Crucial role of P2X7 receptor for effector T cell activation in experimental autoimmune uveitis.

PURPOSE: To investigate the roles of P2X7 receptors (P2RX7) in the pathogenesis of experimental autoimmune uveoretinitis (EAU). STUDY DESIGN: Experimental. METHODS: Either wild-type (P2rx7 +/+ ) or P2rx7-deficient (P2rx7 -∕- ) mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) peptide 1-20. Severity of EAU was evaluated clinically and histopathologically. The induction of IRBP-specific proliferation and cytokines in draining lymph nodes was assessed by enzyme-linked immunosorbent assays (ELISA). The frequency of activation markers was examined by flow cytometry. Furthermore, inhibitory roles of systemic administration of Brilliant Blue G (BBG), an antagonist for P2RX7, in EAU were also assessed in the wild-type mice. RESULTS: The severity of EAU in P2rx7 -∕- mice was reduced as compared with that in P2rx7 +/+ mice, both clinically and histopathologically. IRBP-specific proliferation in P2rx7 -∕- on day 16 was slightly decreased compared to that in P2rx7 +/+ mice. The induction of IRBP-specific interferon (IFN)-γ and interleukin (IL)-17 in P2rx7 -∕- mice on day 16 was lower than that in P2rx7 +/+ mice. The up-regulation of surface expression of activation markers such as CD25, CD44, and CD69 in response to TCR stimulation in P2rx7 -∕- mice was decreased as compared with that in P2rx7 +/+ mice. Furthermore, neutralization of P2RX7 in vivo by BBG suppressed EAU clinically and histopathologically. IRBP-specific IFN-γ and IL-17 induction in BBG-treated mice was significantly lower than that in vehicle-treated mice. CONCLUSION: The results suggest that P2RX7 is a novel preventative therapeutic target for uveitis as it suppresses the effector functions of both Th1 and Th17 cell responses.

Different distributions of M1 and M2 macrophages in a mouse model of laser-induced choroidal neovascularization.

Choroidal neovascularization (CNV) is a serious complication of age­related macular degeneration. The aim of the present study was to investigate the expression and distribution of M1 and M2 macrophages in a laser­induced CNV adult mouse model. The mRNA expression levels of M1, M2 and pan macrophage markers, and macrophage­associated angiogenic cytokines, were determined by reverse transcription­quantitative polymerase chain reaction. Immunofluorescence studies were performed to determine the location of the macrophages. The expression levels of M1 macrophage markers increased to a greater extent compared with M2 markers in the retinal pigment epithelium (RPE)­choroid complexes following laser photocoagulation. By contrast, the expression levels of M2 macrophage markers increased primarily in the retinas. Immunofluorescence studies revealed that the increased number of cluster of differentiation (CD)206­positive cells were located primarily in the retina, whereas the CD80­positive cells were located around the site of CNVs in the RPE­choroid. In addition, the M1­associated cytokines increased to a greater extent in the RPE­choroid complexes, whereas the M2­associated cytokines were highly expressed in the retinas. These findings indicate that M1 and M2 macrophage numbers increased following CNV; however, the locations were different in this mouse model of laser­induced CNV. The results of the present study suggest that M1 macrophages have a more direct role in inhibiting the development of CNV.

Gene Expression Analysis of the Irrigation Solution Samples Collected during Vitrectomy for Idiopathic Epiretinal Membrane.

PURPOSE: The analysis of gene expression in idiopathic epiretinal membranes (iERMs) may help elucidate ERM formation and its pathology. Here, we conducted a case-control study, in order to determine the expression levels of cytokines and other genes in eyes with macular hole (MH) or iERM. METHODS: Twenty eyes, obtained from seven male and 13 female patients, were included in the study. The average age of the study subjects was 69.1 ± 7.67 years, and 15 eyes had iERM, while five eyes had MH. Irrigation solution samples were collected during vitrectomy, centrifuged, and the levels of cytokine and other mRNAs in the sediment were assessed using real-time PCR. The expression level of 11 cytokine genes, four transcription factor genes, two cytoskeletal genes, and genes encoding two extracellular matrix proteins in eyes with MH or iERM were determined and compared. RESULTS: The expression levels of interleukin 6 (IL6), tumor growth factor B2 (TGFB2), vascular endothelial growth factor A (VEGFA), chemokine C-X-C motif ligand 1 (CXCL1), v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), glial fibrillary acidic protein (GFAP), and tenascin C (TNC) were significantly higher in eyes with iERM than in eyes with MH. The expression of these genes was not associated with the preoperative visual acuity of the investigated patients. CONCLUSIONS: The obtained results indicate that real-time PCR analysis of irrigation solution samples collected during vitrectomy can help assess the expression levels of several genes, and that iERM is associated with the expression of pro-inflammatory genes and the genes expressed during angiogenesis and wound healing process (IL6, TGFB2, VEGFA, CXCL1, RELA, GFAP, and TNC).

Dietary Omega-3 Fatty Acids Suppress Experimental Autoimmune Uveitis in Association with Inhibition of Th1 and Th17 Cell Function.

Omega (ω)-3 long-chain polyunsaturated fatty acids (LCPUFAs) inhibit the production of inflammatory mediators and thereby contribute to the regulation of inflammation. Experimental autoimmune uveitis (EAU) is a well-established animal model of autoimmune retinal inflammation. To investigate the potential effects of dietary intake of ω-3 LCPUFAs on uveitis, we examined the anti-inflammatory properties of these molecules in comparison with ω-6 LCPUFAs in a mouse EAU model. C57BL/6 mice were fed a diet containing ω-3 LCPUFAs or ω-6 LCPUFAs for 2 weeks before as well as after the induction of EAU by subcutaneous injection of a fragment of human interphotoreceptor retinoid-binding protein emulsified with complete Freund's adjuvant. Both clinical and histological scores for uveitis were smaller for mice fed ω-3 LCPUFAs than for those fed ω-6 LCPUFAs. The concentrations of the T helper 1 (Th1) cytokine interferon-γ and the Th17 cytokine interleukin-17 in intraocular fluid as well as the production of these cytokines by lymph node cells were reduced for mice fed ω-3 LCPUFAs. Furthermore, the amounts of mRNAs for the Th1- and Th17-related transcription factors T-bet and RORγt, respectively, were reduced both in the retina and in lymph node cells of mice fed ω-3 LCPUFAs. Our results thus show that a diet enriched in ω-3 LCPUFAs suppressed uveitis in mice in association with inhibition of Th1 and Th17 cell function.

M2 Macrophages Enhance Pathological Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy.

PURPOSE: To investigate the roles played by M2 macrophages in a mouse model of oxygen-induced retinopathy (OIR). METHODS: Oxygen-induced retinopathy was induced in C57BL/6J mice by exposing postnatal day seven (P7) pups to 75% oxygen and then returning them to room air at P12. Real-time RT-PCR and immunofluorescence staining were used to assess the levels and distributions of different macrophage markers. Bone marrow-derived M1 and M2 macrophages and mannosylated clodronate liposomes (MCLs) were injected into the vitreous on P12 to examine the effects at P17. M2 macrophages were cocultured with human retinal endothelial cells (HRECs) to examine their effects on proliferation and tube formation. RESULTS: The results showed that the M2 macrophages, rather than M1 phenotype, were highly expressed in OIR mice. The number of M2 macrophages had increased significantly at P17, and the increase was closely associated with the presence of neovascular tufts in the OIR retinas. Selective depletion of M2 macrophages suppressed the pathological neovascularization and promoted physiological revascularization. In contrast, intravitreal injection of bone marrow-derived M2 macrophages or the culture supernatants promoted pathological neovascularization and inhibited physiological revascularization. In an in vitro coculture system, M2-polarized macrophages significantly promoted proliferation and tube formation of HRECs. CONCLUSIONS: These results indicated that M2 macrophages, rather than M1, play an important role in promoting retinal pathological neovascularization probably by producing secreted factors. Thus, targeting M2 macrophages could be a potential therapeutic option for inhibiting retinal pathological neovascularization.

[A case report of malignant lymphoma receiving infliximab therapy with Behçet's disease].

BACKGROUND: To report a case of malignant lymphoma occurring in Behçet's disease (BD) with infliximab therapy. CASE: A 62-year-old man was diagnosed with BD in 1997. Despite treatment with colchicine, cyclosporine and prednisolone, he had frequent bilateral posterior ocular attacks. He was started on infliximab in August 2007 and for 6 months had no ocular attacks. Cyclosporine was therefore reduced. After 4 years of infliximab administration, he had neither ocular attacks nor general symptoms. However, he had general malaise and weight loss from the end of March 2012. Peripheral blood examination showed abnormal cells, so we terminated the infliximab. Bone marrow aspiration showed diffuse proliferation of medium to large lymphoid cells, and the histological diagnosis was diffuse large B-cell lymphoma. He was treated with 8 cycles of chemotherapy and 4 times intrathecal chemotherapy, and is now in remission. After termination of infliximab, he had no further ocular attacks. CONCLUSION: Although malignant lymphoma associated with BD is rare, attending ophthalmologists need to keep it in mind.

EBI3 is pivotal for the initiation of experimental autoimmune uveitis.

Murine experimental autoimmune uveitis (EAU) is a model for human autoimmune uveitis, whose pathogenesis is caused by both Th1 and Th17 cell responses. Epstein-Barr virus-induced gene 3 (EBI3) is a component of the heterodimeric cytokines: interleukin (IL)-27 and IL-35. Although IL-27 was shown to initiate Th1 cell development, it is also recognized as a negative regulator of fully activated CD4+ T cells, including Th17 cells. Recently, IL-35 also has also been reported to play immunosuppressive roles in autoimmunity. To investigate the roles of EBI3 in EAU, EBI3(-/-) mice were immunized with human interphotoreceptor retinoid binding protein peptide 1-20 (IRBP) to induce EAU. We observed that the clinical score in EBI3(-/-) mice was diminished compared with that in EBI3(+/+) mice up to day 22 after immunization, whereas the score in EBI3(-/-) mice reached the same levels as that of EBI3(+/+) mice after day 28. Histological analysis revealed a significant reduction of cellular infiltration into the retina in EBI3(-/-) mice on day 16. Although Th1 cell responses and IRBP-specific IL-10 production were reduced in EBI3(-/-) mice, the development of Th17 cell responses was unaffected on day 9. On day 21, Th1 cell responses and IRBP-specific IL-10 production was restored to the same levels as that in EBI3(+/+) mice, and Th17 cell responses significantly increased in EBI3(-/-) mice. Furthermore, Foxp3 expression in CD4+ T cells was comparable between EBI3(+/+) and EBI3(-/-) mice on days 9 and 21. Therefore, these results indicate that EBI3 may be important in EAU initiation by Th1 cell responses and may suppress EAU by inhibition of both Th1 and Th17 cell responses in the late/maintenance phase.

[Pathophysiology and new treatment of uveitis].

Uveitis is narrow-defined inflammation of the uvea, also clinically include all inflammatory conditions in the eye. Uveitis may occur as a consequence of various causes and background, such as autoimmune diseases, infections, and hematopoietic malignancy. We have to treat uveitis not only controlling the inflammation but also maintaining up the visual function of the eye because the most uveitis is chronic and relapsing inflammatory disorder. Behçét's disease is a systemic disease and results in loss of vision without adequate treatment. Behçét's disease was a representative of vision loss uveitis because Behçét's patient usually had treatment resistance of conventional treatment, such as colchicine and cyclosporine. However, biological therapy with TNF-α, which started from 2007, has revolutionized the treatment strategy of Behçét's disease. It is not too much to say that Behçét's patient is free from fear of vision loss by the dramatic decrease of ocular attach. Biological therapy is not approved as a treatment of uveitis except Behçét's disease. Some protracted cases of Sarcoidosis and Vogt-Koyanagi-Harada disease are resistant to corticosteroid therapy and require new treatment. In this review, we discuss the pathophysiology of uveitis and report new treatment of Behçét's disease by biological therapy.

Melanocyte-secreted fibromodulin promotes an angiogenic microenvironment.

Studies have established that pigmentation can provide strong, protective effects against certain human diseases. For example, angiogenesis-dependent diseases such as wet age-related macular degeneration and infantile hemangioma are more common in light-skinned individuals of mixed European descent than in African-Americans. Here we found that melanocytes from light-skinned humans and albino mice secrete high levels of fibromodulin (FMOD), which we determined to be a potent angiogenic factor. FMOD treatment stimulated angiogenesis in numerous in vivo systems, including laser-induced choroidal neovascularization, growth factor-induced corneal neovascularization, wound healing, and Matrigel plug assays. Additionally, FMOD enhanced vascular sprouting during normal retinal development. Deletion of Fmod in albino mice resulted in a marked reduction in the amount of neovascularization induced by retinal vein occlusion, corneal growth factor pellets, and Matrigel plugs. Our data implicate the melanocyte-secreted factor FMOD as a key regulator of angiogenesis and suggest an underlying mechanism for epidemiological differences between light-skinned individuals of mixed European descent and African-Americans. Furthermore, inhibition of FMOD in humans has potential as a therapeutic strategy for treating angiogenesis-dependent diseases.

IL-10 is significantly involved in HSP70-regulation of experimental subretinal fibrosis.

Subretinal fibrosis is directly related to severe visual loss, especially if occurs in the macula, and is frequently observed in advanced age-related macular degeneration and other refractory eye disorders such as diabetic retinopathy and uveitis. In this study, we analyzed the immunosuppressive mechanism of subretinal fibrosis using the novel animal model recently demonstrated. Both TLR2 and TLR4 deficient mice showed significant enlargement of subretinal fibrotic area as compared with wild-type mice. A single intraocular administration of heat shock protein 70 (HSP70), which is an endogenous ligand for TLR2 and TLR4, inhibited subretinal fibrosis in wild-type mice but not in TLR2 and TLR4-deficient mice. Additionally, HSP70 induced IL-10 production in eyes from wild-type mice but was impaired in both TLR2- and TLR4-deficient mice, indicating that HSP70-TLR2/TLR4 axis plays an immunomodulatory role in subretinal fibrosis. Thus, these results suggest that HSP70-TLR2/TLR4 axis is a new therapeutic target for subretinal fibrosis due to prognostic CNV.

A Novel Platelet-Activating Factor Receptor Antagonist Inhibits Choroidal Neovascularization and Subretinal Fibrosis.

Choroidal neovascularization (CNV) is a critical pathogenesis in age-related macular degeneration (AMD), the most common cause of blindness in developed countries. To date, the precise molecular and cellular mechanisms underlying CNV have not been elucidated. Platelet-activating factor (PAF) has been previously implicated in angiogenesis; however, the roles of PAF and its receptor (PAF-R) in CNV have not been addressed. The present study reveals several important findings concerning the relationship of the PAF-R signaling with CNV. PAF-R was detected in a mouse model of laser-induced CNV and was upregulated during CNV development. Experimental CNV was suppressed by administering WEB2086, a novel PAF-R antagonist. WEB2086-dependent suppression of CNV occurred via the inhibition of macrophage infiltration and the expression of proangiogenic (vascular endothelial growth factor) and proinflammatory molecules (monocyte chemotactic protein-1 and IL-6) in the retinal pigment epithelium-choroid complex. Additionally, WEB2086-induced PAF-R blockage suppresses experimentally induced subretinal fibrosis, which resembles the fibrotic subretinal scarring observed in neovascular AMD. As optimal treatment modalities for neovascular AMD would target the multiple mechanisms of AMD-associated vision loss, including neovascularization, inflammation and fibrosis, our results suggest PAF-R as an attractive molecular target in the treatment of AMD.

Suppression of autoimmune retinal inflammation by an antiangiogenic drug.

Chronic and recurrent uveitis account for approximately 10% of legal blindness in the western world. Autoimmune uveitis is driven by activated CD4(+) T cells that differentiate into effector T helper cells (Th1, Th2, and Th17) which release proinflammatory cytokines that damage the retina. In this study we investigated the effect of the methionine aminopeptidase 2 (MetAP2) inhibitor, Lodamin, on T cell activation and differentiation. MetAp2 is an enzyme which regulates cellular protein synthesis and is highly expressed in T cells. Lodamin was found to suppress T cell receptor (TCR) mediated T cell proliferation and reduced the production of Th1 and Th17 cells. Further, Lodamin suppressed overall inflammation in the mouse model of experimental autoimmune uveitis (EAU) by a six fold. This effect was attributed in part to a reduction in retinal proinflammatory cytokines, down regulation of MetAP2 expression in purified lymph node CD4(+) T cells, and a general normalization of the systemic immune reaction.

IL-23-independent induction of IL-17 from γδT cells and innate lymphoid cells promotes experimental intraocular neovascularization.

Choroidal neovascularization (CNV) is a characteristic of age-related macular degeneration. Genome-wide association studies have provided evidence that the immune system is involved in the pathogenesis of age-related macular degeneration; however, the role of inflammatory cytokines in CNV has not been established. In this study, we demonstrated that IL-17 had a strong potential for promoting neovascularization in a vascular endothelial growth factor-independent manner in laser-induced experimental CNV in mice. Infiltrated γδT cells and Thy-1(+) innate lymphoid cells, but not Th17 cells, were the main sources of IL-17 in injured eyes. IL-23 was dispensable for IL-17 induction in the eye. Instead, we found that IL-1ß and high-mobility group box 1 strongly promoted IL-17 expression by γδT cells. Suppression of IL-1ß and high-mobility group box 1, as well as depletion of γδT cells, reduced IL-17 levels and ameliorated experimental CNV. Our findings suggest the existence of a novel inflammatory cytokine network that promotes neovascularization in the eye.

Involvement of periostin in regression of hyaloidvascular system during ocular development.

PURPOSE: A timely regression of the hyaloid vascular system (HVS) is required for the normal ocular development. Although macrophages have a critical role in this process, the exact mechanism remains undetermined. Periostin is a matricellular protein involved in tissue and vascular remodeling. The purpose of our study was to determine whether periostin is involved in the HVS regression. METHODS: We used wild type (WT) and periostin knockout (KO) mice. Indocyanine green angiography and immunohistochemistry with isolectin B4 were used to evaluate the HVS regression. TUNEL-labeling was used to quantify the number of apoptotic hyaloid vascular endothelial cells. F4/80 and Iba-1 staining was performed to determine the number and location of macrophages in the vitreous. The location of periostin also was investigated by immunohistochemistry. To determine the functional role of periostin, the degree of adhesion of human monocytes to fibronectin was measured by an adhesion assay. RESULTS: The HVS regression and peak in the number of TUNEL-positive apoptotic endothelial cells were delayed in periostin KO mice. The number of F4/80 positive cells in the vitreous was higher in periostin KO mice. Only a small number of Iba-1-positive cells near the hyaloid vessels was co-stained with periostin, and peripheral blood monocytes were not stained with periostin. Adhesion assay showed that periostin increased the degree of attachment of monocytes to fibronectin. CONCLUSIONS: These results suggest that periostin, which is secreted by the intraocular macrophages, enhances the HVS regression by intensifying the adhesion of macrophages to hyaloid vessels.

Simultaneous analysis of multiple cytokines in the vitreous of patients with sarcoid uveitis.

PURPOSE: Levels of some cytokines are significantly higher in the vitreous fluid of patients with acute uveitis than in normal vitreous fluid. The authors sought to determine which proinflammatory cytokines were upregulated in the vitreous fluid of patients with ocular sarcoidosis. METHODS: Samples of vitreous fluid were collected from patients with sarcoid uveitis and from nonsarcoid control patients with idiopathic epiretinal membrane. The levels of 27 proinflammatory cytokines were measured with a multiplex beads array system. Postvitrectomy macular thickness was also measured by using spectral domain optical coherence tomography (SD-OCT). To assess the relationship between cytokine levels and disease stage, the authors divided patients into three groups based on macular thickness 1 month after operation. RESULTS: The vitreous levels of 17 cytokines were significantly higher in patients with ocular sarcoidosis than in nonsarcoid controls. Serum levels of interferon γ-induced protein 10 (IP-10) were also higher in ocular sarcoidosis patients than in nonsarcoid controls. Conversely, serum levels of interleukin (IL) 15 in ocular sarcoidosis patients were lower than in the control group. Analysis of cytokine levels and macular thickness revealed that IL-1ra, IL-4, IL-8, IFN-γ, IP-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1ß, and regulated on activation, normal T-cell expressed and secreted (RANTES) were significantly upregulated in patients with thin cystoid macular edema group. CONCLUSIONS: Patients with ocular sarcoidosis had elevated levels of proinflammatory cytokines in vitreous fluids. Different cytokines might contribute to different stages of macular edema.

Aminoimidazole carboxamide ribonucleotide ameliorates experimental autoimmune uveitis.

PURPOSE: To investigate the anti-inflammatory effect of an adenosine monophosphate (AMP) analog, aminoimidazole carboxamide ribonucleotide (AICAR), in experimental autoimmune uveoretinitis (EAU). METHODS: C57BL/6 mice were injected daily with AICAR (200 mg/kg, intraperitoneally [IP]) from day 0, the day of interphotoreceptor retinoid-binding protein (IRBP) immunization, until day 21. The severity of uveitis was assessed clinically and histopathologically. T-cell proliferation and cytokine production of IFN-γ, IL-17, and IL-10 in response to IRBP stimulation were determined. In addition, regulatory T-cell (Treg) populations were measured. Co-stimulatory molecule expression (CD40, 80, 86, and I-Ab) on dendritic cells (DCs) in EAU and on bone marrow-derived dendritic cells (BMDCs) treated with AICAR was measured. RESULTS: AICAR treatment significantly reduced clinical and histologic severity of EAU as well as ocular cytokine production. An anti-inflammatory effect associated with the inhibition of T-cell proliferation and Th1 and Th17 cytokine production was observed. Increases in the Th2 response and Treg population were not observed with AICAR treatment. AICAR did significantly inhibit BMDC maturation by reducing co-stimulatory molecule expression. CONCLUSIONS: AICAR attenuates EAU by preventing generation of Ag-specific Th1 and Th17 cells. Impaired DC maturation may be an underlying mechanism for this anti-inflammatory effect observed with AICAR.

Chemokines in aqueous humour before and after intravitreal triamcinolone acetonide in eyes with macular oedema associated with branch retinal vein occlusion.

PURPOSE: To determine the aqueous humour levels of chemokines before and after an intravitreal injection of triamcinolone acetonide (IVTA) in eyes with macular oedema associated with a branch retinal vein occlusion (ME-BRVO). DESIGN: Single-centre, prospective, consecutive interventional case series. PARTICIPANTS: Seventeen eyes of 17 consecutive patients with ME-BRVO who underwent IVTA were studied. Seven eyes without retinal vascular disease served as control. INTERVENTION: All patients with ME-BRVO underwent IVTA. MAIN OUTCOME MEASURES: The optical coherence tomographically determined foveal thickness (FT) and the aqueous humour levels of inflammatory chemokines of the C-C subfamily, including eotaxin, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), ß (MIP-1ß), and RANTES was determined before the IVTA (baseline) and at 1 week after the IVTA. RESULTS: At the baseline, only MCP-1 and MIP-1ß were detected in the aqueous, and MIP-1ß was significantly higher in eyes with a ME-BRVO than in controls (p = 0.004). The level of both of these chemokines was not correlated with the FT (p = 0.654 and p = 0.608, respectively). One week after IVTA, the FT was significantly decreased (p < 0.001), and the levels of MCP-1 and MIP-1ß were also significantly reduced (p < 0.001 and p = 0.044, respectively). The decrease in the FT was correlated with the decrease in only MIP-1ß (r = 0.58, p = 0.020). CONCLUSIONS: Alterations of the aqueous level of MIP-1ß reflect the improvement of the macular oedema after IVTA in eyes with ME-BRVO. This indicates that the steroid-dependent ME-BRVO was closely related with the level of MIP-1ß.

Neutrophil-dominant experimental autoimmune uveitis in CC-chemokine receptor 2 knockout mice.

PURPOSE: Murine experimental autoimmune uveitis (EAU) is an animal model of human uveitis. It has been demonstrated that ocular-infiltrating macrophages are crucial for EAU induction, and monocyte chemoattractant protein-1 (MCP-1) was actually upregulated in the eye. CC chemokine receptor-2 (CCR2) is the receptor of MCP-1, and macrophages fail to recruit particular lesions in CCR2 knockout (KO) mice. To confirm the role of macrophages in EAU, we examined EAU in CCR2 KO mice. METHODS: CCR2 KO mice and wild-type (WT) mice that had the same genetic background were immunized with human interphotoreceptor retinoid-binding protein peptide 1-20 emulsified in complete Freund's adjuvant. At multiple time-points, EAU severity was evaluated based on microscopic fundus observation and histological examination. To examine the phenotype of retinal-infiltrating cells, single cells were prepared from the eye and analysed by flow cytometry. RESULTS: In WT mice, EAU was induced at the peak of day 16 and marked macrophage infiltration was observed. Although macrophages failed to be recruited into the eye in CCR2 KO mice, severe uveitis was induced unexpectedly. Flow cytometry and histology revealed that most of the infiltrating cells were neutrophils. We also compared the intraocular chemokine concentrations between WT mice and KO mice. Two CXC chemokine (monokine induced by interferon-γ and interferon-γ-inducible protein-10) were upregulated in KO mice. CONCLUSION: Interphotoreceptor retinoid-binding protein peptide immunization caused neutrophil-dominant uveitis in CCR2 KO mice. In the absence of macrophages, neutrophils can be alternatively recruited and can cause tissue damage.

Broad spectrum antiangiogenic treatment for ocular neovascular diseases.

UNLABELLED: Pathological neovascularization is a hallmark of late stage neovascular (wet) age-related macular degeneration (AMD) and the leading cause of blindness in people over the age of 50 in the western world. The treatments focus on suppression of choroidal neovascularization (CNV), while current approved therapies are limited to inhibiting vascular endothelial growth factor (VEGF) exclusively. However, this treatment does not address the underlying cause of AMD, and the loss of VEGF's neuroprotective can be a potential side effect. Therapy which targets the key processes in AMD, the pathological neovascularization, vessel leakage and inflammation could bring a major shift in the approach to disease treatment and prevention. In this study we have demonstrated the efficacy of such broad spectrum antiangiogenic therapy on mouse model of AMD. METHODS AND FINDINGS: Lodamin, a polymeric formulation of TNP-470, is a potent broad-spectrum antiangiogenic drug. Lodamin significantly reduced key processes involved in AMD progression as demonstrated in mice and rats. Its suppressive effects on angiogenesis, vascular leakage and inflammation were studied in a wide array of assays including; a Matrigel, delayed-type hypersensitivity (DTH), Miles assay, laser-induced CNV and corneal micropocket assay. Lodamin significantly suppressed the secretion of various pro-inflammatory cytokines in the CNV lesion including monocyte chemotactic protein-1 (MCP-1/Ccl2). Importantly, Lodamin was found to regress established CNV lesions, unlike soluble fms-like tyrosine kinase-1 (sFlk-1). The drug was found to be safe in mice and have little toxicity as demonstrated by electroretinography (ERG) assessing retinal and by histology. CONCLUSIONS: Lodamin, a polymer formulation of TNP-470, was identified as a first in its class, broad-spectrum antiangiogenic drug that can be administered orally or locally to treat corneal and retinal neovascularization. Several unique properties make Lodamin especially beneficial for ophthalmic use. Our results support the concept that broad spectrum antiangiogenic drugs are promising agents for AMD treatment and prevention.