Long-term surveillance provides real-world evidences of safety and effectiveness in intravitreal aflibercept treatment for age-related macular degeneration.

This prospective, multicentre, postmarketing surveillance were conducted to report on the long-term safety and effectiveness of intravitreal aflibercept (IVT-AFL) treatment in clinical practice of Japanese patients with neovascular age-related macular degeneration (nAMD) who newly initiated IVT-AFL treatment. The primary outcomes were the incidence of adverse events (AEs) and of adverse drug reactions (ADRs) over 36 months. Number of injections, timing of ADR occurrence, and some effectiveness index were also summarised. A total of 3,872 patients received 7.2 ± 5.8 (mean ± standard deviation) injections, and AEs occurred in 5.73% of patients. ADRs were reported in 2.76% of patients, with ocular and nonocular ADRs in 2.07% and 0.72% of patients, respectively. Most vitreo-retinal events developed within 6 months of initial IVT-AFL treatment, and most instances of increased intraocular pressure and cerebral infarction developed after 6 months of follow-up. Mean best-corrected visual acuity and central retinal thickness were numerically better throughout the follow-up period compared with baseline. These results indicated acceptable tolerability and effectiveness of IVT-AFL treatment in patients with nAMD in clinical practice in Japan. Information regarding the risk and the timing of ADRs is valuable for safe and effective long-term treatment of patients with nAMD.Trial registration number: NCT01756248.

Astaxanthin increases choroidal blood flow velocity.

PURPOSE: Previous studies have reported that astaxanthin (AXT) has antioxidative and anti-inflammatory effects in addition to its ability to shorten blood transit times. As laser speckle flowgraphy (LSFG) can noninvasively visualize the hemodynamics of the choroidal circulation, we used the technique to evaluate whether continuous ingestion of 12 mg of AXT per day could increase quantitative blood flow velocity. METHODS: In this randomized, double-blind, placebo-controlled study, we examined 20 healthy volunteers who ingested 12 mg AXT or placebo capsules over a 4-week period. LSFG was measured in the right eyes of all subjects at pre-ingestion, and at 2 and 4 weeks after the treatment of AXT. LSFG values were used to calculate the square blur rate (SBR), which is a quantitative index of relative blood flow velocity. RESULTS: A significant increase of the macular SBR was seen 4 weeks after AXT ingestion when compared to the pre-ingestion values (Wilcoxon signed-rank test, P = 0.018). In contrast, no statistical difference in the macular SBR was detected in the placebo group (Friedman test, P = 0.598). No subjective or objective adverse events were found after the 12-mg AXT ingestion. CONCLUSIONS: Results suggest that administration of AXT over a 4-week period can elevate the choroidal blood flow velocity without any adverse effects.

Intravitreal anti-vascular endothelial growth factor therapy with bevacizumab for tuberous sclerosis with macular oedema.

PURPOSE: To describe two patients with macular oedema secondary to tuberous sclerosis complex (TSC) who were treated with intravitreal bevacizumab injection. METHODS: Interventional case reports. Bevacizumab 1.25 mg was injected into the vitreous of two patients with TSC-associated macular oedema / exudative retinal detachment. Vascular endothelial growth factor (VEGF) concentration in the vitreous fluid was measured by enzyme-linked immunosorbent assay (ELISA) in one of these patients. RESULTS: Patient 1: a 22-year-old woman with TSC was diagnosed as having multiple retinal hamartomas in both eyes. Eleven years later, the patient developed macular oedema with epiretinal membrane formation in the right eye. The patient underwent pars-plana vitrectomy with retinal photocoagulation for retinal tumours. VEGF concentration in the vitreous fluid was high compared to that in patients without retinal vascular diseases. Recurrent macular oedema disappeared by intravitreal injection of bevacizumab. Patient 2: a 32-year-old woman with TSC-associated retinal hamartoma, temporally showing macular exudative retinal detachment, developed neovascularization originated from the tumour. By intravitreal bevacizumab injection, the tumour size reduced markedly with regression of neovascularization. CONCLUSION: These results suggest that VEGF derived from retinal hamartomas causes macular oedema associated with TSC. Intravitreal injections of bevacizumab may be a useful therapeutic option for macular oedema secondary to TSC.

[Pathogenesis of infectious conjunctivitis in Nepal].

PURPOSE: We investigated human adenovirus (HAdV) and Chlamydia trachomatis in patients with infectious conjunctivitis in Nepal. METHOD: We obtained swabs from 6 patients with infectious conjunctivitis in a remote area near the Indian border (group A), and from 30 patients at the B. P. Koirala Eye Center of Tribhuvan University Teaching Hospital in Kathmandu (group B). Rapid diagnosis of HAdV was conducted in Nepal, using Capilia adeno eye (Capilia), a rapid adenoviral antigen diagnostic kit using immunochromatography. Residual swabs were brought to Japan and examined for HAdV and Chlamydia trachomatis using polymerase chain reaction (PCR). Etiological analysis of 214 patients with trachoma was also investigated by PCR. RESULTS: Capilia results were negative for the six samples of group A and positive for 13 patients (43%) in group B. PCR showed one (17%) as positive in group A and 30 (100%)in group B. The serotype of all HAdV positive samples was HAdV-8. C serovar of Chlamydia trachomatis was detected in ninety seven cases out of 214 patients with trachoma. CONCLUSION: HAdV-8 and Chlamydia trachomatis serotype C seem to be prevalent in Nepal.

Inhibition of choroidal neovascularization with an anti-inflammatory carotenoid astaxanthin.

PURPOSE: Astaxanthin (AST) is a carotenoid found in marine animals and vegetables. The purpose of the present study was to investigate the effect of AST on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms. METHODS: Laser photocoagulation was used to induce CNV in C57BL/6J mice. Mice were pretreated with intraperitoneal injections of AST daily for 3 days before photocoagulation, and treatments were continued daily until the end of the study. CNV response was analyzed by volumetric measurements 1 week after laser injury. Retinal pigment epithelium-choroid levels of IkappaB-alpha, intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2 were examined by Western blotting or ELISA. AST was applied to capillary endothelial (b-End3) cells, macrophages, and RPE cells to analyze the activation of NF-kappaB and the expression of inflammatory molecules. RESULTS: The index of CNV volume was significantly suppressed by treatment with AST compared with that in vehicle-treated animals. AST treatment led to significant inhibition of macrophage infiltration into CNV and of the in vivo and in vitro expression of inflammation-related molecules, including VEGF, IL-6, ICAM-1, MCP-1, VEGFR-1, and VEGFR-2. Importantly, AST suppressed the activation of the NF-kappaB pathway, including IkappaB-alpha degradation and p65 nuclear translocation. CONCLUSIONS: AST treatment, together with inflammatory processes including NF-kappaB activation, subsequent upregulation of inflammatory molecules, and macrophage infiltration, led to significant suppression of CNV development. The present study suggests the possibility of AST supplementation as a therapeutic strategy to suppress CNV associated with AMD.

Macrophage migration inhibitory factor ameliorates UV-induced photokeratitis in mice.

Acute ultraviolet (UV) exposure causes photokeratitis, and induces apoptosis in corneal cells of the eye. Macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine. Today, MIF is considered as an integral component of the host antimicrobial alarm system and stress response that promotes the proinflammatory functions of immune cells. Also, MIF is considered to contribute the wound healing process. The aim of the present study is to determine the effects of MIF expression on UV irradiated corneal damage. MIF transgenic (MIF-Tg), wild type (WT), and MIF deficient (MIF KO) mice were UVB-irradiated of 400mJ/cm2 to induce acute UV-photokeratitis. MIF Tg mice constitutively produce high levels of MIF. Morphological changes were most severe in MIF KO mice, and WT and MIF Tg mice were following. Corneal basement membrane of MIF-Tg was well preserved. Prominent higher level of MIF was observed in MIF-Tg than WT after UVB irradiation in cornea. TUNEL staining showed a significantly smaller number of TUNEL positive nuclei in MIF-Tgm (6.2+/-4.3 cells/section, p<0.01 compared with WT) than WT (30.7+/-9.1) and MIF KO mice (32.1+/-12.7) 24h after UV exposure. The number of c-Jun positive nuclei was significantly higher in MIF Tg (p<0.01) than in WT and MIF KO mice. Serial observation revealed that BrdU incorporation was significantly upregulated in MIF Tg (p<0.01), but downregulated in MIF KO (p<0.01) than WT mice. MIF expression may thus be related to the amelioration of UVB-caused corneal injury, and this association was attributable to the upregulation of cell proliferation after acute UV-induced corneal damage, which involves the c-Jun dependent pathway. In conclusion, UV-damaged cornea is recoverable without MIF, however it takes longer time than normal condition. Cornea is less damaged and can make a quick recovery when ocular tissue is enough supplied with MIF.

Anti-inflammatory effect of angiotensin type 1 receptor antagonist on endotoxin-induced uveitis in rats.

BACKGROUND: Angiotensin II type 1 (AT1) receptor-antagonists are widely used for treatment of hypertension. Recent studies have demonstrated a protective effect of renin angiotensin system (RAS) antagonism against immune-mediated inflammatory diseases such as myocarditis, chronic allograft rejection, antiglomerular basement membrane nephritis, colitis, and arthritis. However, only a few reports have demonstrated the effect of RAS in ocular inflammatory conditions. The purpose of this study was to investigate the anti-inflammatory effect of a selective AT1 receptor antagonist, losartan, on endotoxin-induced uveitis (EIU) and compare the effect on experimental autoimmune uveoretinitis (EAU). METHODS: To induce EIU, 7-week-old Lewis rats were injected subcutaneously with 200 microg lipopolysaccharide (LPS). Losartan was administered intravenously at the same time. The aqueous humor was collected from eyes 24 h after LPS injection. The number of infiltrating cells, protein concentration, and levels of tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein-1 (MCP-1) in the aqueous humor were determined. The collected eyes were immunohistochemically stained with monoclonal antibody for activated nuclear factor (NF)-kappaB. To induce EAU, C57BL/6 mice (6-8 weeks old) were immunized with human interphotoreceptor retinoid binding protein (hIRBP)-derived peptide emulsified in complete Freund's adjuvant (CFA) and concomitantly injected with purified Bordetella pertussis toxin (PTX). Clinical severity of EAU and T cell proliferative response were analyzed. RESULTS: Losartan significantly suppressed the development of EIU. Numbers of aqueous cells of control EIU rats, those from EIU rats treated with 1 or 10 mg/kg of losartan were 75.3+/-45.6 x 10(5), 27.9+/-8.1 x 10(5), or 41.3+/-30.9 x 10(5) cells/ml respectively (p<0.01 vs control). Aqueous protein, TNF-alpha, and MCP-1 levels were also significantly decreased in a manner dependent on the amount of losartan administered (p<0.01). Treatment of EIU rats with losartan suppressed activation of NF-kappaB at the iris ciliary body. Thus, the suppressive effect of losartan on ocular inflammation in EIU appeared to result from down-regulation of NF-kappaB activation and reduction of inflammatory cytokine production. On the other hand, in the EAU model, neither the clinical score nor the antigen-specific T cell proliferative response was significantly influenced by the treatment with losartan. CONCLUSIONS: The present findings indicate that RAS may be involved in the acute inflammation of the eye, but not in T cell-dependent ocular autoimmunity. Antagonism of the RAS may be a potential prophylactic strategy for treatment of the human acute ocular inflammation.

Macular pigment lutein is antiinflammatory in preventing choroidal neovascularization.

BACKGROUND: Choroidal neovascularization (CNV) is a critical pathogenesis in age-related macular degeneration, the most common cause of blindness in the developed countries. The aim of the current study was to investigate the effect of lutein supplementation on the development of the murine model of laser-induced CNV together with underlying molecular mechanisms. METHODS AND RESULTS: Mice were orally pretreated with lutein daily from 3 days before laser photocoagulation until the end of the study. The index of CNV volume was significantly suppressed by the treatment with lutein, compared with vehicle-treated animals. Lutein treatment led to significant inhibition of macrophage infiltration into CNV and of the in vivo and in vitro expression of inflammation-related molecules including vascular endothelial growth factor, monocyte chemotactic protein -1, and intercellular adhesion molecule-1. Importantly, lutein suppressed IkappaB-alpha degradation and nuclear translocation of nuclear factor (NF)-kappaB p65 both in vivo and in vitro. Additionally, the development of CNV was significantly suppressed by inhibiting NF-kappaB p65 nuclear translocation, to the levels seen in the lutein treatment. CONCLUSIONS: Lutein treatment led to significant suppression of CNV development together with inflammatory processes including NF-kappaB activation and subsequent upregulation of inflammatory molecules, providing molecular evidence of potential validity of lutein supplementation as a therapeutic strategy to suppress CNV.

Increased expression of erythropoietin receptor in human pterygial tissues.

Erythropoietin (Epo) induces physiological activities such as cell proliferation, migration, and angiogenesis in Epo receptor (EpoR)-expressing vascular endothelial and tumor cells. Recently, it has been demonstrated that growth factor-independent proliferation is frequently observed during the cell transformation process. Pterygium is a fibrovascular proliferating tissue that includes transformed cells. The aim of this study was to examine the localization of Epo and EpoR proteins in human pterygial tissues. Eleven samples including nine pterygia and two normal bulbar conjunctivas, which were surgically excised, were studied. Formalin-fixed, paraffin-embedded tissue sections were constructed and then were examined by immunohistochemistry with anti-Epo and EpoR antibodies. Cytoplasmic immunoreactivity for EpoR was heterogeneously detected in basal and suprabasal cells of the pterygium epithelium. In the pterygium stroma, a variety of endothelial cells forming vascular cavities showed cytolasmic immunoreactivity for EpoR. In normal conjunctival epithelium, a few basal cells showed a weak homogeneous immunoreactivity for EpoR in the cytoplasm. The number of EpoR-expressing epithelial cells was much higher in the pterygium compared to the normal conjunctiva. EpoR expression was marginally detected in stromal microvessels of the normal conjunctiva. Immunoreactivity for Epo was not noted in pterygium epithelium and stroma, and in normal conjunctiva. These results suggest that the Epo-independent EpoR-signaling pathway plays a potential role in cell proliferation and angiogenesis in human pterygium.

Increased osteopontin levels in the vitreous of patients with diabetic retinopathy.

PURPOSE: Osteopontin (OPN) has diverse functions such as cell adhesion, chemoattraction, immunomodulation, and angiogenesis. The aim of this study is to analyze the OPN levels in vitreous fluid obtained from diabetic retinopathy (DR) and non-DR patients. METHODS: Nineteen patients out of 11 with DR and 8 without DR underwent pars plana vitrectomy and vitreous fluid was obtained simultaneously. Two distinct sandwich enzyme-linked immunosorbent assay systems (systems 1 and 2) were applied, which have been developed in our laboratories to quantify the OPN concentrations in vitreous fluid. RESULTS: The non-thrombin-cleaved full-length OPN levels in the vitreous fluid were 921.63 +/- 45.38 ng/ml in DR and 632.80 +/- 83.43 ng/ml in non-DR using system 1. Also, vitreous thrombin-cleaved and noncleaved OPN levels were increased to 2,109.22 +/- 151.651 and 1,651.13 +/- 229.82 ng/ml in patients with DR and non-DR using system 2. The vitreous OPN levels were significantly higher in DR than those in non-DR (p < 0.01 by system 1 and p < 0.05 by system 2). CONCLUSION: Thrombin-cleaved and noncleaved vitreous OPN levels in patients with DR were increased compared with control subjects, suggesting that OPN plays a potential role in the pathogenesis of diabetic retinal ischemia.

Expression of erythropoietin receptor in human epiretinal membrane of proliferative diabetic retinopathy.

PURPOSE: It is widely accepted that intravitreous levels of erythropoietin (Epo) are elevated in patients with ischaemic retinal diseases such as proliferative diabetic retinopathy (PDR). The aim of this study was to examine the expression of Epo and the Epo receptor (EpoR) in epiretinal membranes with and without diabetes. METHODS: Eighteen epiretinal membranes (PDR (n = 10), idiopathic epiretinal membranes (IERMs) without diabetes (n = 4) and inner limiting membranes (ILMs) (n = 4)) were obtained during pars plana vitrectomy. Formalin-fixed and paraffin-embedded tissues were examined by immunohistochemistry with anti-Epo and EpoR antibodies. RESULTS: The histopathological findings demonstrated that PDR membranes consisted of a variety of endothelial cells forming a microvascular cavity with red blood cells and non-vascular stromal mononuclear cells. Membranous and cytoplasmic immunoreactivity for EpoR was strongly detected in endothelial cells and stromal cells in all PDR patients. Although microvessels were not observed in IERMs and ILMs, immunoreactivity for EpoR was noted in the cellular component of IERMs, and was weakly detected in ILMs. Epo was not expressed in any membrane. CONCLUSION: EpoR was strongly expressed in microvessels of all PDR membranes. The in vivo evidence in this study suggests that Epo in the vitreous binds to EpoR in PDR membranes, which subsequently leads to the proliferation of new retinal vessels. EpoR immunoreactivity in non-vascular stromal cells in PDR membranes, and IERMs and ILMs might be indirectly correlated with ischaemia.

Positive association of pigment epithelium-derived factor with total antioxidant capacity in the vitreous fluid of patients with proliferative diabetic retinopathy.

BACKGROUND: Pigment epithelium-derived factor (PEDF), a glycoprotein with potent neuronal differentiating activity, was recently found to inhibit advanced glycation end product (AGE)-induced retinal hyperpermeability and angiogenesis through its antioxidative properties, suggesting that it may exert beneficial effects on diabetic retinopathy by acting as an endogenous antioxidant. However, the inter-relationship between PEDF and total antioxidant capacity in the eye remains to be elucidated. AIMS: To determine vitreous PEDF and total antioxidant levels in patients with proliferative diabetic retinopathy (PDR), and to investigate the relationship between them. METHODS: Vitreous levels of PEDF and total antioxidant capacity were measured by an ELISA in 39 eyes of 36 patients with diabetes and PDR and in 29 eyes of 29 controls without diabetes. RESULTS: Vitreous levels of total antioxidant capacity were significantly lower in patients with diabetes and PDR than in controls (mean (SD) 0.16 (0.05) vs 0.24 (0.09) mmol/l, respectively, p<0.001). PEDF levels correlated positively with total antioxidant status in the vitreous of patients with PDR (r = 0.37, p<0.05) and in controls (r = 0.41, p<0.05). Further, vitreous levels of PEDF in patients with PDR without vitreous haemorrhage (VH(-)) were significantly (p<0.05) decreased, compared with those in the controls or in patients with PDR with vitreous haemorrhage (VH(+); PDR VH(-), 4.5 (1.1) microg/ml; control, 7.4 (4.1) microg/ml; PDR VH(+) 8.5 (3.6) microg/ml). CONCLUSION: This study demonstrates that PEDF levels are associated with total antioxidant capacity of vitreous fluid in humans, and suggests that PEDF may act as an endogenous antioxidant in the eye and could play a protective role against PDR.

Inhibition of nuclear factor-kappa B activation attenuates hydrogen peroxide-induced cytotoxicity in human lens epithelial cells.

AIMS: Hydrogen peroxide (H(2)O(2)) is the major oxidant involved in cataract formation. Lens epithelial cells have been suggested to be the first site of oxidative damage. The authors investigated the relationship between H(2)O(2)-induced cytotoxicity and activation of nuclear factor kappa B (NF-kappaB) in human lens epithelial (HLE) cells. METHODS: HLE B-3 cells were stimulated by various concentrations of H(2)O(2) in the presence or absence of pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of NF-kappaB. H(2)O(2)-induced cytotoxicity was measured by lactate dehydrogenase cytotoxicity assay. Translocation of NF-kappaB was examined by Western blot and immunocytochemistry using anti-p65 antibody. RESULTS: H(2)O(2)-induced cytotoxicity increased in a concentration-dependent manner. PDTC treatment significantly suppressed the cytotoxicity induced by H(2)O(2). After stimulated with H(2)O(2), NF-kappaB was found translocated from cytoplasm into the nuclei. PDTC treatment also inhibited the translocation of NF-kappaB. CONCLUSIONS: NF-kappaB signal pathway may be important in the development of H(2)O(2)-induced damage in HLE cells that is involved in cataractogenesis.

Phosphorylation of p27(KIP1) in lens epithelial cells after extraction of fiber cells.

Opacification of the posterior capsule depends on replication of the residual lens epithelial cells lining the capsule. However, the mechanisms in the regulation of lens cell proliferation have not been determined. The purpose of this study is to examine the expression of p27(KIP1), a cyclin-dependent kinase inhibitor, and its phosphorylation, and cyclin D1 in lens epithelial cells after extraction of fiber cells. C57Bl6 mice (12 weeks old) were anesthetized, and the lens fiber cells were surgically extracted. Eyeballs were collected and fixed at 15 min and 24 h after extraction with and without injection of a specific phosphorylated extracellular signal-regulated kinase (pERK) 1/2 inhibitor (PD98059) to the anterior chamber. Collected tissues were analyzed using immunohistochemistry with anti-p27(KIP1), anti-phosphorylated p27(KIP1) on serine 10 (s10-phospho-p27) and cyclin D1 antibodies. Human lens epithelial cells were cultured, and then were treated with and without 40 ng/ml human recombinant basic fibroblast growth factor (bFGF), which was analyzed by Western blot analysis. In the untreated lens, p27(KIP1) was not phosphorylated in the lens epithelial cells, although p27(KIP1)-positive nuclei were detected in the lens cells of the equatorial region. Immunoreactivity for cyclin D1 was hardly detected in the lens. Nuclear immunoreactivity for p27(KIP1) and s10-phospho-p27 was observed in several lens cells of the equatorial region 15 min after extraction of fiber cells. Western blotting demonstrated that the p27(KIP1) phosphorylation form was upregulated 15 min after bFGF treatment in cultured lens epithelial cells. Many cyclin D1-positive nuclei were noted 24 h after the surgery. p27(KIP1) phosphorylation and cyclin D1 induction were inhibited by PD98059. s10-phospho-p27 and p27(KIP1) immunoreactivity was undetected in the lens cells 24 h after the extraction of fiber cells. It is possible that the phosphorylation of p27(KIP1), and cyclin D1 expression are regulated by the ERK pathway in lens cells after the extraction of fiber cells.

Captopril suppresses inflammation in endotoxin-induced uveitis in rats.

Captopril is an inhibitor of angiotensin-converting enzyme (ACE) that is largely used in the treatment of cardiovascular diseases. Several previous studies have demonstrated that captopril exhibits a wide variety of biological activities, including an anti-inflammatory action, on which we focused our attention. The aim of the present study was to investigate the efficacy of captopril on endotoxin induced uveitis (EIU) in rats. We investigated its effect upon cellular infiltration and protein leakage, as well as on the concentration of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), prostaglandin E2 (PGE2), monocyte chemoattractant protein-1 (MCP-1) in the anterior chamber. In addition, we checked its effect on activation of nuclear factor kappa B (NF-kappaB) in iris and ciliary body (ICB) cells in vivo. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). One hour after the LPS inoculation, either 1mg/kg, 10mg/kg or 100mg/kg captopril were injected intravenously. 24h later, the aqueous humor was collected from both eyes, and the number of infiltrating cells and protein concentration in the aqueous humor were determined. Levels of TNF-alpha, PGE2, NO and MCP-1 were determined by enzyme-linked immunosorbent assay. On some eyes, after enucleation, immunohistochemical staining with a monoclonal antibody against activated NF-kappaB was performed. Captopril treatment significantly decreased the inflammatory cells infiltration, the level of protein, concentrations of TNF-alpha, PGE2, NO and MCP-1 in the aqueous humor. The number of activated NF-kappaB-positive cells was lower in ICB of the rats treated with captopril 3h after the LPS injection. The present results indicate that captopril suppresses the inflammation in EIU by inhibiting the NF-kappaB-dependent pathway and the subsequent production of pro-inflammatory mediators.

Inhibitory effects of lutein on endotoxin-induced uveitis in Lewis rats.

PURPOSE: Lutein deposits in the macula and lens of human eyes with high concentration and is well known as an eye-protective nutrient for its beneficial effects on eye disease such as age-related macular degeneration and cataract. The purpose of the present study was to investigate the effects of lutein on endotoxin-induced uveitis (EIU) in rats. METHODS: EIU was induced in male Lewis rats by subcutaneous injection of 200 microg lipopolysaccharide. Lutein or dexamethasone was administered intravenously at 30 minutes before, at the same time as, and at 30 minutes after LPS treatment. The aqueous humor was collected at 24 hours after LPS injection, the number of infiltrating cells, the protein concentration, and the levels of nitric oxide (NO), tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, prostaglandin (PG)-E2, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein (MIP)-2 in the aqueous humor were determined. Immunohistochemical staining with a monoclonal antibody against activated nuclear factor (NF)-kappaB was performed to evaluate the effect of lutein on NF-kappaB activation in the iris-ciliary body (ICB) of rats. A mouse macrophage cell line (RAW264.7 cells) was stimulated with LPS in the presence or absence of lutein. Expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and degradation of inhibitor kappaB (IkappaB) were analyzed by Western blot analysis. RESULTS: Lutein suppressed the development of EIU in a dose-dependent fashion. The anti-inflammatory effect of 100 mg/kg lutein was as strong as that of 1 mg/kg dexamethasone. Treatment with lutein reduced the concentrations of NO, TNF-alpha, IL-6, PGE2, MCP-1, and MIP-2 in aqueous humor. Lutein also suppressed the activation of NF-kappaB in the ICB as well as iNOS and COX-2 expression and IkappaB degradation in RAW cells. CONCLUSIONS: These findings indicate that lutein has anti-inflammatory effects on EIU by inhibiting the NF-kappaB dependent signaling pathway and the subsequent production of proinflammatory mediators.

Phosphorylation of p27(KIP1) in the mitotic cells of the corneal epithelium.

PURPOSE: The mechanism in regulation of the cell cycle and proliferation of corneal epithelium in the homeostatic ocular surface remains unclear. The aim of this study is to examine the expression of p27(KIP1) and its phosphorylation in corneal epithelium. METHODS: The eyes of C57BL/6 mice (7 weeks old) were enucleated. Formalin-fixed and paraffin-embedded tissue sections were examined using immunohistochemistry with anti-p27(KIP1), threonine 187 phosphorylated p27(KIP1) (T187-phospho-p27), and phosphorylated Histon H3 (pHiston H3) antibodies. Anti-T187-phospho-p27 and anti-pHiston H3 polyclonal antibodies were used for parallel immunofluorescent staining. RESULTS: pHiston H3-immunopositive cells were noted in basal cells of the corneal epithelium. At high magnification of DAPI nuclear staining, mitotic and non-mitotic cells were observed in corneal basal layer. p27(KIP1)-positive nuclei were detected in corneal basal cells, where non-mitotic basal cells were located. In contrast, mitotic cells showed under detectable level on p27(KIP1) immunoreactivity. Immunoreactivity for T187-phospho-p27 was detected in basal cells of the corneal epithelium. At high magnification, it was confirmed that the immunopositive cells were mitotic cells. Immunoreactivity of T187-phospho-p27 as well as pHiston H3 was localized in the same corneal basal cells using double-staining immunohistochemistry. CONCLUSIONS: These results suggested that degradation of p27(KIP1) regulates progression into mitosis in corneal basal cells.

Expression of cdc2 and p27(KIP1) phosphorylation in mitotic cells of the human retinoblastoma.

It was recently demonstrated that a lack of p27(KIP1) degradation resulted in the suppression of cdc2 activity and consequent inhibition of entry into the M-phase. The aim of this study was to examine the distribution of phosphorylated p27(KIP1) on threonine 187 (T187-phospho-p27) and cdc2 in mitotic cells of human retinoblastoma, a malignant retinal neoplasm. Several T187-phospho-p27-immunopositive cells were observed in mitotic retinoblastoma cells, but not in the normal retina. Immunoreactivity for T187-phospho-p27 was located in the prophase and metaphase of mitotic tumor cells. In contrast, tumor cells in the anaphase showed no immunoreactivity for T187-phospho-p27. Nuclear expression of cdc2 was detected in many retinoblastoma cells, including mitotic cells. The immunoreactivity in mitotic cells was located in the prophase, as well as metaphase. In contrast, anaphase cells did not show immunoreactivity. Double staining demonstrated the same localization of T187-phospho-p27 and cdc2 in mitotic cells. These results suggest that p27(KIP1) interacts with cdc2 in the M-phase of human retinoblastoma cells.

Phosphorylation of extracellular signal-regulated kinase and p27(KIP1) after retinal detachment.

PURPOSE: The roles of the extracellular signal-regulated kinase (ERK) pathway in the expression of cyclin D1 and p27(KIP1), the phosphorylation of p27(KIP1), and proliferation activity were examined after retinal detachment. METHODS: Normal eyes and eyes at 15 min, 2 and 4 days after retinal detachment in C57Bl6 mice were examined by immunohistochemistry using anti-phosphorylated (p) ERK1/2, anti-cyclin D1, anti-p27(KIP1), anti-p27(KIP1) phosphorylated at serine 10 (S10-phospho-p27), and anti-proliferating cell nuclear antigen (PCNA) antibodies with or without treatment with a specific ERK inhibitor, PD98059. Mouse Müller cells were isolated and examined for alteration of p27(KIP1) and cyclin D1 after exposure of basic fibroblast growth factor (bFGF) with and without treatment of PD98059 using Western blotting. RESULTS: In the normal retina, nuclear immunoreactivity for p27(KIP1), but not S10-phospho-p27 or pERK1/2, was observed in the middle sublayer of the inner nuclear layer (INL), where Müller glial cells are situated. At 15 min after the retinal detachment, p27(KIP1), S10-phospho-p27 and pERK1/2-positive nuclei were noted in the INL, whereas immunoreactivity for pERK1/2 or S10-phospho-p27 was not observed after treatment with PD98095. Cyclin D1 was induced in the INL 2 days after the retinal detachment, and the induction was inhibited by PD98059. At 4 days after the detachment, p27(KIP1) immunoreactivity was not observed, and cyclin D1 and PCNA were expressed. The disappearance of p27(KIP1) was suppressed, whereas expression of cyclin D1 and PCNA was not observed in mice treated with PD98059. Exposure of bFGF relatively decreased the expression level of p27(KIP1) and increased the level of cyclin D1 in mouse Müller cells, compared with control level. Induction of cyclin D1 and decrease in p27(KIP1) were inhibited with treatment of PD98059. CONCLUSION: Phosphorylation of ERK and expression of p27(KIP1) and cyclin D1 are involved in the proliferation of Müller cells after retinal detachment.