffold for artificial corneas prepared by using ultrahigh hydrostatic pressure to decellularize porcine corneas 
Molecular Vision  2009;15:2022-2028.
To evaluate the stability and biocompatibility of artificial corneal stroma that was prepared by using ultrahigh hydrostatic pressurization treatment to decellularize corneas.
The porcine cornea was decellularized by two methods, a detergent method and an ultrahigh hydrostatic pressure (UHP) method. Either 1% w/v Triton® X-100 or sodium dodecyl sulfate (SDS) was used for the detergent method, and 10,000 atmospheres (atm; 7.6×106 mmHg) was applied to the cornea for 10 min at 10 °C by a high-pressure machine for the UHP method. Hematoxylin-eosin staining was performed to confirm the removal of the corneal cells, and then decellularized porcine corneal stroma was implanted into rabbit corneal pockets. After eight weeks, the rabbit eyes were enucleated to examine the tissue compatibility of the implanted stroma.
Complete decellularization was confirmed only in corneas treated by the UHP method, and little inflammation was seen when they were implanted into the rabbit corneal pockets.
Porcine corneal stroma completely decellularized by the UHP method has extremely high biocompatibility and is a possible corneal scaffold for an artificial cornea.
PMCID: PMC2763123  PMID: 19844587
20.  Detection of the bcl-2 t(14;18) Translocation and Proto-Oncogene Expression in Primary Intraocular Lymphoma 
Primary intraocular lymphoma (PIOL) is a diffuse large B cell lymphoma that initially infiltrates the retina, vitreous, or optic nerve head, with or without central nervous system involvement. This study examined the expression of the bcl-2 t(14;18) translocation, the bcl-10 gene, and high expression of bcl-6 mRNA in PIOL cells.
Microdissection and PCR analysis were used to examine vitreous specimens in patients with PIOL for the presence of bcl-2 t(14;18) translocations, the bcl-10 gene, and expression of bcl-6 mRNA. A medical record review was also conducted to determine whether the bcl-2 t(14;18) translocation correlated with prognosis.
Forty of 72 (55%) PIOL patients expressed the bcl-2 t(14;18) translocation at the major breakpoint region. Fifteen of 68 (22%) patients expressed the translocation at the minor cluster region. The bcl-10 gene was detected in 6 of 26 (23%) patients, whereas 4 of 4 (100%) PIOL patients expressed higher levels of bcl-6 mRNA compared with inflammatory lymphocytes. An analysis of clinical outcome in 23 PIOL patients revealed no significant association between bcl-2 t(14;18) translocations and survival or relapse. However, patients with the translocation were significantly younger.
PIOL has unique molecular patterns of bcl-2, bcl-10, and bcl-6 when compared with other systemic lympho-mas. This study lays the foundation for future studies aimed at exploring the genotypic classification of PIOL based on the quantitative molecular framework of gene expression profil-ing, with the goal of providing useful adjuncts to the pathologic diagnosis of this complex disease.
PMCID: PMC1945012  PMID: 16799010
Primary orbital non-Hodgkin’s lymphoma is a mucosa-associated lymphoid tissue (MALT)-type extranodal marginal zone lymphoma. Chronic antigen stimulation is implicated as a causative agent in the development of some mature B-cell proliferations; for example, there are associations involving Helicobacter pylori with gastric or conjunctival MALT lymphoma and Chlamydia psittaci with ocular adnexal lymphoma. We examined the molecular signatures of H pylori and Chlamydia in eight orbital lymphomas.
Polymerase chain reaction (PCR) was performed on DNA extracted from microdissected lymphoma cells. H pylori was detected with the urease B and vac/m2 primers. A multiplex touchdown enzyme time-release PCR assay designed to simultaneously detect Chlamydia trachomatis, Chlamydia pneumoniae, and C psittaci was performed. Authenticity of the PCR-amplified products was verified by Southern blot hybridization.
H pylori DNA was detected in an orbital lymphoma of a French patient who had positive serum H pylori titer. C pneumoniae, but neither C psittaci nor C trachomatis, DNA was detected in another orbital lymphoma of a Chinese patient from Hong Kong. H pylori, C pneumoniae, and C psittaci genes were not found in the other six orbital lymphomas.
H pylori or C pneumoniae genomic fingerprints were detected in two of seven primary orbital MALT lymphomas. These findings provide evidence for a possible involvement of particular infectious microorganisms such as H pylori and Chlamydia in primary orbital lymphoma. These different microorganisms may play similar roles in the etiology of orbital MALT lymphomas from different geographic regions. Antibiotic therapy could be considered for orbital MALT lymphomas associated with positive infection.
PMCID: PMC1809900  PMID: 17471326
22.  SUV39H1 interacts with HTLV-1 Tax and abrogates Tax transactivation of HTLV-1 LTR 
Retrovirology  2006;3:5.
Tax is the oncoprotein of HTLV-1 which deregulates signal transduction pathways, transcription of genes and cell cycle regulation of host cells. Transacting function of Tax is mainly mediated by its protein-protein interactions with host cellular factors. As to Tax-mediated regulation of gene expression of HTLV-1 and cellular genes, Tax was shown to regulate histone acetylation through its physical interaction with histone acetylases and deacetylases. However, functional interaction of Tax with histone methyltransferases (HMTase) has not been studied. Here we examined the ability of Tax to interact with a histone methyltransferase SUV39H1 that methylates histone H3 lysine 9 (H3K9) and represses transcription of genes, and studied the functional effects of the interaction on HTLV-1 gene expression.
Tax was shown to interact with SUV39H1 in vitro, and the interaction is largely dependent on the C-terminal half of SUV39H1 containing the SET domain. Tax does not affect the methyltransferase activity of SUV39H1 but tethers SUV39H1 to a Tax containing complex in the nuclei. In reporter gene assays, co-expression of SUV39H1 represses Tax transactivation of HTLV-1 LTR promoter activity, which was dependent on the methyltransferase activity of SUV39H1. Furthermore, SUV39H1 expression is induced along with Tax in JPX9 cells. Chromatin immunoprecipitation (ChIP) analysis shows localization of SUV39H1 on the LTR after Tax induction, but not in the absence of Tax induction, in JPX9 transformants retaining HTLV-1-Luc plasmid. Immunoblotting shows higher levels of SUV39H1 expression in HTLV-1 transformed and latently infected cell lines.
Our study revealed for the first time the interaction between Tax and SUV39H1 and apparent tethering of SUV39H1 by Tax to the HTLV-1 LTR. It is speculated that Tax-mediated tethering of SUV39H1 to the LTR and induction of the repressive histone modification on the chromatin through H3 K9 methylation may be the basis for the dose-dependent repression of Tax transactivation of LTR by SUV39H1. Tax-induced SUV39H1 expression, Tax-SUV39H1 interaction and tethering to the LTR may provide a support for an idea that the above sequence of events may form a negative feedback loop that self-limits HTLV-1 viral gene expression in infected cells.
PMCID: PMC1363732  PMID: 16409643
23.  The potential role of amyloid β in the pathogenesis of age-related macular degeneration 
Journal of Clinical Investigation  2005;115(10):2793-2800.
Drusen are extracellular deposits that lie beneath the retinal pigment epithelium (RPE) and are the earliest signs of age-related macular degeneration (AMD). Recent proteome analysis demonstrated that amyloid β (Aβ) deposition was specific to drusen from eyes with AMD. To work toward a molecular understanding of the development of AMD from drusen, we investigated the effect of Aβ on cultured human RPE cells as well as ocular findings in neprilysin gene–disrupted mice, which leads to an increased deposition Aβ. The results showed that Aβ treatment induced a marked increase in VEGF as well as a marked decrease in pigment epithelium-derived factor (PEDF). Conditioned media from Aβ-exposed RPE cells caused a dramatic increase in tubular formation by human umbilical vein endothelial cells. Light microscopy of senescent neprilysin gene–disrupted mice showed an increased number of degenerated RPE cells with vacuoles. Electron microscopy revealed basal laminar and linear deposits beneath the RPE layer, but we did not observe choroidal neovascularization (CNV). The present study demonstrates that Aβ accumulation affects the balance between VEGF and PEDF in the RPE, and an accumulation of Aβ reproduces features characteristic of human AMD, such as RPE atrophy and basal deposit formation. Some other factors, such as breakdown of integrity of Bruch membrane, might be necessary to induce CNV of AMD.
PMCID: PMC1201663  PMID: 16167083
24.  A study of comparison between the nationwide epidemiological survey in 1991 and previous surveys on behçet’s disease in Japan 
The 4th nationwide epidemiological survey on Behçet disease (BD), which included all patients with BD at 1,200 hospitals selected at random from 10,081 hospitals in Japan, was carried out by the BD Research Committee of the Ministry of Health and Welfare in 1991 to examine the epidemiological features of BD in Japan by comparing with previous surveys. 3,938 patients from these hospitals were examined by the Japanese diagnostic criteria of BD (JCBD) revised in 1987 and the International criteria for classification of BD (ICBD). Among these 3,938 patients, 622 patients were only suspected of having BD or clinical signs of the disease were unknown, and most of these patients were incompatible with the ICBD. So these patients were excluded from the study of epidemiological features. The average patients age has risen 7–8 years over the last 20 years and the average age of onset in both sexes increased by about 3 years from 1972 to 1991. While a decrease in the sex ratio was seen in the complete-type and the incomplete-type BD without ocular symptoms, a sustained high sex ratio was shown in incomplete-type BD with ocular symptoms. The positive rate of HLA-B51 antigen was 54.9% (men: 56.9%, women: 52.2%) significantly higher than die 15–16% in healthy subjects but it might have been gradually decreasing. Also the clinical course of BD has become too mild for prognosis. According to diese epidemiological features of BD, the clinical manifestation of BD in Japan might have become the Western type of BD.
PMCID: PMC2723522  PMID: 21432185
Behçet’s disease; epidemiological features; sex ratio; HLA-B51; ocular symptoms
25.  Detection of Borna Disease Virus-Reactive Antibodies from Patients with Psychiatric Disorders and from Horses by Electrochemiluminescence Immunoassay 
The prevalence of Borna disease virus (BDV)-specific antibodies among patients with psychiatric disorders and healthy individuals has varied in several reports using several different serological assay methods. A reliable and specific method for anti-BDV antibodies needs to be developed to clarify the pathological significance of BDV infections in humans. We developed a new electrochemiluminescence immunoassay (ECLIA) for the antibody to BDV that uses two recombinant proteins of BDV, p40 and p24 (full length). Using this ECLIA, we examined 3,476 serum samples from humans with various diseases and 917 sera from blood donors in Japan for the presence of anti-BDV antibodies. By ECLIA, 26 (3.08%) of 845 schizophrenia patients and 9 (3.59%) of 251 patients with mood disorders were seropositive for BDV. Among 323 patients with other psychiatric diseases, 114 with neurological diseases, 75 with chronic fatigue syndrome, 85 human immunodeficiency virus-infected patients, 50 with autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosis and 17 with leprosy, there was no positive case except one case each with alcohol addiction, AIDS, and dementia. Although 19 (1.36%) of 1,393 patients with various ocular diseases, 10 (1.09%) of 917 blood donors, and 3 (4.55%) of 66 multitransfused patients were seropositive for BDV-specific antigen, high levels of seroprevalence in schizophrenia patients and young patients (16 to 59 years old) with mood disorders were statistically significant. The immunoreactivity of seropositive sera could be verified for specificity by blocking with soluble p40 and/or p24 recombinant protein. Anti-p24 antibody was more frequent than p40 antibody in most cases, and in some psychotic patients antibody profiles showed only p40 antibody. Although serum positive for both p40 and p24 antibodies was not found in this study, the p40 ECLIA count in schizophrenia patients was higher than that of blood donors. Furthermore, we examined 90 sera from Japanese feral horses. Antibody profiles of control human samples are similar to that of naturally BDV-infected feral horses. We concluded that BDV infection was associated in some way with psychiatric disorders.
PMCID: PMC95757  PMID: 10473520

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