A Case Study

OK Publisher Copyright: © 2023 American Academy of OphthalmologyPurpose: To investigate intraretinal neovascularization and microvascular anomalies by correlating in vivo multimodal imaging with corresponding ex vivo histology in a single patient. Design: A case study comprising clinical imaging from a community-based practice, and histologic analysis at a university-based research laboratory (clinicopathologic correlation). Participants: A White woman in her 90s treated with numerous intravitreal anti-VEGF injections for bilateral type 3 macular neovascularization (MNV) secondary to age-related macular degeneration (AMD). Methods: Clinical imaging comprised serial infrared reflectance, eye-tracked spectral-domain OCT, OCT angiography, and fluorescein angiography. Eye tracking, applied to the 2 preserved donor eyes, enabled the correlation of clinical imaging signatures with high-resolution histology and transmission electron microscopy. Main Outcome Measures: Histologic/ultrastructural descriptions and diameters of vessels seen in clinical imaging. Results: Six vascular lesions were histologically confirmed (type 3 MNV, n = 3; deep retinal age-related microvascular anomalies [DRAMAs], n = 3). Pyramidal (n = 2) or tangled (n = 1) morphologies of type 3 MNV originated at the deep capillary plexus (DCP) and extended posteriorly to approach without penetrating persistent basal laminar deposit. They did not enter the subretinal pigment epithelium (RPE)–basal laminar space or cross the Bruch membrane. Choroidal contributions were not found. The neovascular complexes included pericytes and nonfenestrated endothelial cells, within a collagenous sheath covered by dysmorphic RPE cells. Deep retinal age-related microvascular anomaly lesions extended posteriorly from the DCP into the Henle fiber and the outer nuclear layers without evidence of atrophy, exudation, or anti-VEGF responsiveness. Two DRAMAs lacked collagenous sheaths. External and internal diameters of type 3 MNV and DRAMA vessels were larger than comparison vessels in the index eyes and in aged normal and intermediate AMD eyes. Conclusions: Type 3 MNV vessels reflect specializations of source capillaries and persist during anti-VEGF therapy. The collagenous sheath of type 3 MNV lesions may provide structural stabilization. If so, vascular characteristics may be useful in disease monitoring in addition to fluid and flow signal detection. Further investigation with longitudinal imaging before exudation onset will help determine if DRAMAs are part of the type 3 MNV progression sequence. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references.publishersversionpublishe

Abundant Lipid and Protein Components of Drusen

Drusen are extracellular lesions characteristic of aging and age-related maculopathy, a major retinal disease of the elderly. We determined the relative proportions of lipids and proteins in drusen capped with retinal pigment epithelium (RPE) and in RPE isolated from non-macular regions of 36 human retinas with grossly normal maculas obtained <6 hr after death.Druse pellets were examined by light and electron microscopy. Component proteins were extracted using novel methods for preserved tissues, separated, subjected to tryptic digestion and LC-MS(MS)(2) analysis using an ion trap mass spectrometer, and identified with reference to databases. Lipid classes were separated using thin layer chromatography and quantified by densitometry. Major druse components were esterified cholesterol (EC), phosphatidylcholine (PC), and protein (37.5+/-13.7, 36.9+/-12.9, and 43.0+/-11.5 ng/druse, respectively). Lipid-containing particles (median diameter, 77 nm) occupied 37-44% of druse volume. Major proteins include vitronectin, complement component 9, apoE, and clusterin, previously seen in drusen, and ATP synthase subunit beta, scavenger receptor B2, and retinol dehydrogenase 5, previously seen in RPE. Drusen and RPE had similar protein profiles, with higher intensities and greater variability in drusen. C8, part of the complement membrane attack complex, was localized in drusen by immunofluorescence.At least 40% of druse content is comprised by lipids dominated by EC and PC, 2 components that are potentially accounted for by just one pathway, the secretion of lipoproteins by RPE. Manipulating genes encoding apolipoprotein pathways would be a fruitful approach to producing drusen with high EC content in laboratory animals. Therapies that directly mitigate drusen should prepare for the substantial volume of neutral lipids. The catalog of major druse proteins is nearing completion

Darwin's Duchenne: Eye constriction during infant joy and distress

Darwin proposed that smiles with eye constriction (Duchenne smiles) index strong positive emotion in infants, while cry-faces with eye constriction index strong negative emotion. Research has supported Darwin's proposal with respect to smiling, but there has been little parallel research on cry-faces (open-mouth expressions with lateral lip stretching). To investigate the possibility that eye constriction indexes the affective intensity of positive and negative emotions, we first conducted the Face-to-Face/Still-Face (FFSF) procedure at 6 months. In the FFSF, three minutes of naturalistic infant-parent play interaction (which elicits more smiles than cry-faces) are followed by two minutes in which the parent holds an unresponsive still-face (which elicits more cry-faces than smiles). Consistent with Darwin's proposal, eye constriction was associated with stronger smiling and with stronger cry-faces. In addition, the proportion of smiles with eye constriction was higher during the positive-emotion eliciting play episode than during the still-face. In parallel, the proportion of cry-faces with eye constriction was higher during the negative-emotion eliciting still-face than during play. These results are consonant with the hypothesis that eye constriction indexes the affective intensity of both positive and negative facial configurations. A preponderance of eye constriction during cry-faces was observed in a second elicitor of intense negative emotion, vaccination injections, at both 6 and 12 months of age. The results support the existence of a Duchenne distress expression that parallels the more well-known Duchenne smile. This suggests that eye constriction-the Duchenne marker-has a systematic association with early facial expressions of intense negative and positive emotion. © 2013 Mattson et al

Lipoprotein Particles of Intraocular Origin in Human Bruch Membrane: An Unusual Lipid Profile

PURPOSE. Throughout adulthood, Bruch membrane (BrM) accumulates esterified cholesterol (EC) associated with abundant 60-to 80-nm-diameter lipoprotein-like particles (LLP), putative apolipoprotein B (apoB) lipoproteins secreted by the retinal pigment epithelium (RPE). In the present study, neutral lipid, phospholipids, and retinoid components of human BrM-LLP were assayed. METHODS. Particles isolated from paired choroids of human donors were subjected to comprehensive lipid profiling (preparative liquid chromatography [LC] gas chromatography [GC]), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), Western blot analysis, and negative stain electron microscopy. Results were compared to plasma lipoproteins isolated from normolipemic volunteers and to conditioned medium from RPE-J cells supplemented with palmitate to induce particle synthesis and secretion. RESULTS. EC was the largest component (32.4 Ϯ 7.9 mol%) of BrM-LLP lipids. EC was 11.3-fold more abundant than triglyceride (TG), unlike large apoB lipoproteins in plasma. Of the fatty acids (FA) esterified to cholesterol, linoleate (18:2n6) was the most abundant (41.7 Ϯ 4.7 mol%). Retinyl ester (RE) was detectable at picomolar levels in BrM-LLP. Notably scarce in any BrM-LLP lipid class was the photoreceptor-abundant FA docosahexaenoate (DHA, 22:6n3). RPE-J cells synthesized apoB and numerous EC-rich spherical particles. CONCLUSIONS. BrM-LLP composition resembles plasma LDL more than it does photoreceptors. An EC-rich core is possible for newly synthesized lipoproteins as well as those processed in plasma. Abundant EC could contribute to a transport barrier in aging and lesion formation in age-related maculopathy (ARM). Analysis of BrM-LLP composition has revealed new aspects of retinal cholesterol and retinoid homeostasis. (Invest Ophthalmol Vis Sci. 2009;50:870 -877 2 Early ARM is characterized by drusen (focal extracellular debris), basal linear deposit (BlinD; a diffusely distributed drusenoid material), and altered RPE morphology and pigmentation. This disease stage has limited treatment options, including antioxidant nutritional supplements, and, in its later stages, loss of eyesight is possible. Although some gene sequence variants increase ARM risk, 3 the largest risk factor for early ARM remains advanced age. It is therefore important to understand how age-related changes in the affected tissues impel some individuals toward severe disease. Lipoproteins are naturally occurring nanoparticles composed of lipid and protein held together by noncovalent forces. Each particle is a microemulsion consisting of a surface of phospholipids (PLs), unesterified cholesterol (UC), and apolipoproteins and a core of neutral lipids, principally esterified cholesterol (EC) and triglyceride (TG). Lipoprotein classes differ in relative amount of lipids, protein/lipid ratio, and apolipoprotein species present, resulting in differences in size, density, and electrophoretic mobility. Lipoprotein classes containing apoB are chylomicrons (CM; from intestine), very-lowdensity lipoproteins (VLDL; from liver), and LDL (metabolite of VLDL). ApoB lipoproteins must be properly lipidated by their source cells in order for particle maturation and secretion to proceed. Core lipid composition reflects the availability of input FA and the substrate preferences of catalytic enzymes in upstream pathways. 10 Rather, recent evidence implicates EC as part of an apoB lipoprotein constitutively produced within the eye by the RPE and secreted into BrM, where it participates in ARM progression. Native human RPE expresses apolipoprotein mRNA transcripts, the proteins of apos B-100 and E, and notably, microsomal triglyceride transfer protein (MTP), required for apoB secretion and the product of the abetalipoproteinemia gene. 11,12 Isolated BrM-LLP segregate into the appropriate band of a density gradient but differ from plasma lipoproteins in cholesterol profile. 13 Cultured RPE secretes apoE, primarily into a high-density fraction. 14 Size and lipid composition are strongly related for apoB lipoproteins, in that particles Ͼ25 nm diameter (CM and VLDL) have TG-rich cores and smaller particles (including LDL) have EC-rich cores. 15 BrM-LLP, as large as VLDL or small CM, are expected to be TG-rich. Indeed, an early assay of BrM/choroid From the Departments of 1 Ophthalmology

DICER1 mutations in childhood cystic nephroma and its relationship to DICER1-renal sarcoma

The pathogenesis of cystic nephroma of the kidney has interested pathologists for over 50 years. Emerging from its initial designation as a type of unilateral multilocular cyst, cystic nephroma has been considered as either a developmental abnormality or a neoplasm or both. Many have viewed cystic nephroma as the benign end of the pathologic spectrum with cystic partially differentiated nephroblastoma and Wilms tumor, whereas others have considered it a mixed epithelial and stromal tumor. We hypothesize that cystic nephroma, like the pleuropulmonary blastoma in the lung, represents a spectrum of abnormal renal organogenesis with risk for malignant transformation. Here we studied DICER1 mutations in a cohort of 20 cystic nephromas and 6 cystic partially differentiated nephroblastomas, selected independently of a familial association with pleuropulmonary blastoma and describe four cases of sarcoma arising in cystic nephroma, which have a similarity to the solid areas of type II or III pleuropulmonary blastoma. The genetic analyses presented here confirm that DICER1 mutations are the major genetic event in the development of cystic nephroma. Further, cystic nephroma and pleuropulmonary blastoma have similar DICER1 loss of function and ‘hotspot' missense mutation rates, which involve specific amino acids in the RNase IIIb domain. We propose an alternative pathway with the genetic pathogenesis of cystic nephroma and DICER1-renal sarcoma paralleling that of type I to type II/III malignant progression of pleuropulmonary blastoma

As in Real Estate, Location Matters: Cellular Expression of Complement Varies Between Macular and Peripheral Regions of the Retina and Supporting Tissues.

The cellular events that dictate the initiation of the complement pathway in ocular degeneration, such as age-related macular degeneration (AMD), is poorly understood. Using gene expression analysis (single cell and bulk), mass spectrometry, and immunohistochemistry, we dissected the role of multiple retinal and choroidal cell types in determining the complement homeostasis. Our scRNA-seq data show that the cellular response to early AMD is more robust in the choroid, particularly in fibroblasts, pericytes and endothelial cells. In late AMD, complement changes were more prominent in the retina especially with the expression of the classical pathway initiators. Notably, we found a spatial preference for these differences. Overall, this study provides insights into the heterogeneity of cellular responses for complement expression and the cooperation of neighboring cells to complete the pathway in healthy and AMD eyes. Further, our findings provide new cellular targets for therapies directed at complement

Lysolipids are prominent in subretinal drusenoid deposits, a high-risk phenotype in age-related macular degeneration

IntroductionAge related macular degeneration (AMD) causes legal blindness worldwide, with few therapeutic targets in early disease and no treatments for 80% of cases. Extracellular deposits, including drusen and subretinal drusenoid deposits (SDD; also called reticular pseudodrusen), disrupt cone and rod photoreceptor functions and strongly confer risk for advanced disease. Due to the differential cholesterol composition of drusen and SDD, lipid transfer and cycling between photoreceptors and support cells are candidate dysregulated pathways leading to deposit formation. The current study explores this hypothesis through a comprehensive lipid compositional analysis of SDD.MethodsHistology and transmission electron microscopy were used to characterize the morphology of SDD. Highly sensitive tools of imaging mass spectrometry (IMS) and nano liquid chromatography tandem mass spectrometry (nLC-MS/MS) in positive and negative ion modes were used to spatially map and identify SDD lipids, respectively. An interpretable supervised machine learning approach was utilized to compare the lipid composition of SDD to regions of uninvolved retina across 1873 IMS features and to automatically discern candidate markers for SDD. Immunohistochemistry (IHC) was used to localize secretory phospholipase A2 group 5 (PLA2G5). ResultsAmong the 1873 detected features in IMS data, three lipid classes, including lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE) and lysophosphatidic acid (LysoPA) were observed nearly exclusively in SDD while presumed precursors, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidic acid (PA) lipids were detected in SDD and adjacent photoreceptor outer segments. Molecular signals specific to SDD were found in central retina and elsewhere. IHC results indicated abundant PLA2G5 in photoreceptors and retinal pigment epithelium (RPE). DiscussionThe abundance of lysolipids in SDD implicates lipid remodeling or degradation in deposit formation, consistent with ultrastructural evidence of electron dense lipid-containing structures distinct from photoreceptor outer segment disks and immunolocalization of secretory PLA2G5 in photoreceptors and RPE. Further studies are required to understand the role of lipid signals observed in and around SDD

The genomic landscape of juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm (MPN) of childhood with a poor prognosis. Mutations in NF1, NRAS, KRAS, PTPN11 and CBL occur in 85% of patients, yet there are currently no risk stratification algorithms capable of predicting which patients will be refractory to conventional treatment and therefore be candidates for experimental therapies. In addition, there have been few other molecular pathways identified aside from the Ras/MAPK pathway to serve as the basis for such novel therapeutic strategies. We therefore sought to genomically characterize serial samples from patients at diagnosis through relapse and transformation to acute myeloid leukemia in order to expand our knowledge of the mutational spectrum in JMML. We identified recurrent mutations in genes involved in signal transduction, gene splicing, the polycomb repressive complex 2 (PRC2) and transcription. Importantly, the number of somatic alterations present at diagnosis appears to be the major determinant of outcome

Biometrics, Impact, and Significance of Basal Linear Deposit and Subretinal Drusenoid Deposit in Age-Related Macular Degeneration.

PurposeBasal linear deposit (BLinD) is a thin layer of soft drusen material. To elucidate the biology of extracellular deposits conferring age-related macular degeneration (AMD) progression risk and inform multimodal clinical imaging based on optical coherence tomography (OCT), we examined lipid content and regional prevalence of BLinD, soft drusen, pre-BLinD, and subretinal drusenoid deposit (SDD) in AMD and non-AMD aged eyes. We estimated BLinD volume and illustrated its relation to type 1 macular neovascularization (MNV).MethodsDonor eyes were classified as early to intermediate AMD (n = 25) and age-matched controls (n = 54). In high-resolution histology, we assessed BLinD/soft drusen thickness at 836 and 1716 locations in AMD and control eyes, respectively. BLinD volume was estimated using solid geometry in donor eyes, one clinically characterized.ResultsBLinD, drusen, type 1 MNV, and fluid occupy the sub-RPE-basal laminar space. BLinD volume in a 3-mm diameter circle may be as much as 0.0315 mm3. Osmophilic lipid was more concentrated in BLinD/drusen than SDD. In the fovea, BLinD/drusen was prevalent in AMD eyes; pre-BLinD was prevalent in control eyes. SDD was low in the fovea and high in perifovea, especially in AMD eyes.ConclusionsAlthough invisible, BLinD may presage type 1 MNV. BLinD volume approaches the criterion OCT drusen volume of 0.03 mm3 for AMD progression risk. BLinD culminates years of subfoveal lipid accumulation. SDD is detected relatively late in life, with currently unknown precursors. Deposit topography suggests one outer retinal lipid recycling system serving specialized cone and rod physiology, and its dysregulation in AMD is due to impaired transfer to the circulation

Biometrics, Impact, and Significance of Basal Linear Deposit and Subretinal Drusenoid Deposit in Age-Related Macular Degeneration.

PurposeBasal linear deposit (BLinD) is a thin layer of soft drusen material. To elucidate the biology of extracellular deposits conferring age-related macular degeneration (AMD) progression risk and inform multimodal clinical imaging based on optical coherence tomography (OCT), we examined lipid content and regional prevalence of BLinD, soft drusen, pre-BLinD, and subretinal drusenoid deposit (SDD) in AMD and non-AMD aged eyes. We estimated BLinD volume and illustrated its relation to type 1 macular neovascularization (MNV).MethodsDonor eyes were classified as early to intermediate AMD (n = 25) and age-matched controls (n = 54). In high-resolution histology, we assessed BLinD/soft drusen thickness at 836 and 1716 locations in AMD and control eyes, respectively. BLinD volume was estimated using solid geometry in donor eyes, one clinically characterized.ResultsBLinD, drusen, type 1 MNV, and fluid occupy the sub-RPE-basal laminar space. BLinD volume in a 3-mm diameter circle may be as much as 0.0315 mm3. Osmophilic lipid was more concentrated in BLinD/drusen than SDD. In the fovea, BLinD/drusen was prevalent in AMD eyes; pre-BLinD was prevalent in control eyes. SDD was low in the fovea and high in perifovea, especially in AMD eyes.ConclusionsAlthough invisible, BLinD may presage type 1 MNV. BLinD volume approaches the criterion OCT drusen volume of 0.03 mm3 for AMD progression risk. BLinD culminates years of subfoveal lipid accumulation. SDD is detected relatively late in life, with currently unknown precursors. Deposit topography suggests one outer retinal lipid recycling system serving specialized cone and rod physiology, and its dysregulation in AMD is due to impaired transfer to the circulation