Outcomes of Eyes With Diabetic Macular Edema That Are Lost to Follow-up After Anti-Vascular Endothelial Growth Factor Therapy.

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 nonproliferative 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 at the return and final visits compared with the visit before LTFU. RESULTS: The mean age was 64.5 years, the mean LTFU duration was 322 days, and the mean follow-up duration after return was 502 days. Compared with 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 = .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), or 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 central foveal thickness increased when comparing the visit before LTFU (270 µm) with the return visit (305 µm, P = .012), but no difference was found by the final visit (247 µm, P = .07). CONCLUSIONS: Anti-VEGF-treated patients with DME who were LTFU for a prolonged period experienced a modest decline in VA that recovered after restarting treatment.

The Timing of Large Submacular Hemorrhage Secondary to Age-Related Macular Degeneration Relative to Anti-VEGF Therapy.

PURPOSE: To characterize the timing of large submacular hemorrhage (SMH) secondary to neovascular age-related macular degeneration (AMD) relative to anti-vascular endothelial growth factor (VEGF) therapy. DESIGN: Retrospective, consecutive case series. PARTICIPANTS: The study included 46 eyes of 46 patients with large SMH resulting from neovascular AMD selected to undergo pars plana vitrectomy with subretinal tissue plasminogen activator at the Mid Atlantic Retina group of the Wills Eye Hospital. METHODS: Patient charts were reviewed to identify baseline characteristics and anti-VEGF treatment details. OCT was used to evaluate pigmented epithelial detachments, SMH, and subretinal fluid before and after SMH. MAIN OUTCOME MEASURES: The timing of SMH in relation to last anti-VEGF injection, the anti-VEGF treatment status (i.e., naive, stable, or recently extended or shortened) at the time of SMH, and the length of the anti-VEGF treatment interval at the time of bleeding. RESULTS: Submacular hemorrhage occurred in 15 patients (36%) who were treatment naive. In patients treated with anti-VEGF, 19 (45%) had a stable treatment interval, 5 (12%) had a recently extended interval, and 3 (7%) had a shortened interval. The average treatment interval at the time of SMH was 6.8 weeks with a median of 7 total injections before SMH. Seven treated patients (26%) experience an SMH while having a 4-week dosing interval. The average time between last injection and SMH was 29 days. Forty-eight percent of patients treated with anti-VEGF agents experienced an SMH within 30 days of anti-VEGF injection. Chi-square analysis found SMH more likely to occur within 30 days of anti-VEGF injection than after 30 days. CONCLUSIONS: Large SMH in neovascular AMD in a treat-and-extend regimen does not seem to be associated with prolonged dosing intervals or recent interval extension, and a large proportion of such hemorrhages are likely to be a result of mechanisms other than loss of effective VEGF inhibition.

Extensive "stalagmite" epi-iridic juvenile xanthogranuloma simulating diffuse melanoma in a 16-year-old girl.

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.

Endogenous Bacterial Endophthalmitis: A Five-Year Retrospective Review at a Tertiary Care Academic Center.

PURPOSE: To better characterize the demographics and outcomes of endogenous bacterial endophthalmitis (EBE). METHODS: Retrospective observational case series of 49 eyes of 41 patients with EBE evaluated at a single academic center. RESULTS: Gram stain (p = .395), symptom duration prior to treatment (p = .483), and number of intravitreal antimicrobial injections (p = .421) did not correlate with mean change in VA. There was no significant difference between mean LogMAR VA at initial evaluation (1.7 ± 0.9) and last available follow up (1.4 ± 1.1, p = .15). Patients with bilateral EBE had 7.25 times greater odds of having cancer (95% CI 1.28, 41.14; p = .025). CONCLUSION: EBE in a North American population showed poor visual recovery despite treatment. It was most commonly due to gram-positive bacteria with the most common infectious source being endocarditis. Malignancy should be considered in patients with bilateral EBE.

Humanin Protects RPE Cells from Endoplasmic Reticulum Stress-Induced Apoptosis by Upregulation of Mitochondrial Glutathione.

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.

Optical Coherence Tomography Angiography of Diabetic Retinopathy in Human Subjects.

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.

MULTIMODAL IMAGING OF GEOGRAPHIC AREAS OF RETINAL DARKENING.

PURPOSE: To describe the clinical and multimodal imaging characteristics of a patient with unilateral asymptomatic dark retinal lesions corresponding to ellipsoid zone hyporeflectivity on optical coherence tomography (OCT). METHODS: The authors report a case of a 35-year-old man with HIV who presents with asymptomatic dark geographic retinal lesions corresponding to ellipsoid zone hyporeflectivity on OCT. Multimodal imaging techniques, including fundus color and widefield photographs, autofluorescence, spectral domain OCT (Spectralis, Heidelberg, Germany), prototype spectral domain OCT (Carl Zeiss Meditec, Dublin, CA) with OCT angiography, and en face images, were performed to evaluate and characterize the morphology of these lesions. RESULTS: Clinical examination and multimodal imaging reveal geographic darkening of the retina. Optical coherence tomography conveys hyporeflectivity of the ellipsoid zone band, which occurs abruptly and is present only in the areas of geographic retinal darkening. Optical coherence tomographic angiography shows a qualitatively similar appearance of the vasculature from the superficial retina through the avascular retina and the choriocapillaris, on both sides of the demarcation of retinal darkening, and also as compared to the unaffected eye. En face images from spectral domain OCT reveal an abrupt darkening of the tissue that localizes and is limited to the ellipsoid zone, with similar topographic appearance across the demarcation line. CONCLUSION: Geographic areas of darkening with photoreceptor hyporeflectivity have been described previously as "dark without pressure." In this case, the authors demonstrate photoreceptor hyporeflectivity that localizes to the clinically darkened areas, without topographic qualitative differences on en face spectral domain OCT images. The authors term these dark areas as geographic areas of retinal darkening because of ellipsoid nonreflectivity (GARDEN) spots.

OCT angiography in healthy human subjects.

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.

Exposure to a histone deacetylase inhibitor has detrimental effects on human lymphocyte viability and function.

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 <20 nmol/L compared with >600 nmol/L in melanoma cell lines; >40% apoptotic cell death in PBMCs versus <10% in melanoma cell lines was seen at the same concentration. Phospho-histone variant H2A.X (pH2A.X) increased 2-fold in healthy donor PBMCs at 1 nmol/L, whereas the same effect in the melanoma cell line M229 required 10 nmol/L. pH2A.X was inhibited slightly in the PBMCs of 3 patients with metastatic melanoma at 1 nmol/L and in the melanoma cell line M370 at 10 nmol/L. Panobinostat inhibited phospho-STAT1/3/5/6, -p38, -ERK, -p53, -cyclin D3, and -histone H3 in flow cytometry-gated healthy donor B and T cells, whereas it induced up to 6-fold activation in patients with metastatic melanoma and bone marrow samples. In human lymphocytes, panobinostat alters key lymphocyte activation signaling pathways and is cytotoxic at concentrations much lower than those required for melanoma antitumor activity, resulting in an adverse therapeutic window.

The oncogenic BRAF kinase inhibitor PLX4032/RG7204 does not affect the viability or function of human lymphocytes across a wide range of concentrations.

PURPOSE: PLX4032 (RG7204), an oncogenic BRAF kinase inhibitor undergoing clinical evaluation, has high response rates in early clinical trials in patients with advanced BRAF(V600E) 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 BRAF(V600E) 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 (IC(50)) of PLX4032 for resting human PBMC was between 50 and 150 µmol/L compared with an IC(50) below 1 µmol/L for sensitive BRAF(V600E) 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 BRAF(V600)(E) mutant melanoma.

Modulation of cell signaling networks after CTLA4 blockade in patients with metastatic melanoma.

BACKGROUND: The 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/PRINCIPAL: Findings 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/SIGNIFICANCE: The 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 REGISTRATION: clinicaltrials.gov; Registration numbers NCT00090896 and NCT00471887.