SIX-YEAR OUTCOMES OF INTRAVITREAL BEVACIZUMAB FOR CHOROIDAL NEOVASCULARIZATION IN PATIENTS WITH PATHOLOGIC MYOPIA.

PURPOSE: To investigate the 6-year outcome of intravitreal bevacizumab (IVB) to treat eyes with active choroidal neovascularization (CNV) due to pathologic myopia. METHODS: Medical records of 36 eyes of 35 consecutive patients with high myopia (refractive error ≥8 D or axial length ≥26.5 mm) and active CNV, who had been treated with IVB and followed for ≥6 years were analyzed. The factors that predicted the best-corrected visual acuity (BCVA) at 6 years after IVB were determined by multiple regression analyses. RESULTS: The mean age of the subjects was 58 years, and the mean axial length was 29 mm. Twenty-one eyes had subfoveal CNV and 15 eyes had nonsubfoveal CNV. During the 6-year follow-up, the mean number of IVB was 1.78. The mean BCVA logMAR (equivalent Snellen visual acuity) was 0.50 (20/63), 0.31 (20/40), 0.39 (20/50), and 0.45 (20/63) at the baseline, and at 2, 4, and 6 years after the IVB. The BCVA was significantly improved at 2 and 4 years compared with baseline values but not at 6 years. Stepwise multiple regression analyses showed that the BVCA at 6 years was significantly correlated with the size of the CNV-related macular atrophy, and the baseline BCVA and CNV size. CONCLUSION: The significant correlation between the BCVA at 6 years and the size of the macular atrophy indicates that treatments to prevent the development of macular atrophy are important for the long-term visual outcome in eyes with active CNV.

Polypoidal choroidal vasculopathy in a case with retinitis pigmentosa.

There have been no reports describing polypoidal choroidal vasculopathy (PCV) in eyes with retinitis pigmentosa (RP). A 63-year-old woman who had been diagnosed as having RP was referred to us because of sudden onset of blurred vision in her right eye. Funduscopic examination revealed retinal findings typical of RP in both eyes. The macular area of the right fundus showed polypoidal lesions with massive hemorrhages. Fluorescein angiography and indocyanine green angiography showed multiple polypoidal lesions. Optical coherence tomography showed a large hemorrhagic retinal pigment epithelial (RPE) detachment and polypoidal lesions. The PCV subsided after three applications of anti-vascular endothelial growth factor (VEGF) therapy and a single application of photodynamic therapy, but "mottled lesions" with hyper- and hypofluorescence appeared temporal to the macula after disappearance of hemorrhage. We present a case of PCV in an eye with RP. Further studies are necessary to clarify whether anti-VEGF therapies could affect RPE status in eyes with RP.

Identification of genes and proteins specifically regulated by costimulation of mast cell Fcε Receptor I and chemokine receptor 1.

Mast cell function is a critical component of allergic reactions. Mast cell responses mediated by the high-affinity immunoglobulin E receptor FcεRI can be enhanced by co-activation of additional receptors such as CC chemokine receptor 1 (CCR1). To examine the downstream effects of FcεRI-CCR1 costimulation, rat basophilic leukemia cells stably transfected with CCR1 (RBL-CCR1 cells) were sensitized and activated with antigen and/or the CCR1 ligand CC chemokine ligand (CCL) 3. Gene and protein expression were determined at 3h and 24h post-activation, respectively, using GeneChip and Luminex bead assays. Gene microarray analysis demonstrated that 32 genes were differentially regulated in response to costimulation, as opposed to stimulation with antigen or CCL3 alone. The genes most significantly up-regulated by FcεRI-CCR1 costimulation were Ccl7, Rgs1, Emp1 and RT1-S3. CCL7 protein was also expressed at higher levels 24h after dual receptor activation, although RGS1, EMP1 and RT1-S3 were not. Of the panel of chemokines and cytokines tested, only CCL2, CCL7 and interleukin (IL)-6 were expressed at higher levels following costimulation. IL-6 expression was seen only after FcεRI-CCR1 costimulation, although the amount expressed was very low. CCL7, CCL2 and IL-6 might play roles in mast cell regulation of late-phase allergic responses.

The role of TRB3 in mast cells sensitized with monomeric IgE.

Mast cells play a key role in immunoglobulin E (IgE)-associated allergic disorders; however, the cellular effects of sensitization remain poorly understood. Using gene microarrays and the multiplexing ELISA method, we examined the effects of sensitization on RBL-CCR1 cell transcription and chemokine/cytokine secretion. Sensitization most prominently increased transcription of Trb3, the gene for tribbles homolog 3 (TRB3), and also increased the release of most of the cytokines and chemokines tested. Knockdown of TRB3 via RNAi significantly induced the production of tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6), and the chemokine mast cell protease-1 (MCP-1). TRB3 deficiency also induced IκBα phosphorylation. TRB3 may therefore serve as a negative regulator of pro-inflammatory cytokines and chemokines, controlling the extent of the inflammatory response.

Identification of anti-retinal antibodies in patients with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrial counties. Recent findings indicate that the autoimmunity is involved in the pathogenesis of the disease. However, there is no autoantibody biomarker applied in a clinical setting for diagnosis and prognosis of AMD. In order to reveal retinal antigens targeted by serum IgG from AMD patients, mouse retinal tissue proteins were separated by 2-dimensional electrophoresis and the proteins in the immunoblots that were specific for dry and wet AMD patients IgG were identified by LC-MS/MS. Retinol-binding protein 3 and aldolase C (ALDOC) were mainly recognized by IgG form wet AMD patients. Pyruvate kinase M2 (PKM2) was targeted by both dry and wet AMD and level of anti-PKM2 IgG antibody was correlated best with the stage of AMD. Expression of ALDOC and PKM2 was decreased in mouse retina from aging whereas PKM2 deposit on RPE was increased in aged mice. Our data demonstrate that sera of AMD patients contain autoantibodies against retinal proteins and anti-PKM2 IgG serves as a biomarker for diagnosis and prognosis of AMD. Further investigation of the association of anti-retinal antibody level with expression level of antigens in retina will be needed to reveal the disease pathogenesis.

Serum autoantibody biomarkers for age-related macular degeneration and possible regulators of neovascularization.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrial counties. Its pathogenesis is at least partially mediated by immunological factors, including a possible autoimmune response. To date, only a few antibodies have been identified in sera from patients with AMD. In order to reveal an autoantibody profile for AMD and identify biomarkers for progression of this disease, we have performed an antigen microarray analysis of serum samples from patients with AMD and healthy controls. Sera from the AMD groups contained high levels of IgG and IgM autoantibodies to some systemic antigens when compared to the normal group. Targeted antigens included cyclic nucleotide phosphodiesterase, phosphatidylserine (PS) and proliferating cell nuclear antigen. The IgG/IgM ratio for antibodies to PS was notably elevated in the AMD group compared to the normal group, and this ratio correlated best with the stage of AMD patients with an anti-PS ratio greater than the cut-off value had a 44-fold risk for advanced AMD with choroidal neovascularization. PS immunoreactivity was also elevated in AMD retina. Moreover, IgG autoantibodies purified from sera of AMD patients induced more tube formation on choroidal-retinal endothelial cells compared to those of healthy donors. Hence, sera from patients with AMD contain specific autoantibodies which may be used as biomarkers for AMD, and the IgG/M ratio for autoantibodies to PS might allow better monitoring of AMD progression.

Modeling gas permeation through membranes by kinetic Monte Carlo: applications to H2, O2, and N2 in hydrated Nafion®.

We present a simulation tool in order to predict gas permeation through heterogeneous, microphase separated structures. The method combines dissipative particle dynamics (DPD) with kinetic Monte Carlo (KMC). Morphologies obtained from DPD are mapped onto a high density grid on which gas diffusion takes place. Required input parameters for the KMC calculations are the gas solubility and gas diffusion constant within each of the pure phase components. Our method was tested and validated for permeation of H(2), O(2), and N(2) gasses through hydrated Nafion membranes at various temperatures and water contents. We predict that membranes that contain an equal volume fraction of water, those with the highest ion exchange capacity exhibit the largest N(2) and O(2) permeation rates. For membranes of the same ion exchange capacity the H(2), O(2), and N(2) and permeability increases approximately linearly with Bragg spacing. We also predict that O(2) gas permeation depends much more on bottleneck phenomena within the phase separated morphologies than H(2) gas permeation. Overall, the calculated H(2) and O(2) permeability is found to be slightly lower than experimental values. This is attributed to the robustness of DPD resulting in ∼7% larger Bragg spacing as compared with experiment and∕or increased gas solubility within the polymer phase with water uptake.

The role of histamine in ocular allergy.

Ocular allergy is a disorder affecting increasing numbers of individuals worldwide. Among the inflammatory mediators that contribute to ocular allergy, histamine is perhaps the best characterized. This monoamine is released by sensitized mast cells upon exposure to allergen and causes symptoms such as redness and tearing. Histamine may also recruit immune cells that can cause long-term damage to ocular surfaces. In this chapter we will discuss the known functions of histamine and histamine receptors in ocular allergy and will describe promising therapies targeting the histamine-signaling pathway.

Critical role of IgE-dependent mast cell activation in a murine model of allergic conjunctivitis.

BACKGROUND: Allergic conjunctivitis is characterized by allergen-specific IgE in the serum and infiltration of eosinophils into the conjunctiva. The role of IgE and mast cells in allergic conjunctivitis is largely unknown, however. OBJECTIVES: We investigated the importance of conjunctival mast cells in a murine model of IgE-mediated allergic conjunctivitis. METHODS: IgE-mediated allergic conjunctivitis was initiated in C57BL/6-Kit(+/+) wild-type mice, mast cell-deficient Kit(W-sh/W-sh) mice, and Kit(W-sh/W-sh) mice that had been subconjunctivally or systemically engrafted with bone marrow-derived, cultured mast cells (BMCMCs) from Kit(+/+) wild-type mice, and clinical symptoms and infiltration of eosinophil of the eyes were evaluated. Total numbers of mast cells in the conjunctiva were counted, and the phenotypes of these cells were characterized by means of immunostaining and PCR analysis of murine mast cell proteases. RESULTS: No mast cells were detected in the conjunctiva or eyelid dermis of adult Kit(W-sh/W-sh) mice. Subconjunctival injection of BMCMCs resulted in local mast cell reconstitution, with the numbers of reconstituted mast cells in the conjunctiva and eyelid dermis comparable with those observed in wild-type Kit(+/+) littermates. Reconstituted and naive conjunctival mast cells expressed proteases ascribed to connective tissue-type mast cells but not mucosal mast cells. Passive transfer of ragweed-specific IgE followed by antigen challenge resulted in both early-phase clinical symptoms and late-phase eosinophilic inflammation in Kit(+/+) mice. These responses, which were significantly decreased in Kit(W-sh/W-sh) mice, were restored on reconstitution of the conjunctival mast cell population. CONCLUSIONS: These results suggest a direct contribution of IgE-activated mast cells to both the early-phase reaction and late-phase inflammation during ocular allergy.

Autoimmunity in retinal degeneration: autoimmune retinopathy and age-related macular degeneration.

Autoantibody production is associated with a variety of ocular disorders, including autoimmune retinopathy (AIR) and age-related macular degeneration (AMD). A breakdown of immunologic tolerance (ocular immune privilege), including the blood-retinal barrier, anti-immune and anti-inflammatory proteins, and anterior chamber-associated immune deviation may play important roles in these disorders. Although the exact triggers for ocular autoimmunity are unknown, autoimmune targeting of retinal tissue is clearly associated with and may contribute to the pathogenesis of both AIR and AMD. Autoantibody production has long been associated with AIR, a collection of disorders that includes cancer-associated retinopathy, melanoma-associated retinopathy and non-paraneoplastic autoimmune retinopathy. A growing body of evidence indicates that AMD pathogenesis, too, involves ocular inflammation and autoimmunity. Identification and quantification of autoantibodies produced in patients with AIR and AMD may assist with diagnosis, prognosis, and choice of treatments. Animal models that allow investigation of ocular autoimmunity will also be needed to better understand the disease processes and to develop novel therapies. In this review we discuss ocular immune privilege and potential mechanisms of autoimmunity in the eye. We describe how autoimmunity relates to the pathogenesis of AIR and AMD. We explain how the antigen microarray technique is used to detect autoantibodies in patient serum samples, and discuss how current animal models for AMD can be used to investigate autoimmune pathogenesis. Finally, we outline unanswered questions and exciting areas of future study related to autoimmune retinal degeneration.

Multiscale prediction of functional self-assembled materials using machine learning: high-performance surfactant molecules.

Various physical properties of functional materials can be induced by controlling their chemical molecular structures. Therefore, molecular design is crucial in the fields of engineering and materials science. With its remarkable development in various fields, machine learning combined with molecular simulation has recently been found to be effective at predicting the electronic structure of materials (Nat. Commun., 2017, 8, 872 and Nat. Commun., 2017, 8, 13890). However, previous studies have used similar microscale information as input and output data for machine learning, i.e., molecular structures and electronic structures. In this study, we determined whether multiscale data can be predicted using machine learning via a self-assembly functional material system. In particular, we investigated whether machine learning can be used to predict dispersion and viscosity, as the representative physical properties of a self-assembled surfactant solution, from the chemical molecular structures of a surfactant. The results showed that relatively accurate information on these physical properties can be predicted from the molecular structure, suggesting that machine learning can be used to predict multiscale systems, such as surfactant molecules, self-assembled micelle structures, and physical properties of solutions. The results of this study will aid in further development of the application of machine learning to materials science and molecular design.

5'-end SAGE for the analysis of transcriptional start sites.

Identification of the mRNA start site is essential in establishing the full-length cDNA sequence of a gene and analyzing its promoter region, which regulates gene expression. Here we describe the development of a 5'-end serial analysis of gene expression (5' SAGE) that can be used to globally identify transcriptional start sites and the frequency of individual mRNAs. Of the 25,684 5' SAGE tags in the HEK293 human cell library, 19,893 matched to the human genome. Among 15,448 tags in one locus of the genome, 85.8%-96.1% of the 5' SAGE tags were assigned within -500 to +200 nt of mRNA start sites using the RefSeq, UniGene and DBTSS databases. This technique should facilitate 5'-end transcriptome analysis in a variety of cells and tissues.

Optical Coherence Tomographic Imaging of Posterior Episclera and Tenon's Capsule.

Purpose: To investigate structural features of the posterior episclera and Tenon's capsule in patients with high myopia. Methods: This hospital-based observational study included highly myopic eyes (myopic refractive error > -8 diopters or axial length ≥26.5 mm) in which the posterior sclera in its full thickness could be visualized on swept-source optical coherence tomography (OCT) images in all 12 radial scans centered on the fovea. We assessed the posterior episclera and Tenon's capsule. Results: The study included 278 eyes of 175 patients (mean age, 60.9 ± 11.4 years; range, 32-89 years; axial length, 30.7 ± 1.9 mm; range, 26.5-36.6 mm). The episclera was detected outside of the sclera in 164 eyes (59.0%) and appeared as a relatively uniform structure with a reflectivity slightly lower than scleral reflectivity. In these eyes, mean scleral thickness was 197 ± 73 µm in the foveal region and 164 ± 64 µm and 146 ± 59 µm at 1000 and 2500 µm temporal to the fovea, respectively. The posterior episclera was visualized in the region temporal to the fovea. Mean episcleral thickness detected in 77 eyes was 80 ± 27 µm and 82 ± 30 µm at 1000 µm and 2500 µm temporal to the fovea, respectively. Tenon's capsule was detected in 11 of the 278 eyes (4.0%) as structurally, loosely connected tissue with a meshwork-like appearance. The measured thickness in four eyes was 60 ± 32 µm. In 25 eyes with extremely thin sclera (<100 µm at 1000 µm temporal to the foveola), the mean thickness of the sclera and episclera were 87 ± 11 µm and 65 ± 15 µm, respectively. Conclusions: Swept-source OCT applied to a subset of highly myopic eyes with significant thinning of the retina and choroid allowed visualization of the posterior sclera and episclera, and in some cases, also Tenon's capsule.

Swept-source optical coherence tomographic findings in eyes with metastatic choroidal tumor.

PURPOSE: To report the swept-source optical coherence tomographic (OCT) findings in two eyes with choroidal metastases. OBSERVATIONS: Two patients with choroidal metastasis were studied. The metastasis was from a breast cancer in Case 1 and from a lung cancer in Case 2. In Case 1, swept-source OCT showed a highly reflective solid tumor with low optical reflective tissues that had replaced the choroidal tissue. Swept-source OCT was able to image the choroidal mass where other fundus imaging methods such as fluorescein angiography did not show the mass. Ophthalmoscopy of Case 2 showed hemorrhages in the inner retina, on the tumor, and in the vitreous. Swept-source OCT showed a subretinal mass with a steeple-crowned cap and a ruptured Bruch's membrane on the tumor. CONCLUSION AND IMPORTANCE: Swept-source OCT imaging can detect the inner structure of a choroidal mass and retina around it in good detail.

Glaucomatous-Type Optic Discs in High Myopia.

PURPOSE: To assess the prevalence of glaucoma in patients with high myopia defined as myopic refractive error of >-8 diopters or axial length ≥26.5 mm. METHODS: The hospital-based observational study included 172 patients (336 eyes) with a mean age of 61.9±12.3 years and mean axial length of 30.1±2.3 mm (range: 24.7-39.1mm). Glaucomatous-type optic discs were defined by glaucomatous optic disc appearance. Glaucoma was defined by glaucomatous optic disc appearance and glaucomatous Goldmann visual field defects not corresponding with myopic macular changes. RESULTS: Larger disc area (mean: 3.18±1.94 mm2) was associated with longer axial length (P<0.001; standardized correlation coefficient: 0.45). Glaucoma was detected in 94 (28%; 95% Confidence intervals: 23%, 33%) eyes. In multivariate analysis, glaucoma prevalence was 3.2 times higher (P<0.001) in megalodiscs (>3.79 mm2) than in normal-sized discs or small discs (<1.51 mm2) after adjusting for older age. Axial length was not significantly (P = 0.38) associated with glaucoma prevalence in that model. Glaucoma prevalence increased by a factor of 1.39 for each increase in optic disc area by one mm2. Again, axial length was not significantly (P = 0.38) associated with glaucoma prevalence when added to this multivariate model. CONCLUSION: Within highly myopic individuals, glaucoma prevalence increased with larger optic disc size beyond a disc area of 3.8 mm2. Highly myopic megalodiscs as compared to normal sized discs or small discs had a 3.2 times higher risk for glaucomatous optic nerve neuropathy. The increased glaucoma prevalence in axial high myopia was primarily associated with axial myopia associated disc enlargement and not with axial elongation itself.

Chorioretinal folds in eyes with myopic staphyloma.

PURPOSE: To determine the frequency and clinical characteristics of chorioretinal folds emanating from the edge of a staphyloma in highly myopic patients. DESIGN: Observational case series. METHODS: Eight hundred and eighty-three eyes of 463 patients with an axial length ≥26.5 mm in at least 1 eye were studied. The fellow eyes of patients with unilateral high myopia were also included. Wide-field fundus images and fundus autofluorescence images were used to detect chorioretinal folds emanating from the staphyloma edge. In 100 patients, the eye shape was analyzed by 3-dimensional magnetic resonance imaging (3D MRI). RESULTS: A posterior staphyloma was found in 459 of the 883 eyes (52.0%). Choroidal folds radiating from the staphyloma edge were found in 6 of the 459 eyes (1.3%) with a posterior staphyloma in wide-field autofluorescent images. The axial length varied greatly from 24.3 mm to 32.5 mm. Regardless of the axial length, all of the 6 eyes had a wide, macular type of staphyloma. Chorioretinal folds emanated from the upper or upper-temporal staphyloma edge. 3D MRI images showed the presence of a notch along the upper or temporal edge of the outpouching, and the eye curvature became flatter toward the steep edge of the outpouching. CONCLUSIONS: Chorioretinal folds can emanate from the staphyloma edge in highly myopic patients even though the edge was away from the macula. Some directional force toward the steeper edge of the staphyloma might be related to the development of chorioretinal folds.

Serum inflammatory markers after rupture retinal laser injury in mice.

BACKGROUND AND OBJECTIVE: To characterize the cellular, immunological, and inflammatory response to retinal photocoagulation of intense rupture laser lesions as a model of retinal degenerative diseases. MATERIALS AND METHODS: Seven C57BL/6 mice were irradiated using a 532-nm laser to induce 10 retinal burns per eye that ruptured Bruch's membrane. Blood was drawn from the saphenous vein before and 2 months after laser treatment. The serum was run on antigen microarrays with 85 molecular markers associated with retinal degenerative diseases. RESULTS: Rupture laser resulted in dramatic changes in the immunoglobulin reactivity of most inflammatory markers 2 months after laser injury. Approximately two-thirds increased expression and one-third decreased expression. Notable markers that were increased included complement C3, CRP, PKM2, and aldolase. CONCLUSION: Rupture laser injury causes a change in the serum inflammatory markers after 2 months similar to macular degeneration, diabetic retinopathy, and cancer-associated retinopathy. This animal model could be used as a biomarker for disease stage and activity in retinal degenerations.

Lipopolysaccharide-inducible Gene Expression Profile in Human Monocytes.

Monocytes/macrophages play a key role in host defense by phagocytosing invaded pathogens, presenting antigens to immune cells and producing numerous inflammatory mediators. Although the expression of many proteins and genes has been described to be up-regulated in activated human monocytes, a complete picture of the pathophysiological function of activated human monocytes has not yet been drawn. In this study the serial analysis of gene expression (SAGE) procedure was applied to lipopolysaccharide (LPS)-stimulated human monocytes. A total of 35,874 tags corresponding to more than 12,000 different transcripts was sequenced. In addition, the Long SAGE procedure was conducted in LPS-stimulated monocytes to increase the accuracy of corresponding gene identification. Comparison of the gene expression profile with that of resting monocytes revealed the whole LPS-inducible gene expression profile. The functional classifications of LPS-inducible genes (≥8-fold increase compared with resting monocytes) in monocytes showed that 25% of inducible genes were identified to encode cytokines and chemokines, followed by proteins related to metabolism (11%), cell surface antigens (9%), nuclear proteins (8%), proteases (6%), proteins related to extracellular transport (4%) and intracellular transducers (4%). Moreover, 14% of LPS-inducible genes still encode proteins with unknown function. This study represents the first global analysis of LPS-inducible genes in human monocytes and provides tremendous novel information for the function of LPS-activated monocytes and targets for diagnosing, monitoring and treating sepsis and various human infectious and inflammatory diseases.

The murine CCR3 receptor regulates both eosinophilia and hyperresponsiveness in IgE-mediated allergic conjunctivitis.

BACKGROUND/AIMS: Allergic conjunctivitis is characterised by early-phase clinical symptoms and late-phase inflammation in the conjunctiva. The role of different chemokine receptors in allergic conjunctivitis, especially during the early-phase reaction, is still unclear. We investigated the importance of CC chemokine receptor (CCR) 3 in a murine model of IgE-mediated allergic conjunctivitis using CCR3-deficient (CCR3(-/-)) mice. METHODS: Allergic conjunctivitis was initiated in wild-type (WT) and CCR3(-/-) mice by passive transfer of ragweed (RW)-specific IgE, followed by topical challenge with RW in eye drops. Early-phase reactions including clinical symptoms and vascular leakage, as well as late-phase eosinophil infiltration of the conjunctiva were evaluated. The expression of mRNAs in the conjunctiva was quantified by real-time PCR analysis. RESULTS: The number of infiltrated eosinophils in the conjunctiva following RW challenge, was significantly higher in RW-IgE-sensitised WT mice compared with those sensitised with phosphate-buffered saline for WT, but this was not observed in similarly treated CCR3(-/-) mice. The early-phase clinical symptoms and vascular leakage were also suppressed in CCR3(-/-) mice. The number of conjunctival mast cells were not different between CCR3(-/-) mice and WT mice, and the mRNAs for FcεRІα and the connective tissue-type mast cell proteases were detected in the conjunctiva of both WT and CCR3(-/-) mice. RW-IgE-sensitised CCR3(-/-) mice displayed significantly reduced expression of CCL2, CCL3, and IL-6 compared with WT mice. CONCLUSIONS: These results demonstrate a direct contribution of CCR3 to both the early-phase reaction and late-phase inflammation during ocular allergy.