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Purpose : To evaluate the effect of loss to follow up (LTFU) on outcomes in eyes with diabetic macular edema (DME) treated with anti-vascular endothelial growth factor (VEGF). Design : Retrospective cohort study Methods : Single-center study of 90 eyes of 73 patients with non-proliferative diabetic retinopathy (NPDR) and DME treated with anti-VEGF injections who were LTFU for >6 months. Main outcomes were the change in mean visual acuity (VA) and central foveal thickness (CFT) at the return and final visits compared to the visit before LTFU. Results : Mean age was 64.5 years, mean LTFU duration was 322 days, and mean follow-up duration after return was 502 days. Compared to the mean VA at the visit before LTFU (0.42, Snellen ∼20/52), mean VA worsened at the return visit (0.54, Snellen ∼20/69, p=0.004). No significant change in the mean VA was noted at the 3-month after return visit (0.50, Snellen ∼20/63), the 6-month after return visit (0.46, Snellen ∼20/57), the 12-month after return visit (0.42, Snellen ∼20/52) and the final follow up (0.47, Snellen ∼20/59). When analyzed by NPDR severity before LTFU, no difference in VA was found from the visit before LTFU to the final visit. Mean CFT increased when comparing the visit before LTFU (270 μm) to the return visit (305 μm, p=0.012), but no difference was found by the final visit [247 μm, p=0.07]. Conclusions : Anti-VEGF treated DME patients who were LTFU for a prolonged period experienced a modest decline in VA that recovered after restarting treatment.

A 16-year-old girl noted worsening redness and photophobia of the right eye that had previously been treated unsuccessfully with sequential courses of topical antibiotics and topical corticosteroids. Clinical examination revealed diffuse flakelike thickening of the iris surface, pupillary margin, and anterior chamber angle, suggesting diffuse iris melanoma. Anterior segment optical coherence tomography depicted the mass as an epi-iridic deposit with "stalagmite" surface appearance. Fine-needle aspiration biopsy confirmed an atypical histiocytic proliferative disorder consistent with juvenile xanthogranuloma. Aggressive topical corticosteroid treatment was started. There were no systemic findings. Following therapy, the lesion resolved completely.

BACKGROUND AND OBJECTIVE: Optical coherence tomography angiography (OCTA) is a novel, non-invasive OCT technique capable of imaging the retinal vasculature. This study aims to evaluate the retinal microvasculature in diabetic human subjects with OCTA and assess potential clinical applications. PATIENTS AND METHODS: Cross-sectional study of 33 subjects with diabetic retinopathy. OCTA was performed on 3 mm × 3 mm sections using a swept-source OCTA prototype and a phase- and intensity-based contrasting algorithm. OCT angiograms were studied with corresponding clinical examination and fluorescein angiograms, when available, to assess accuracy and clinical utility. RESULTS: OCTA was able to demonstrate most clinically relevant vascular changes in subjects with diabetic retinopathy, including microaneurysms, impaired vascular perfusion, some forms of intraretinal fluid, vascular loops, intraretinal microvascular abnormalities, neovascularization, and cotton-wool spots that were largely consistent with fluorescein angiography. CONCLUSION: OCTA generates high-resolution angiograms that illustrate many of the clinically relevant findings in diabetic retinopathy and offers a novel complement or alternative to fluorescein angiography. Although currently an investigational technique, OCTA in combination with standard OCT imaging is at least as good as fluorescein angiography in the evaluation of the macular complications of diabetic retinopathy. [ Ophthalmic Surg Lasers Imaging Retina. 2015;46:796–805.]

BACKGROUND AND OBJECTIVE: To noninvasively evaluate the retinal microvasculature in healthy human subjects with optical coherence tomography angiography (OCTA). PATIENTS AND METHODS: Cross-sectional, observational study of five healthy subjects. OCTA was performed on 3 × 3 mm 2 sections centered on the fovea, nasal macula, and temporal macula. Retinal vasculature was assessed within three horizontal slabs consisting of the inner, middle, and outer retina. The vasculature within each retinal slab was reconstructed using phase-based and intensity contrast-based algorithms and visualized as separate en face images. RESULTS: OCTA in healthy subjects demonstrates capillary networks consistent with previous histological studies. No retinal vessels were found in the outer retina. OCT angiography of the inner and middle retinal layers showed region-specific vascular patterns that consistently corroborated qualitative findings from past histological studies. CONCLUSION: OCTA generates high-resolution, noninvasive angiograms qualitatively similar to conventional fluorescein angiography. OCTA may serve as a bridge to assess some features of the retinal microvasculature between conventionally performed angiograms. [ Ophthalmic Surg Lasers Imaging Retina . 2014;45:510–515.]

Humanin (HN) is a small mitochondrial-encoded peptide with neuroprotective properties. We have recently shown protection of retinal pigmented epithelium (RPE) cells by HN in oxidative stress; however, the effect of HN on endoplasmic reticulum (ER) stress has not been evaluated in any cell type. Our aim here was to study the effect of HN on ER stress-induced apoptosis in RPE cells with a specific focus on ER-mitochondrial cross-talk. Dose dependent effects of ER stressors (tunicamycin (TM), brefeldin A, and thapsigargin) were studied after 12 hr of treatment in confluent primary human RPE cells with or without 12 hr of HN pretreatment (1–20 μg/mL). All three ER stressors induced RPE cell apoptosis in a dose dependent manner. HN pretreatment significantly decreased the number of apoptotic cells with all three ER stressors in a dose dependent manner. HN pretreatment similarly protected U-251 glioma cells from TM-induced apoptosis in a dose dependent manner. HN pretreatment significantly attenuated activation of caspase 3 and ER stress-specific caspase 4 induced by TM. TM treatment increased mitochondrial superoxide production, and HN co-treatment resulted in a decrease in mitochondrial superoxide compared to TM treatment alone. We further showed that depleted mitochondrial glutathione (GSH) levels induced by TM were restored with HN co-treatment. No significant changes were found for the expression of several antioxidant enzymes between TM and TM plus HN groups except for the expression of glutamylcysteine ligase catalytic subunit (GCLC), the rate limiting enzyme required for GSH biosynthesis, which is upregulated with TM and TM+HN treatment. These results demonstrate that ER stress promotes mitochondrial alterations in RPE that lead to apoptosis. We further show that HN has a protective effect against ER stress-induced apoptosis by restoring mitochondrial GSH. Thus, HN should be further evaluated for its therapeutic potential in disorders linked to ER stress.

Histone deacetylase inhibitors (HDACi) have been reported to increase tumor antigen expression, and have been successfully tested as adjuvants for melanoma immunotherapy in mouse models. In this work, we tested the effects of a pan-HDACi on human lymphocytes and melanoma cell lines. Effects of the pan-HDACi panobinostat (LBH589) on cell viability, cell cycle, apoptosis, and DNA damage were determined in peripheral blood mononuclear cells (PBMC) from 2 healthy donors, 13 patients with metastatic melanoma, 2 bone marrow samples from patients with different malignances, and 12 human melanoma cell lines. Intracellular signaling in lymphocytes, with or without cytokine stimulation, was analyzed by phospho-flow cytometry in one of each type. The IC50 in PBMCs was 600 nmol/L in melanoma cell lines; >40% apoptotic cell death in PBMCs versus

Purpose: PLX4032 (RG7204), an oncogenic BRAF kinase inhibitor undergoing clinical evaluation, has high response rates in early clinical trials in patients with advanced BRAFV600E mutant melanoma. Combining PLX4032 with immunotherapy may allow expanding the durability of responses. The effects of PLX4032 on immune cells were studied to explore the feasibility of future combinatorial approaches with immunotherapy for melanoma. Experimental Design: Peripheral blood mononuclear cells (PBMC) and BRAFV600E mutant melanoma cells were exposed to increasing concentrations of PLX4032 and the cell viability, proliferation, cell cycle, apoptosis, and phosphorylation of signaling proteins were analyzed. Effects of PLX4032 on antigen-specific T-cell function were analyzed by specific cytokine release and cytotoxicity activity. Results: The 50% inhibition concentration (IC50) of PLX4032 for resting human PBMC was between 50 and 150 μmol/L compared with an IC50 below 1 μmol/L for sensitive BRAFV600E mutant melanoma cell lines. Activated lymphocytes were even more resistant with no growth inhibition up to concentrations of 250 μmol/L. PLX4032 had a marginal effect on cell-cycle arrest, apoptotic cell changes or alteration of phosphorylated signaling molecules in lymphocytes. Functional analysis of specific antigen recognition showed preserved T-cell function up to 10-μmol/L concentration of PLX4032, whereas the cytotoxic activity of PLX4032 was maintained up to high concentrations of 50 μmol/L. Conclusions: The preserved viability and function of lymphocytes exposed to high concentrations of PLX4032 suggest that this agent could be a potential candidate for combining with immunotherapy strategies for the treatment of patients with BRAFV600E mutant melanoma. Clin Cancer Res; 16(24); 6040–8. ©2010 AACR.

Author(s): Comin-Anduix, Begona; Sazegar, Hooman; Chodon, Thinle; Matsunaga, Douglas; Jalil, Jason; von Euw, Erika; Escuin-Ordinas, Helena; Balderas, Robert; Chmielowski, Bartosz; Gomez-Navarro, Jesus; Koya, Richard C; Ribas, Antoni | Abstract: BackgroundThe effects on cell signalling networks upon blockade of cytotoxic T lymphocyte-associated antigen-4 (CTLA4) using the monoclonal antibody tremelimumab were studied in peripheral blood mononuclear cell (PBMC) samples from patients with metastatic melanoma.Methodology/principalFindings Intracellular flow cytometry was used to detect phosphorylated (p) signaling molecules downstream of the T cell receptor (TCR) and cytokine receptors. PBMC from tremelimumab-treated patients were characterized by increase in pp38, pSTAT1 and pSTAT3, and decrease in pLck, pERK1/2 and pSTAT5 levels. These changes were noted in CD4 and CD8 T lymphocytes but also in CD14 monocytes. A divergent pattern of phosphorylation of Zap70, LAT, Akt and STAT6 was noted in patients with or without an objective tumor response.Conclusions/significanceThe administration of the CTLA4-blocking antibody tremelimumab to patients with metastatic melanoma influences signaling networks downstream of the TCR and cytokine receptors both in T cells and monocytes. The strong modulation of signaling networks in monocytes suggests that this cell subset may be involved in clinical responses to CTLA4 blockade.Clinical trial registrationclinicaltrials.gov; Registration numbers NCT00090896 and NCT00471887.

Background:Histone deacetylase inhibitors (HDACi) have been shown to increase cancer-testing and melanomasomal tumor antigen expression, which may allow their use as adjuvants to immunotherapy for melanoma. For this use, a key question is to test the effects of HDACi on lymphocytes compared to melanoma cells. Methods: We tested the effects on cell viability, cell cycle, apoptosis and DNA damage of the pan-HDACi LBH589 on peripheral blood mononuclear cells (PBMC) from a healthy donor (HD), four patients with metastatic melanoma (MM), two previously established human melanoma cell lines (M229 and M370), and two bone marrow samples of patients with multiple myeloma previously treated with GM-CSF. In addition, phospho-flow cytometry was used to study the effects of the HDACi on intracellular signalling in lymphocytes with or without pulsing with IL2 (400IU) or IFNα (10,000). For some experiments, HD PBMC were genetically modified to express the T cell receptor (TCR) for the melanoma antigen MART-1 using retroviral transduction for testing in in vitro cytotoxicity assays. Results. In replicate experiments the 50% inhibition concentration (IC50) of LBH589 for PBMC was low (< 20 nM) in comparison to the melanoma cells (> 600 nM). LBH589 induced > 20% (10 nM) and > 40% (1-10 mM) apoptotic cell death demonstrated by double presence of a sub-G0/G1 peak and cleaved poly (ADP-ribose) polymerase (PARP) in PBMC samples, while it was < 10% in melanoma cell lines at these same concentrations. In a DNA damage assay, there was around 2-fold increase in the phosphorylation of the histone variant H2A.X in HD PBMC at 1 nM, while it required 10 nM for the similar effect in the melanoma cell line M229. However, the phosphorylation of H2A.X in PBMCs of three patients with MM (1 nM LBH589) was slightly inhibited. The pH2A.X of the other cell line and one of the MM patients was a 1.5- fold increase. The maximal effects on signaling pathways were seen after 30 minutes of treatment. LBH589 slightly inhibited phosphorylation of STATs 1, 3, 5 and 6 and MAPK proteins (p38, ERK), p53, cyclin D3 and histone H3 in flow gated B and T cells from the HD. On the contrary, the same phosphoproteins were activated up to six times higher in the MM patient samples and in a bone marrow sample. Conclusions. The HDACi LBH589 induced cytotoxic effects at nanomolar concentrations on human lymphocytes and altered key signaling pathways involved in lymphocyte activation. These effects are at lower concentrations than the antitumor activity in melanoma in vitro, resulting in an adverse therapeutic window. Therefore, LBH589 should be used with caution if intended to sensitize melanoma to immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4713. doi:1538-7445.AM2012-4713