Takahiro Kawaji

Targeted conversion of the transthyretin gene in vitro and in vivo

Familial amyloidotic polyneuropathy (FAP) is the common form of hereditary generalized amyloidosis and is characterized by the accumulation of amyloid fibrils in the peripheral nerves and other organs. Liver transplantation has been utilized as a therapy for FAP, because the variant transthyretin (TTR) is predominantly synthesized by the liver, but this therapy is associated with several problems. Thus, we need to develop a new treatment that prevents the production of the variant TTR in the liver. In this study, we used HepG2 cells to show in vitro conversion of the TTR gene by single-stranded oligonucleotides (SSOs), embedded in atelocollagen, designed to promote endogenous repair of genomic DNA. For the in vivo portion of the study, we used liver from transgenic mice whose intrinsic wild-type TTR gene was replaced by the murine TTR Val30Met gene. The level of gene conversion was determined by real-time RCR combined with mutant-allele-specific amplification. Our results indicated that the level of gene conversion was approximately 11 and 9% of the total TTR gene in HepG2 cells and liver from transgenic mice, respectively. Gene therapy via this method may therefore be a promising alternative to liver transplantation for treatment of FAP.

Elevated erythropoietin in vitreous with ischemic retinal diseases

The aim of the current study was to measure the concentrations of erythropoietin in the vitreous fluid and analyze its association with vascular endothelial growth factor (VEGF) in ischemic vitreoretinal diseases. Vitreous fluid samples were collected from patients with proliferative diabetic retinopathy, branch retinal vein occlusion and idiopathic macular hole. Concentrations of erythropoietin and VEGF in vitreous fluid were significantly elevated in patients with proliferative diabetic retinopathy and branch retinal vein occlusion as compared to patients with macular hole. There were no differences in serum concentrations of erythropoietin and VEGF among patient groups. There was significant correlation between erythropoietin and VEGF concentrations in vitreous fluid. Erythropoietin was up-regulated in ischemic disorders and may act as an endogenous neuroprotective factor against ischemic retinal disorders.

Persistent subretinal indocyanine green induces retinal pigment epithelium atrophy

PURPOSE To describe a case of a patient with macular hole with subretinal indocyanine green (ICG) during vitrectomy. DESIGN Interventional case report. METHODS A 66-year-old woman with macular hole underwent a vitrectomy with ICG. RESULTS After application of ICG into the vitreous, ICG was introduced in the subretinal space. Indocyanine green was found to be present for more than 6 months. Retinal pigment epithelium atrophy appeared at the site of the lesion. CONCLUSIONS Although ICG may be a useful tool for distinguishing the internal limiting membrane and other tissues careful application is required to prevent side effects.

Simultaneous increases in multiple proinflammatory cytokines in the aqueous humor in pseudophakic glaucomatous eyes

PURPOSE: To evaluate cytokine and growth factor levels in the aqueous humor in patients with open‐angle glaucoma (OAG). SETTING: Kumamoto University Hospital, Kumamoto, Japan. DESIGN: Cross‐sectional cohort study. METHODS: Aqueous humor samples were collected from cataract cases and OAG cases. Aqueous levels of cytokines and growth factors were determined by multiplex immunoassay. The data were analyzed using the Tukey‐Kramer honestly‐significant‐difference test and multiple regression analyses. RESULTS: The study evaluated 52 cataract cases and 73 OAG cases. In the cataract cases, the mean interleukin (IL)‐6, IL‐8, monocyte chemotactic protein (MCP)‐1, tumor necrosis factor‐α, epidermal growth factor, vascular endothelial growth factor (VEGF), platelet‐derived growth factor (PDGF)‐AA, PDGF‐AB/BB, and VEGF levels (all pg/mL) were 21.4, 4.6, 829.4, 0.8, 0.5, 33.8, 1.6, and 77.9, respectively. In 23 phakic primary OAG (POAG) and 26 phakic exfoliation glaucoma cases, the corresponding values were 15.1 and 8.3, 15.2 and 12.3, 1142.5 and 1253.9, 0.3 and 0.5, 1.5 and 1.4, 57.7 and 58.0, 2.5 and 3.3, 37.4 and 59.7, respectively. In pseudophakic OAG cases, the IL‐8 levels for exfoliation glaucoma (P=.0002) and MCP‐1 for POAG (P=.0008) and exfoliation glaucoma (P<.0001) were significantly different compared with phakic OAG. Interleukin‐6, IL‐8, and MCP‐1 levels were correlated (P<.0001). Multiple regression analyses showed the highest association of pseudophakic status with IL‐8 and MCP‐1 levels (P=.0002 and P<.0001, respectively). CONCLUSION: The OAG patients, especially those with pseudophakic eyes, had simultaneous cytokine level increases, suggesting the aqueous humor microenvironment is altered in pseudophakic glaucomatous eyes. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Frequency and risk factors for intraocular pressure elevation after posterior sub-Tenon capsule triamcinolone acetonide injection.

PurposeThis study investigated the effects of posterior sub-Tenon capsule (PST) injection of triamcinolone acetonide (TA) on intraocular pressure (IOP) in the human eye. MethodsThe study included 115 patients who received PST injections of 40-mg TA to treat macular edema with diabetic retinopathy (n=57), branch retinal vein occlusion (n=35), central retinal vein occlusion (n=13), or other disorders (n=10). IOP measurements were performed on the day of injection, and 0.5, 1, 2, 3, 6, 9, and 12 months later. ResultsIn 26 (22.6%) of the 115 eyes, an IOP of 24 mm Hg or higher was observed during the 12-month follow-up period after PST TA injection. IOP elevation significantly correlated with young age, but not with past history of diabetes mellitus or systemic hypertension, sex, or type of retinal disease with macular edema. In total, 23 eyes were treated with antiglaucoma medications to control elevated IOP (24 mm Hg or higher). External trabeculotomy was performed in 1 case where medications failed to correct elevated IOP. ConclusionsPST TA injection is associated with high rates of steroid-induced IOP elevation in eyes with previously normal IOP. However, IOP elevation may be less common after PST injection than after intravitreal injection. Our findings indicate that IOP must be carefully monitored after PST TA injection.

Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina.

Thioredoxin (TRX) plays a variety of redox‐related roles in organisms. To investigate its function as an endogenous redox regulator in NMDA‐induced retinal neurotoxicity, we injected NMDA with TRX, mutant TRX or saline into the vitreous cavity of rat eyes. Retinal ganglion cells were rescued by TRX, compared with saline, when evaluated by retrograde labeling analysis at 7 days after NMDA injection. TRX, but not its mutant form, prevented NMDA‐induced apoptosis in the retina, as measured by terminal deoxynucleotidyl transferase‐mediated UTP nick‐end labeling. The induction of caspase 3 and 9, but not caspase 8, by NMDA was significantly lower in TRX‐treated eyes than in saline‐treated eyes. NMDA‐induced activation of the MAPKs, p38 kinase and c‐Jun N‐terminal kinase after 6 h and of the MAPK kinases (MKKs) MKK3/6 and MKK4 after 3 h was markedly suppressed in retinal ganglion cells by TRX but not by the mutant form. NMDA‐induced increases in protein carbonylation, nitrosylation and lipid peroxidation were also suppressed in TRX‐treated eyes. We concluded that the intravitreous injection of TRX effectively attenuated NMDA‐induced retinal cell damage and that suppression of oxidative stress and inhibition of apoptotic signaling pathways were involved in this neuroprotection.

Combined intravitreal bevacizumab and trabeculectomy with mitomycin C versus trabeculectomy with mitomycin C alone for neovascular glaucoma.

PurposeTo evaluate the effects of intravitreal bevacizumab (IVB) before mitomycin C trabeculectomy (MMCT) for neovascular glaucoma (NVG). MethodsThe study is a retrospective, comparative, consecutive case series. The study group consisted of 57 eyes from 50 patients with NVG who underwent a first MMCT: 33 eyes were treated with MMCT alone between June 1, 2005 and May 17, 2007 (Control Group); and, 24 eyes were treated with a combination of IVB and MMCT after May 18, 2007 (IVB Group). Surgical complications, intraocular pressure (IOP), and the probability of success were compared between the 2 groups. Surgical failure was defined as IOP ≥22 mm Hg for 2 consecutive follow-up visits, a deterioration of visual acuity to no light perception, or additional glaucoma surgeries. ResultsThere were no significant differences in preoperative data between the groups. Hyphema associated with MMCT occurred significantly less often in the IVB Group (P=0.006). IOPs at 7 and 10 days after MMCT were significantly lower in the IVB Group (P=0.01 and 0.02, respectively). However, Kaplan-Meier survival-curve analysis showed the probability of success 120, 240, and 360 days after MMCT of 87.5%, 79.2%, and 65.2% in the IVB Group, and 75.0%, 71.9%, and 65.3% in the Control Group. No significant difference in survival times was found between the groups (P=0.76). ConclusionsIVB before MMCT reduced hyphema associated with MMCT for NVG. IVB provided further IOP reduction immediately after MMCT, but did not significantly improve surgical outcomes over longer periods.

Impact of liver transplantation on transthyretin-related ocular amyloidosis in Japanese patients

OBJECTIVE To evaluate the long-term impact of liver transplantation on ocular manifestations of familial amyloid polyneuropathy (FAP) in Japanese patients. METHODS Medical records were retrospectively reviewed in a long-term follow-up study. Of 52 patients with FAP amyloidogenic transthyretin Val30Met, 22 patients underwent liver transplantation. We assessed ocular manifestations, including amyloid deposition at the pupillary border, pupillary border with irregularity, vitreous opacities, and glaucoma, in patients who underwent liver transplantation. In addition, we compared the clinical characteristics of vitreous opacities-the most common ocular manifestation of FAP-in patients who underwent liver transplantation and those who did not to determine the effect of transplantation on the progression of ocular amyloidosis. RESULTS Mean time after FAP onset was 10 years and after liver transplantation was 7 years in patients who underwent liver transplantation. All ocular manifestations increased with time after transplantation. Eight patients (36%) developed vitreous opacities and 4 patients (18%) developed glaucoma during follow-up. Mean time from FAP onset to vitreous opacities onset was significantly shorter in patients with early-onset disease who underwent liver transplantation than in those who did not. CONCLUSIONS Patients with FAP who undergo liver transplantation continue to have a long-term risk of severe ocular manifestations, especially vitreous opacities and glaucoma, which can restrict their daily lives, even after liver transplantation.

Intraocular Pressure Elevation after Injection of Triamcinolone Acetonide : A Multicenter Retrospective Case-Control Study

PURPOSE To determine the risk factors for intraocular pressure (IOP) elevation after the injection of triamcinolone acetonide (TA). DESIGN Retrospective interventional case-control study. METHODS SETTING Multicenter. PATIENT POPULATION Four hundred and twenty-seven patients. OBSERVATION PROCEDURES Intraocular pressure levels after TA treatment by the sub-Tenon capsule injection (STI; 12 mg, 20 mg, or 40 mg), intravitreal injection (IVI; 4 mg or 8 mg), or the combination of STI (20 mg) and IVI (4 mg), and IOP levels after two TA treatments. MAIN OUTCOME MEASURE Risk factors for IOP levels of 24 mm Hg or higher. RESULTS Younger age (hazards ratio [HR], 0.96/year; P < .0001), IVI (HR, 1.89/year; P < .0001), and higher baseline IOP (HR, 1.15/mm Hg; P = .003) were identified as risk factors. Dose dependency was shown in STI-treated eyes (HR, 1.07/mg; P = .0006), as well as after IVI (HR, 1.64/mg; P = .013). The combination of STI and IVI was a significant risk factor (HR, 2.27; P = .003) compared with STI alone. In eyes receiving two TA treatments, IVI (HR, 2.60; P = .010), higher IOP elevation after the first injection (HR, 1.18/mm Hg; P = .011), and increased dosage of STI (HR, 1.07/mm Hg; P = .033) were risk factors. CONCLUSIONS Younger age, higher baseline IOP, IVI, and increased TA dosage were associated with TA-induced IOP elevation. IOP elevation after repeated TA injection was frequently associated with eyes treated with IVI, high IOP elevation after the first injection, and high doses of STI.

Lactoferrin Glu561Asp facilitates secondary amyloidosis in the cornea

Aim: To elucidate the pathogenic mechanism of amyloid formation in corneal amyloidosis with trichiasis. Methods: Ophthalmological examination was performed in nine patients to determine secondary corneal amyloidosis with trichiasis. Congo red staining and immunohistochemistry using anti-human lactoferrin antibody were used for biopsied corneal samples. For genetic analyses, single strand conformation polymorphism (SSCP), direct DNA sequence analysis, and polymerase chain reaction (PCR) induced mutation restriction analysis (IMRA) were employed to detect lactoferrin gene polymorphism. Results: All patients had had trichiasis at least for 1 year, and all amyloid-like deposits were found in one eye with trichiasis. Ophthalmological examination revealed that eight patients showed gelatinous type of amyloid deposition and one showed lattice type of amyloid deposition. Studies of biopsied corneal samples with Congo red stain revealed positive staining just under the corneal epithelial cells. Immunoreactivity of anti-human lactoferrin antibodies was recognised in all tissues with positive Congo red staining. Lactoferrin gene analysis revealed that seven patients were heterozygotic and two were homozygotic for lactoferrin Glu561Asp. The frequency of the polymorphism in the patients was significantly different from that in 56 healthy control subjects. Conclusion: Lactoferrin Glu561Asp is a key polymorphism related to facilitating amyloid formation in corneal amyloidosis with trichiasis.