Abstract: OBJECTIVE: To examine the expression of alpha-crystallin, a small heat-shock protein family, and apoptosis in retinal neoplastic cells. METHODS: Thirteen enucleated globes were included in this study, 1 with retinocytoma and 12 with retinoblastoma. Formalin-fixed paraffin-embedded tissue sections were processed for immunohistochemistry with alpha-crystallin antibodies. Apoptotic cells were detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. RESULTS: In the retinocytoma, alphaA-crystallin was expressed in the cytoplasm of all tumor cells, whereas alphaB-crystallin immunoreactivity was only weakly positive. Apoptotic cells were rarely noted in retinocytoma cells; the apoptotic index was 0.29. Examination of the retinoblastoma globes revealed 6 cases (50%) that were strongly positive for alphaA-crystallin. The mean (SD) apoptotic indices in the strongly and weakly positive cases were 3.55 (2.61) and 7.50 (2.61), respectively. The apoptotic index was significantly higher in those cases that were weakly positive for alphaA-crystallin than in those that were strongly positive (P < .05). No correlation was observed between apoptotic index and alphaB-crystallin immunoreactivity, although 50% of retinoblastomas were strongly positive for alphaB-crystallin. CONCLUSIONS: The alphaA- and alphaB-crystallins are expressed in retinoblastomas, and alphaA-crystallin expression may prevent apoptosis of neoplastic cells. Clinical Relevance Suppression of alphaA-crystallin may be useful in controlling tumor growth.
Abstract: AIM: Small heat shock proteins (sHSP) play an important role in the resistance to anticancer drugs. We examined the expression of the sHSP family, HSP27 and alpha-crystallins, in human retinoblastoma with and without preoperative chemotherapy. METHODS: Eighteen enucleated eyes from patients with retinoblastoma were used. Six patients had undergone chemotherapy before enucleation. Formalin-fixed, paraffin-embedded tissue sections were processed for H&E staining and examined by immunohistochemistry using anti-HSP27 and alpha-crystallins antibodies. RESULTS: Eleven of 12 cases with no history of preoperative chemotherapy showed weakly positive or negative staining for HSP27, whereas six and five cases were strongly positive for alphaA and alphaB-crystallin, respectively. In the six cases with a history of chemotherapy, several viable retinoblastoma cells remained. Immunoreactivity for HSP27 and alphaB-crystallin was strongly detected in the cytoplasm of viable retinoblastoma cells, while alphaA-crystallin immunoreactivity was less marked. Immunoreactivity for HSP27 was significantly higher in retinoblastoma cases with preoperative chemotherapy than in those without chemotherapy (p<0.0001). In contrast, immunoreactivity for alphaA-crystallin was significantly lower in cases with chemotherapy than in cases without chemotherapy (p<0.01). CONCLUSIONS: HSP27 and alphaB-crystallin, but not alphaA-crystallin, were highly expressed in viable tumour cells after chemotherapy, suggesting that HSP27 and alphaB-crystallin may protect tumour cells from apoptotic signals produced by anticancer drugs in retinoblastoma.
Abstract: BACKGROUND: Variants in the gene for complement factor H (CFH) have been implicated as a major risk factor for the development of age-related macular degeneration (AMD). Little is known, however, about the factors regulating local expression and secretion of CFH by retinal pigment epithelial cells (RPE). METHODS: Cultured human early passage RPE cells, highly differentiated, polarized human RPE cultures, and bovine RPE explants were incubated in the presence or absence of recombinant human or bovine interferon-gamma (IFN-gamma; 25 ng/ml). CFH expression in cell lysates, and secretion into culture supernatants were examined by Western blot. CHF expression and localization was analyzed by confocal microscopy. Migration assay was performed in a modified Boyden chamber with early passage human RPE cells after stimulation with recombinant CFH protein (1-100 ng/ml). RESULTS: CFH was expressed in the cell lysates of RPE cells, and this expression was significantly upregulated by IFN-gamma. Immunoreactivity for CFH was detected in RPE cells of bovine explants and highly differentiated human RPE monolayers, and the level of immunoreactivity increased after IFN-gamma stimulation. Confocal microscopy revealed that CFH was predominantly localized in the apical cytoplasm of polarized human RPE. Western blot confirmed that IFN-gamma increased CFH secretion into RPE supernatants. Dose-dependent RPE cell chemotactic migration was induced by CFH. CONCLUSION: IFN-gamma promotes CFH expression in the apical compartment of RPE cells and increases secretion of CFH into RPE culture supernatants. Furthermore, CFH promotes chemotactic migration of RPE. This study suggests that interactions between CFH and IFN-gamma have the potential to play a role in the pathogenesis of AMD.
Abstract: Purpose: The aim of this study was to analyze lymphocyte infiltration using immunohistochemistry in proliferative diabetic retinopathy (PDR) membranes. Methods: Sixteen patients, thirteen with PDR and three without diabetes, underwent pars plana vitrectomy and the epiretinal membrane was peeled. Formalin-fixed, paraffin-embedded epiretinal membrane tissues were processed for immunohistochemistry with anti-leukocyte common antigen (LCA), CD3, and CD20 antibodies. The lymphocyte density was determined by direct counting at a high magnification under a light microscope, which was compared with the patients' visual prognosis. Results: The lymphocyte density ranged from 1 to 52 (mean: 9.5) in high-power fields. Of 13 membranes, five showed a lymphocyte density of more than 5 cells, while the other eight membranes showed a cell number of less than 2. The former type is defined as a lymphocyte-rich epiretinal membrane (LERM). Infiltrated lymphocytes were immunohistochemically positive for CD3, a T-cell marker, but not for CD20, a B-cell marker. All patients with LERM showed a poor visual prognosis after vitrectomy. In contrast, the visual prognosis in 7 patients with non-LERM improved or remained unchanged. A significant association was observed between high-level lymphocyte infiltration in the epiretinal membrane and a poor visual prognosis (P<0.001). LCA-positive mononuclear cells were not observed in epiretinal membranes in the absence of diabetes. Conclusion: These results suggest that the high-level infiltration of T lymphocytes into the PDR membrane is well correlated with a poor visual prognosis. Histopathological observation of the epiretinal membrane in PDR patients may provide significant prognostic information.
Abstract: OBJECTIVE: To analyze the histopathology and expression of transforming growth factor beta (TGF-beta) in retinoblastoma with and without cataractous changes. METHODS: Twenty patients with unilateral retinoblastoma underwent enucleation. None of these patients had received preoperative chemotherapy or radiotherapy. Formalin-fixed, paraffin-embedded tissue sections were examined histologically for the presence of morgagnian globules or liquefaction of lens fibers; TGF-beta was immunolocalized using an anti-TGF-beta antibody. RESULTS: Two globes showed several morgagnian globules and liquefaction of the lens fibers, representing cataractous changes. One patient had posterior subcapsular cataract; the other, anterior polar cataract. In both cases, prominent cytoplasmic immunoreactivity for TGF-beta was detected in retinoblastoma cells. In contrast, 3 patients showed histologic evidence of minor cataractous changes. The globes with either minor or no cataractous changes revealed minimal to no expression of TGF-beta. CONCLUSIONS: These results suggest that TGF-beta produced by retinoblastoma cells may induce cataract formation. Clinical Relevance The growth factors produced by retinoblastoma cells may lead to associated pathologies, such as cataracts, in the ocular structures. This study implies that when a child presents with a unilateral cataract, retinoblastoma should be excluded as the primary diagnosis.
Abstract: BACKGROUND: Nectins are Ca2+-independent immunoglobulin (Ig)-like cell-cell-adhesion molecules. We have generated transgenic mice expressing a series of soluble forms of nectin-1, and investigated special effects of each soluble form of nectin-1 in vivo. In the course of generating transgenic mice expressing a soluble form of nectin-1 consisting of the first Ig-like domain of nectin-1 and the Fc portion of human IgG1 (PHveC-VhIg), we found that all of the transgenic founder mice showed a microphthalmia. The purpose of this study is to examine functions of the extracellular domains of nectin-1 in eye development using transgenic technology. METHODS: Eyes of four different transgenic mouse lines expressing each soluble form of nectin-1 were analyzed histologically. Tissue sections were processed with hematoxylin-eosin staining and indirect immunoperoxidase technique. RESULTS: All of five transgenic mouse founders expressing PHveC-VhIg, and of three lines expressing PHveC-VpIg made of the first Ig-like domain fused to porcine Fc portions at 5 weeks showed a microphthalmia, but not all of the transgenic mouse lines expressing PHveCIg or PHveCpIg made of the entire ectodomain fused to human or porcine Fc portions. In the abnormal eyes, the vitreous body was almost absent. In PHveC-VhIg-expressing mice at postnatal day 6, each vitreous space was very small. In the neonatal transgenic mice, the vitreous body was almost the same as that of control mice, and PHveC-VhIg was expressed in the optic nerve, conjunctival epithelium, ciliary body, corneal and lens epithelium. At this stage, nectin-1, -3 and -4 were stained in the optic nerve of control mice as well as in that of the transgenic mice. Nectin-1 is faintly stained in the epithelium of the cornea and lens epithelium, but not in the ciliary body. CONCLUSION: Soluble forms of the first Ig-like domain of nectin-1 (PHveC-VhIg and PHveC-VpIg), but not those of the entire ectodomain (PHveCIg and PHveCpIg), lead to microphthalmia and lack of vitreous body in the transgenic mice.
Abstract: 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.
Abstract: BACKGROUND: Peripapillary subretinal neovascularization (PSRNV) is a rare type of choroidal neovascularization. Herein we report a case of retinoblastoma complicating PSRNV, and discuss the histopathological findings. METHODS: A 1-year-old male underwent enucleation of his right eyeball based on the clinical diagnosis of bilateral retinoblastoma after chemotherapy. RESULTS: There was a mass arising from the retina showing highly calcified and necrotic retinoblastoma. The peripapillary region revealed neovascular membrane extending from the optic nerve head to the subretinal space. The membrane included retinal pigment epithelial (RPE) cells and glial cells, as well as endothelial cells. Immunohistochemistry revealed cytokeratin 18-positive RPE cells situated beneath glial fibrillary acidic protein-positive glial cells and their processes. The neovascular membrane did not have a connection with vessels arising from the optic nerve head. There were multiple mound foci made up of proliferated RPE cells in the globe. CONCLUSION: These results suggest that migration of RPE cells and glial cells plays a crucial role in the pathogenesis of PSRNV, which might be directly or indirectly mediated by retinoblastoma.
Abstract: 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.
Abstract: 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.
Abstract: HLA-B27 associated uveitis is characterized by recurrent alternating acute unilateral attacks of intraocular inflammation in the anterior chamber. The aim of this study was to report an unusual case of repeated exacerbations with vitreous hemorrhage in HLA-B27 associated uveitis. Thirty four-year-old man was diagnosed as HLA-B27 associated uveitis in his right eye. He showed repeated exacerbation of ocular inflammation with retinal vein dilation and small retinal hemorrhage following vitreous hemorrhage. Fluorescein fundus angiography a week before the appearance of vitreous hemorrhage showed no neovascularization. Oral prednisolone administration was started from 40mg/day with gradual tapering. About 3 weeks after the onset, most of the vitreous hemorrhage disappeared and visual acuity was improved to 20/20. Through the decreased vitreous hemorrhage, Weiss ring was detected later. The vitreous hemorrhage found in this patient is a severe exacerbation, and might be a consequence of the vitritis that leads to posterior vitreous detachment.
Abstract: 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.
Abstract: 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.
Abstract: Cell proliferation mainly depends on cell cycles. p27(KIP 1) was cloned as a cell cycle inhibitor in 1994. In the retina, a variety of cells come to express p27(KIP 1) as development progresses, while the number of proliferating cells decreases. Human retinal cells show proliferation activity during embryonic stages, and subsequently differentiate into three neural retinal layers at the neonatal phase, when proliferating cells are no longer detected. The pathogenesis of retinoblastoma and proliferative vitreoretinopathy, major fundus disorders, is correlated with aberration of retinal cell proliferation. Recent research has shown that p27(KIP 1) plays important roles in this pathogenesis. In this review, we discuss the role of p27 (KIP 1) in retinal development, and in the pathology of fundus disorders. We conclude that the clarification of the mechanisms of p27 (KIP 1) expression might contribute to the discovery of a novel therapy targeting for human retinal diseases.
