A ROCK Inhibitor Promotes Graft Survival during Transplantation of iPS-Cell-Derived Retinal Cells.

Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.

Dynamics of Cyclooxygenase-1 Positive Microglia/Macrophage in the Retina of Pathological Model Mice as a Biomarker of the Retinal Inflammatory Diseases.

In an intraocular inflammatory state, microglia residing in the retina become active and migrate inside the retina. In this study, we investigated whether cyclooxygenase-1 (COX-1) expressed by retinal microglia/macrophage can be a biomarker for the diagnosis of retinal diseases. COX-1 was immunopositive in microglia/macrophage and neutrophils, while COX-2 was immunopositive in astrocytes and neurons in the inner layer of normal retina. The number of COX-1 positive cells per section of the retinal tissue was 14 ± 2.8 (mean ± standard deviation) in normal mice, which showed significant increase in the lipopolysaccharide (LPS)-administrated model (62 ± 5.0, p = 8.7 × 10-9). In addition to microglia, we found neutrophils that were positive for COX-1. In the early stage of inflammation in the experimental autoimmune uveoretinitis (EAU), COX-1 positive cells, infiltrating from the ciliary body into the retinal outer nuclear layer, were observed. The number of infiltrating COX-1 positive cells correlated with the severity of EAU. Taken together, the increased number of COX-1 positive microglia/macrophage with morphological changes were observed in the retinas of retinal inflammatory disease models. This suggests that COX-1 can be a marker of disease-related activities of microglia/macrophage, which should be useful for the diagnosis of retinal diseases.

Low Immunogenicity and Immunosuppressive Properties of Human ESC- and iPSC-Derived Retinas.

ESC- and iPSC-derived retinal transplantation is a promising therapeutic approach for disease with end-stage retinal degeneration, such as retinitis pigmentosa and age-related macular degeneration. We previously showed medium- to long-term survival, maturation, and light response of transplanted human ESC- and iPSC-retina in mouse, rat, and monkey models of end-stage retinal degeneration. Because the use of patient hiPSC-derived retina with a disease-causing gene mutation is not appropriate for therapeutic use, allogeneic transplantation using retinal tissue/cells differentiated from a stocked hESC and iPSC line would be most practical. Here, we characterize the immunological properties of hESC- and iPSC-retina and present their three major advantages: (1) hESC- and iPSC-retina expressed low levels of human leukocyte antigen (HLA) class I and little HLA class II in vitro, (2) hESC- and iPSC-retina greatly suppressed immune activation of lymphocytes in co-culture, and (3) hESC- and iPSC-retina suppressed activated immune cells partially via transforming growth factor ß signaling. These results support the use of allogeneic hESC- and iPSC-retina in future clinical application.

Microglia dynamics in retinitis pigmentosa model: formation of fundus whitening and autofluorescence as an indicator of activity of retinal degeneration.

In patients with retinitis pigmentosa (RP), color fundus photography and fundus autofluorescence (FAF) have been used to estimate the disease progression. To understand the origin and the diagnostic interpretation of the fundus color and FAF, we performed in vivo imaging of fundus color and FAF together with histological analyses of the retinal degeneration process using the RP model mice, rd10. FAF partly represented the accumulation of microglia in the photoreceptor outer segments. Fundus whitening suggested the presence of apoptotic cells, which spatiotemporally preceded increase in FAF. We observed two patterns of FAF localization, arcuate and diffuse, each indicating different pattern of apoptosis, wavy and diffuse, respectively. Diffuse pattern of apoptosis was suppressed in dark-raised rd10 mice, in which outer nuclear layer (ONL) loss was significantly suppressed. The occupancy of FAF correlated with the thinning rate of the ONL. Fractalkine, a microglia chemotactic factor, was detected in apoptotic photoreceptors, suggesting chemokine-induced recruitment of microglia into the ONL, which paralleled with accelerated ONL loss and increased FAF occupancy. Thus, we propose that the degree of photoreceptor apoptosis and the rate of ONL thinning in RP patients might be read from the fundus color and the FAF.

Influence of cataract surgery for the first or second eye on vision-related quality of life (VR-QOL) and the predictive factors of VR-QOL improvement.

PURPOSE: To assess improvements in vision-related quality of life (VR-QOL) in patients undergoing their first or second eye cataract surgery, as well as clinical factors related to VR-QOL. STUDY DESIGN: Prospective case series. METHODS: We examined 282 patients undergoing their first (222) or second (60) eye cataract surgery. VR-QOL was evaluated before and after surgery using the 25-item National Eye Institute visual function questionnaire (VFQ-25), along with the best-corrected visual acuity (BCVA), uncorrected visual acuity, and the lens opacities classification system III (LOCSIII). The resulting VFQ-25 subscale scores were compared between patients undergoing their first or second eye cataract surgery, including multiple regression analysis. RESULTS: The mean VFQ-25 composite score (CS) was 71.5 ± 14.2 before and 84.0 ± 10.2 after the first eye cataract surgery and 73.5 ± 12.7 before and 85.4 ± 10.2 after the second eye cataract surgery. VFQ-25 scores improved significantly, with reduced disparity among patients after surgery in both groups. Preoperative CS was related to the preoperative sum of the BCVA (standardized partial regression coefficient (ß) = - 0.254, P < 0.001). Improvement in the CS was related to a preoperative poor BCVA (ß = 0.203, P < 0.001), low CS (ß = - 0.693, P < 0.001), and high general health score (ß = 0.118, P = 0.025). CONCLUSIONS: VR-QOL improved after the first and second eye surgery. Many VFQ-25 subscales were related to the BCVA or LOCSIII scores. Low preoperative VR-QOL and BCVA were related to an improved postoperative VR-QOL.

A Strategy for Personalized Treatment of iPS-Retinal Immune Rejections Assessed in Cynomolgus Monkey Models.

Recently, we successfully transplanted an autograft, or major histocompatibility complex (MHC)-matched allografts, from induced-pluripotent-stem-cell-derived retinal pigment epithelial (iPSC-RPE) cells in patients with age-related macular degeneration. However, there was an issue regarding immune rejection after transplantation. In this study, we established a preoperational in vitro "drug-lymphocytes-grafts immune reaction (Drug-LGIR)" test to determine the medication for immune rejection using host immunocompetent cells (lymphocytes) and transplant cells (target iPSC-RPE cells) together with different medications. The adequacy of the test was assessed by in vivo transplantation in monkey models together with medication based on in vitro data. In the results of Drug-LGIR tests, some drugs exhibited significant suppression of RPE cell-related allogeneic reactions, while other drugs did not, and the efficacy of each drug differed among the recipient monkeys. Based on the results of Drug-LGIR, we applied cyclosporine A or local steroid (triamcinolone) therapy to two monkeys, and successfully suppressed RPE-related immune rejections with RPE grafts, which survived without any signs of rejection under drug administration. We propose that our new preoperational in vitro Drug-LGIR test, which specifies the most efficacious medication for each recipient, is useful for controlling immune attacks with personalized treatment for each patient after retinal transplantation.

Mycoplasma Ocular Infection in Subretinal Graft Transplantation of iPS Cells-Derived Retinal Pigment Epithelial Cells.

Purpose: To report occurrence of acute severe inflammation after surgical implantation of mycoplasma-infected induced pluripotent stem cell-derived RPE (iPS-RPE) cells into the eyes of healthy primates, and determine the immunopathological mechanisms of the inflammation. Methods: Ophthalmic allogeneic transplantation of iPS-RPE cells was performed in the subretina of major histocompatibility complex (MHC)-matched (two eyes) and MHC-mismatched (one eye) healthy cynomolgus monkeys. The clinical course after transplantation was observed using color fundus photography, fluorescence angiography, and optical coherence tomography. After the animals were killed at 1 month after surgery, eyeballs were removed and pathologically examined. Microorganisms were analyzed by PCR methods and BLAST analysis using preserved graft iPS-RPE cells and the recipients' vitreous humor. Mixed lymphocyte-RPE assay was performed on the mycoplasma-infected and noninfected iPS-RPE cells in vitro. Results: In tested eyes, abnormal findings were observed in the grafted retina 2 weeks after surgery. Here, we observed retinal vasculitis and hemorrhage, retinal detachment, and infiltration of inflammatory cells into the retina of the eyes. One month after surgery, animals were killed due to the severe immune responses observed. Using PCR methods, sequence analysis detected mycoplasma-DNA (Mycoplasma arginini species) in both the grafted RPE cells and the collected vitreous fluids of the monkeys. Mixed lymphocyte-RPE assay revealed that the infected iPS-RPE cells enhanced the proliferation of inflammatory cells in vitro. Conclusions: Transplantation of graft iPS-RPE cells contaminated with mycoplasma into the subretina caused severe ocular inflammation. Mycoplasma possesses the ability to cause immune responses in the host.

Detection of Complement Activators in Immune Attack Eyes After iPS-Derived Retinal Pigment Epithelial Cell Transplantation.

Purpose: To determine whether human induced pluripotent stem (iPS) cell-derived retinal pigment epithelial (RPE) cells (iPS-RPE) can express complement factors. Methods: To confirm expression of complement factors in human iPS-RPE cells, we performed flow cytometry, immunohistochemistry, ELISA, and quantitative RT-PCR for the following: C3, C5, CFB (Factor B), C5b-9 (membrane attack complex [MAC]), CFH (Factor H), CFI (Factor I), CD46, CD55, CD59, clusterin, and vitronectin. We also prepared iPS-RPE cells in the presence of recombinant IFN-γ, recombinant TNF-α, lipopolysaccharide, supernatants of naïve T cells, and T helper 1 (Th1) cells. For the transplantation, after preparation of iPS-RPE cells from cynomolgus monkeys, the iPS-RPE cells (allografts) were transplanted into the subretinal space in monkeys. After surgery, monkeys were euthanized for IHC evaluation of the retinal section and determination of complement factors (C3, C5, CFB, MAC, and C1q), cytokines, and immunoglobulin G (IgG). Results: Human iPS-RPE cells expressed complement activators and inhibitors. iPS-RPE cells highly expressed complement factors during inflammatory conditions, especially IFN-γ exposure including Th1 cell supernatants. In immune attack eyes after allogeneic iPS-RPE cell transplantation, complement activators such as C3, CFB, C5, and MAC were detected around the host RPE layer, grafted RPE cells, inflammatory retinal lesions, and transplanted subretinal space. In addition, we observed a large number of C1q and IgG double positive and IFN-γ positive inflammatory cells in the retinal sections. Conclusions: iPS-derived RPE cells greatly expressed complement factors. Thus, RPE cells might be activated and produce complement factors after exposure to infiltrating inflammatory cells in the eye.

Natural Killer Cell Inhibition by HLA-E Molecules on Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelial Cells.

Purpose: To determine whether human induced pluripotent stem (iPS) cell-derived retinal pigment epithelial (RPE) cells (iPS-RPE) can suppress natural killer (NK) cell activation. Methods: iPS-RPE cells were cocultured with peripheral blood mononuclear cells (PBMCs) or purified NK cells from healthy donors after stimulation with cytokines. To confirm expression of NK cell-specific markers, flow cytometry and quantitative RT-PCR (qRT-PCR) were performed. NK cells (or PBMCs) cocultured with iPS-RPE cells were assessed for proliferation by Ki-67 expression with flow cytometry, and NK suppression by RPE cells was assessed for granzyme B production with ELISA. Human leukocyte antigen (HLA) expression including HLA-E on iPS-RPE cells was evaluated with flow cytometry and qRT-PCR. The effect of HLA-E downregulation was also investigated using small interfering RNA (siRNA) systems. Following iPS-RPE cell transplantation in vivo, we evaluated NK cell invasion in the retina with immunohistochemistry. Results: Activated NK cells expressed NK-related markers such as CD16, CD56, and CD11b, and NK cells produced cytotoxic agents such as granzyme B, perforin, and TNF-α. Human iPS-RPE cells inhibited cell proliferation and production of these cytotoxic agents by activated NK cells in vitro. iPS-RPE cells constitutively expressed HLA-E and suppressed NK cell activation through an interaction between HLA-E and CD94/NKG2A. Moreover, immunohistochemical evaluation of monkey RPE transplantation into in vivo immune rejection models showed no NK cell invasion in the retina in allografts or xenografts except for one xenografted eye. Conclusions: Cultured iPS cell-derived RPE cells greatly suppress NK cell activation. Thus, NK cells might be inactivated when exposed to this type of retinal cell.

Detection of Retinal Pigment Epithelium-Specific Antibody in iPSC-Derived Retinal Pigment Epithelium Transplantation Models.

Antibody-mediated rejection is characterized by donor-specific antibody produced by B cells. However, to our knowledge, B cell invasion and antibody in the inflamed retina after transplantation of retinal pigment epithelial (RPE) cells has not been reported. To determine if RPE transplantation could be performed using allografts, we established in vivo immune rejection models with induced pluripotent stem cell (iPSC)-RPE allografts and determined whether RPE-specific antibody could be detected in these models. We detected alloantibodies in the serum from recipient monkeys that had immune attacks in the retina in an immunofluorescent assay using the transplanted iPSC-RPE cells as the antigen. In addition to T cell and antigen-presenting cell immunity, peripheral blood cells and lymph nodes in animal models with allogeneic iPSC-RPE cells also had activated B cells, which were probably secreting alloantibodies. Using serum and transplanted cells, alloreactive antibody can be detected for the diagnosis of immune rejection after transplantation.

Inhibition of T cell-mediated inflammation in uveitis by a novel anti-CD3 antibody.

BACKGROUND: A novel anti-mouse CD3ε antibody, Dow2, recognizes mouse CD3ε without activating T cells and suppresses T-cell activation. The purpose of this study was to determine whether Dow2 can inhibit T cells in uveitis. METHODS: Experimental autoimmune uveitis (EAU) was induced in mice by immunization with retinal peptides, followed by administration of Dow2. Inflammation was evaluated by color fundus photography, optical coherence tomography, fluorescein angiography, and histology. Intraocular cells from EAU mice were used to examine the effect of Dow2 on retinal antigen-specific T cells. The effects of Dow2, conventional CD3ε antibodies, and isotype control immunoglobulin G (IgG) on splenic T cells were compared by assessing cell proliferation by the mixed lymphocyte reaction assay, inflammatory cytokine production by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, and gene expression by quantitative reverse-transcription polymerase chain reaction (RT-PCR). T-cell subpopulations were characterized by flow cytometry to evaluate the expression of CD4, CD8, CD44, CD62L, and Foxp3. RESULTS: Dow2 significantly reduced T-cell activation and counteracted activation associated with anti-CD3ε antibodies. Unlike conventional CD3ε antibodies, Dow2 treatment did not upregulate T helper (Th)1-/Th17-associated gene expression and cytokine production in splenic T cells. Interferon (IFN)-γ production by retinal antigen-specific T cells was also significantly reduced. Ocular inflammation was significantly reduced in Dow2-treated EAU mice compared to control EAU mice, with fewer T cells infiltrating into the retinas of Dow2-treated EAU mice. In immunohistochemistry, Th1 and Th17 cells invaded the retina in control EAU mice but not Dow2-treated EAU mice. No effects on peripheral T-cell numbers were observed following systemic administration of Dow2. CONCLUSION: The novel anti-CD3 antibody Dow2 can inhibit T cell-mediated inflammation in uveitis models. Thus, inhibition of T-cell activation by anti-CD3 therapy with this new antibody may protect uveitis patients from severe ocular inflammation.

Lack of T Cell Response to iPSC-Derived Retinal Pigment Epithelial Cells from HLA Homozygous Donors.

Allografts of retinal pigment epithelial (RPE) cells have been considered for the treatment of ocular diseases. We recently started the transplantation of induced pluripotent stem cell (iPSC)-derived RPE cells for patients with age-related macular degeneration (autogenic grafts). However, there are at least two problems with this approach: (1) high cost, and (2) uselessness for acute patients. To resolve these issues, we established RPE cells from induced iPSCs in HLA homozygote donors. In vitro, human T cells directly recognized allogeneic iPSC-derived RPE cells that expressed HLA class I/II antigens. However, these T cells failed to respond to HLA-A, -B, and -DRB1-matched iPSC-derived RPE cells from HLA homozygous donors. Because of the lack of T cell response to iPSC-derived RPE cells from HLA homozygous donors, we can use these allogeneic iPSC-derived RPE cells in future clinical trials if the recipient and donor are HLA matched.

Successful Transplantation of Retinal Pigment Epithelial Cells from MHC Homozygote iPSCs in MHC-Matched Models.

There is an ongoing controversy as to whether major histocompatibility complex (MHC) matching is a solution for allogeneic stem cell transplantation. In the present study, we established retinal pigment epithelial (RPE) cells from induced pluripotent stem cells (iPSCs) in MHC homozygote donors. We observed no rejection signs in iPSC-derived RPE allografts of MHC-matched animal models without immunosuppression, whereas there were immune attacks around the graft and retinal tissue damage in MHC-mismatched models. In an immunohistochemical examination of MHC-mismatched allografts, the transplanted RPE sheets/cells were located in the subretinal space, but the RPE exhibited inflammatory and hypertrophic changes, and many inflammatory cells, e.g., Iba1+ cells, MHC class II+ cells, and CD3+ T cells, invaded the graft area. Conversely, these inflammatory cells poorly infiltrated the area around the transplanted retina if MHC-matched allografts were used. Thus, cells derived from MHC homozygous donors could be used to treat retinal diseases in histocompatible recipients.

Treatment of choroid metastasis from lung adenocarcinoma with bevacizumab-containing chemotherapy: A case report.

The occurrence of ocular metastasis from lung cancer is uncommon. The present study reports the case of a 69-year-old female patient with lung adenocarcinoma who was found to have a metastatic lesion in the left choroid at the time of presentation. As the patient was found to have a mutation in the epidermal growth factor receptor, treatment with gefitinib was administered; however, the response was evaluated as a progressive disease. Thereafter, the patient received chemotherapy with carboplatin, pemetrexed and bevacizumab. Radiological imaging revealed shrinkage of the primary lesion and choroidal metastasis, and the visual power of the left eye was also shown to improve. Therefore, the present case report demonstrated the efficacy and safety of systemic bevacizumab therapy in combination with a platinum doublet for the treatment of choroid metastasis, with morphological and functional improvements observed with regard to the choroidal metastatic tumor.

Longitudinal relationship between retinal nerve fiber layer thickness parameters assessed by scanning laser polarimetry (GDxVCC) and visual field in glaucoma.

PURPOSE: To investigate the longitudinal relationship between retinal nerve fiber layer (RNFL) thickness parameters assessed by scanning laser polarimetry with variable corneal compensation (GDxVCC) and visual field parameters obtained with the Humphrey field analyzer (HFA) in patients with glaucoma, and to assess the usefulness of GDxVCC in longitudinal follow-up. METHODS: A total of 242 eyes in 122 patients with glaucoma were periodically assessed using GDxVCC and HFA program SITA fast 30­2 for 3­5 years. Eyes with more than four times of reliable HFA and GDxVCC data were obtained from during the follow-up periods were included in the analysis. Changes in HFA parameters (mean deviation [MD], pattern standard deviation [PSD]) and those in GDxVCC parameters (superior average, inferior average, temporal-superior-nasal-inferior-temporal [TSNIT] average, TSNIT standard deviation [SD], nerve fiber indicator [NFI]) were determined by regression analysis. The relationship between HFA and GDxVCC parameters at the initial point and their annual changes were analyzed with canonical correlation analysis and Pearson's correlation coefficients. RESULTS: Twenty-four eyes (9.9%) of 19 patients that met inclusion criteria were statistically analyzed. Longitudinal progression was 0.039 ± 0.971 dB/year in MD, 0.156 ± 0.644 dB/year in PSD, −0.197 ± 0.970 µm/year in TSNIT average, −0.503 ± 1.341 µm/year in superior average, −0.282 ± 0.974 µm/year in inferior average, −0.284 ± 1.013/year in TSNIT SD and 1.269 ± 2.560/year in NFI. In canonical correlation analysis at the initial point, first canonical variates were not statistically significant between HFA and GDxVCC parameters. First canonical variates of annual changes significantly correlated between HFA and GDxVCC parameters (p < 0.01), with correlation coefficient of 0.85. In Pearson's correlation analysis of each parameter, there was a significant relationship between MD and NFI at the initial point (r = −0.46, p < 0.05). There was a statistically significant relationship between progressions in MD and NFI (r = −0.54, p < 0.01) and in PSD and NFI (r = 0.53, p < 0.01). CONCLUSIONS: Longitudinal progression in NFI obtained with GDxVCC was significantly correlated with that in HFA parameters, such as MD and PSD. GDxVCC is a useful tool for longitudinal follow-up assessment of glaucoma.

Hypersalivation induced by olanzapine with fluvoxamine.

Hypersalivation has been reported as a side effect of atypical antipsychotics such as clozapine and olanzapine. As it is very common for antipsychotics to cause dry mouth due to anticholinergic effects, hypersalivation seems to be paradoxical. We present the case of a 34-year-old Japanese man with delusional disorder, somatic type (DSM-IV). He had chronic neck pain as well as somatic hallucination with hypochondriacal delusion for 4 years. Since combination therapy with atypical antipsychotics and selective serotonin reuptake inhibitors (SSRIs) has been introduced in the treatment of refractory psychiatric disorders such as schizophrenia, olanzapine (10 mg/day) was added to fluvoxamine treatment (200 mg/day) in this case. Subsequently, hypersalivation was induced without any extrapyramidal symptoms. It is suggested that hypersalivation was an adverse effect of olanzapine. Possible interaction olanzapine with fluvoxamine might increase the risk of the adverse effect. When combination therapy of atypical antipsychotics and SSRI is introduced, it should be used cautiously with careful observation. Underlying pharmacological and clinical problems will be discussed.