Dedifferentiated liposarcoma of the orbit.

Purpose: To present a rare case of dedifferentiated liposarcoma of the orbit. Observations: A 61-year-old male complained of left-sided proptosis, diplopia, and limited ocular motility for two years. Biopsy results at that time were suggestive of an atypical lipomatous neoplasm. Ten years later, he presented with increase in size of the mass and worsening of his symptoms. Imaging showed a multi-lobulated mass in the left orbit involving the intraconal, medial, and anterior orbit. Decompression and orbitotomy with biopsy were performed to debulk the mass. Pathology showed a low-grade well-differentiated liposarcoma and the patient was monitored thereafter annually. Eight years later, he complained of persistent proptosis and mass effect from the tumor resulting in ptosis and diplopia and underwent orbital exenteration. Histopathological analysis of the exenterated orbit revealed a focal area of dedifferentiated liposarcoma. Conclusions and importance: Dedifferentiation of an orbital mass can occur as a late complication years after the diagnosis of well-differentiated liposarcoma. Compared to the previously published cases of orbital liposarcoma, this presentation shows a prolonged timeline prior to dedifferentiation (18 years after initial diagnosis). Symptoms of growth or invasive features could indicate dedifferentiation and should warrant a biopsy.

Inadvertent Viscoelastic Separation of the Pre-Descemet (Dua) Layer in Deep Anterior Lamellar Keratoplasty With Structural Features Revealed by Polarization Microscopy.

INTRODUCTION: Deep anterior lamellar keratoplasty (DALK) is a corneal transplant technique that removes the host stroma down to either the pre-Descemet (Dua) layer or Descemet membrane. It is most common for the big bubble technique to create a cleavage plane between the posterior stroma and the pre-Descemet layer. This is advantageous because the pre-Descemet layer has been found to be much stronger than Descemet membrane, and makes the procedure easier to perform. In this report, we present an uncommon viscoelastic-related complication of DALK that resulted in excising the pre-Descemet layer and allowing it to be studied using polarization microscopy. METHODS: DALK was performed using a standard big bubble technique. Postoperatively, a double anterior chamber was found to have been created by the inadvertent passage of an ophthalmic viscoelastic device (OVD) through the pre-Descemet layer. This resulted in the OVD being trapped between the pre-Descemet layer and Descemet membrane. The pre-Descemet layer was then resected in a subsequent operation. The pre-Descemet layer and posterior stroma were studied by polarization microscopy using Sirius Red histochemical staining to elucidate the orientation of the collagen fibers. RESULTS: The pre-Descemet layer is composed of lamellar arrays of collagen that have consistent polarization properties within each layer but show variable polarization of the strands, indicative of anisotropic strand orientation. The degree of variable polarization of the pre-Descemet layer is distinct from the overlying posterior stroma. CONCLUSIONS: Injecting an OVD into a big bubble in DALK may result in it being trapped between the pre-Descemet layer and Descemet membrane. The pre-Descemet layer shows alternating layers of varying polarization of collagen. This anisotropic structure helps explain the basis for the additional strength that the pre-Descemet layer is known to have.

Novel DCN Mutation in Armenian Family With Congenital Stromal Corneal Dystrophy.

PURPOSE: Congenital stromal corneal dystrophy (CSCD) is a rare congenital, dominantly inherited disorder characterized by diffuse stromal opacification associated with mutations in the decorin gene ( DCN ). As only 5 families with genetically confirmed CSCD have been reported, the identification of a novel pedigree provides the opportunity to better characterize the phenotype and genetic basis. METHODS: An Armenian family with individuals in 4 consecutive generations demonstrated clinical features consistent with CSCD. Consented individuals underwent slit lamp examination, optical coherence tomography, and confocal microscopy. Genomic DNA was collected from saliva and all coding and adjacent intronic regions of DCN were sequenced. In silico analysis was performed for identified mutation(s). Excised corneal tissue underwent light, electron microscopic, and immunohistochemical evaluation. RESULTS: Affected individuals demonstrated bilateral, diffuse, panstromal corneal opacification. Three of the 6 individuals diagnosed with CSCD underwent genetic analysis; all demonstrated a novel heterozygous frameshift deletion in exon 8 of DCN (p.His317Thrfs*11), predicted to cause a 33 amino acid truncation and to be damaging and disease causing by SIFT and MutationTaster. Light and electron microscopic examination of an excised cornea demonstrated increased corneal thickness, stromal scarring, keratocyte loss, and an irregularity of lamellar collagen spacing and fibril formation. Immunofluorescent examination demonstrated increased DCN immunostaining, predominantly in the widened interlamellar spaces. CONCLUSIONS: We report only the sixth pedigree with genetically confirmed CSCD, associated with a novel DCN frameshift mutation. The clinical evaluation, multimodal imaging, and histopathologic assessment in this family with CSCD broaden our understanding of this rare corneal disease.

Dendritic Melanocytic Hyperplasia in Pterygia: A Potential Source of Diagnostic Confusion with Primary Acquired Melanosis.

Introduction: The aim of this study was to report the nearly ubiquitous prevalence of melanocytic hyperplasia in benign pterygia/pingueculae and establish that the entity is insufficiently recognized. Methods: This is a retrospective immunohistochemical pathology case series of 30 consecutive pterygia/pingueculae samples selected from an ophthalmic pathology database at a single institution. Histopathologic and immunohistochemistry analyses with anti-SOX-10 and anti-MART-1 antibodies were used for identifying melanocytes. The number of squamous cells intervening between melanocytes was determined. Results: The frequency of dendritic melanocytes was found to meet the criteria for dendritic melanocytic hyperplasia in 29 of 30 pterygia/pingueculae samples using specific antibodies. Melanocytes were found in several patterns: diffuse (28%), multifocal (28%), and focal (44%). In each case, the melanocytes were distributed as single melanocytes at the base; clusters of melanocytes were seen in 17% of samples. There were an average of about two intervening epithelial cells between melanocytes at the base. Conclusion: When diagnosed with immunohistochemistry, dendritic melanocytic hyperplasia is nearly ubiquitous in pterygia and pingueculae. Melanocytic hyperplasia may have a distribution that includes nests and single melanocytes above the basal layer, which can be confused with forms of primary acquired melanosis. It is important for pathologists to recognize these lesions as a distinct benign clinicopathologic entity.

Low-grade fibromyxoid sarcoma of the orbit.

A 70-year-old male presented with diplopia and painless proptosis of the left eye for 5 months. Examination showed 6 mm of axial proptosis and restriction of supraduction, abduction and adduction, and mild limitation of infraduction of the left eye. Magnetic resonance imaging demonstrated a large, moderately well-circumscribed intraconal mass in the left lateral orbit, and excisional biopsy was performed. Histopathologic features of mixed fibrous and myxoid areas in a whorl-like pattern and immunohistochemical staining for MUC4 confirmed the diagnosis of low-grade fibromyxoid sarcoma (LGFMS). Next-generation sequencing revealed genetic fusion of EWSR1-CREB3L1. LGFMS is an extremely rare neoplasm with only two prior documented cases of orbital involvement. Here, we report the third case of orbital LGFMS.

Hyperostosis associated with orbital vascular malformation.

A previously healthy adult male presented with a slowly enlarging orbital mass associated with 5 mm of non-pulsatile proptosis. On imaging, a soft tissue lesion with avid contrast enhancement and associated bony hyperostosis was noted. The lesion and hyperostotic bone were surgically debulked, and significant arterial bleeding was noted intraoperatively consistent with an arteriovenous malformation. Histopathologic analysis revealed a vascular malformation with enhanced microvasculature infiltrating the periosteum. While vascular lesions elsewhere in the body can be associated with skeletal changes, bony hyperostosis is a rare feature of orbital vascular malformations.

Late Onset Interface Calcium Deposition After Laser In Situ Keratomileusis.

PURPOSE: The purpose of this study was to report a novel clinical entity characterized by bilateral calcium deposits in the flap interface after uncomplicated laser in situ keratomileusis (LASIK). METHODS: Slit-lamp examination, anterior segment optical coherence tomography imaging, and histopathologic analysis of an interface opacity were performed to characterize and identify the origin of the interface opacities. RESULTS: Two unrelated healthy young men who underwent LASIK in both eyes at 20 (case 1) and 44 (case 2) years of age were diagnosed with bilateral, white anterior stromal opacities 5 years after LASIK surgery. Slit-lamp examination and anterior segment optical coherence tomography imaging demonstrated that the opacities were located at the level of the LASIK interface in both eyes of both cases, with most of the opacities located at the temporal edge of the flap in each eye of case 2. An opacity from case 2 demonstrated birefringence using polarization microscopy and staining with Alizarin red, indicative of calcium deposition. The serum calcium level was borderline elevated in case 1 and within normal limits in case 2. CONCLUSIONS: Intrastromal calcium deposition can occur after LASIK surgery, with the deposits resembling dystrophic deposits located in the LASIK flap interface in individuals with granular corneal dystrophy type 2. Because the etiology and management of calcific and dystrophic interface deposition after LASIK are distinct, it is important for clinicians to differentiate the 2 entities based on the examination, diagnostic imaging, and, if necessary, molecular genetic analysis.

Tear Lipocalin and Lipocalin-Interacting Membrane Receptor.

Tear lipocalin is a primate protein that was recognized as a lipocalin from the homology of the primary sequence. The protein is most concentrated in tears and produced by lacrimal glands. Tear lipocalin is also produced in the tongue, pituitary, prostate, and the tracheobronchial tree. Tear lipocalin has been assigned a multitude of functions. The functions of tear lipocalin are inexorably linked to structural characteristics that are often shared by the lipocalin family. These characteristics result in the binding and or transport of a wide range of small hydrophobic molecules. The cavity of tear lipocalin is formed by eight strands (A-H) that are arranged in a ß-barrel and are joined by loops between the ß-strands. Recently, studies of the solution structure of tear lipocalin have unveiled new structural features such as cation-π interactions, which are extant throughout the lipocalin family. Lipocalin has many unique features that affect ligand specificity. These include a capacious and a flexible cavity with mobile and short overhanging loops. Specific features that confer promiscuity for ligand binding in tear lipocalin will be analyzed. The functions of tear lipocalin include the following: antimicrobial activities, scavenger of toxic and tear disruptive compounds, endonuclease activity, and inhibition of cysteine proteases. In addition, tear lipocalin binds and may modulate lipids in the tears. Such actions support roles as an acceptor for phospholipid transfer protein, heteropolymer formation to alter viscosity, and tear surface interactions. The promiscuous lipid-binding properties of tear lipocalin have created opportunities for its use as a drug carrier. Mutant analogs have been created to bind other molecules such as vascular endothelial growth factor for medicinal use. Tear lipocalin has been touted as a useful biomarker for several diseases including breast cancer, chronic obstructive pulmonary disease, diabetic retinopathy, and keratoconus. The functional possibilities of tear lipocalin dramatically expanded when a putative receptor, lipocalin-interacting membrane receptor was identified. However, opposing studies claim that lipocalin-interacting membrane receptor is not specific for lipocalin. A recent study even suggests a different function for the membrane protein. This controversy will be reviewed in light of gene expression data, which suggest that tear lipocalin has a different tissue distribution than the putative receptor. But the data show lipocalin-interacting membrane receptor is expressed on ocular surface epithelium and that a receptor function here would be rational.

Lipocalin-1 is the acceptor protein for phospholipid transfer protein in tears.

Phospholipid transfer protein, ∼80 kDa, transfers phospholipids from micelles to lipid binding proteins. The acceptor protein in plasma is apolipoprotein-A1, 28 kDa. Previously, phospholipid transfer protein was found in tears but an acceptor protein was not identified. To search for the acceptor protein(s) in tears a fluorescent phospholipid transfer assay was altered to omit the extrinsic acceptor. Human tears were incubated with fluorescent micelles and showed marked transfer activity verifying a native acceptor protein must be present. Reconstituted tears without tear lipocalin (lipocalin-1) eliminated the transfer of phospholipids. To determine if phospholipid transfer protein is involved in carrying phospholipid to the surface of tears from tear lipocalin, a fraction enriched in phospholipid transfer protein was injected into the subphase of a tear mimicking buffer in which tear lipocalin was present. The addition of phospholipid transfer protein did not increase the thickness of the surface layer regardless of the presence of lipid bearing tear lipocalin. The data show that phospholipid transfer protein transfers phospholipid from micelles to tear lipocalin. Phospholipid transfer protein does not transport the phospholipid. While tear lipocalin has no intrinsic transfer activity from micelles, it is the acceptor protein for phospholipid transfer protein in tears.

Evidence for Phospholipids on the Surface of Human Tears.

Purpose: The structure of tears has been theoretically considered three tiers with lipids at the air interface, aqueous and proteins in the subphase, and anchored mucins on the corneal epithelial surface. While many lipid and protein species have been identified in tears by mass spectrometry, the localization of the major components within the tear film structure remains speculative. The most controversial components are phospholipids. Although surface active, phospholipids have been presumed to be bound entirely to protein in the aqueous portion of tears or reside at the aqueous-lipid interface. Herein, the possibility that phospholipids are adsorbed at the air-surface interface of tears is interrogated. Methods: Polarization-modulated Fourier transform infrared reflective absorption spectroscopy (PM-IRRAS) was used to study the presence of phosphate signals at the tear surface. In order to constrain the depth of signal detection to the surface, an extreme grazing angle of incident radiation was employed. Nulling ellipsometry was used to confirm the presence of monolayers and surface thicknesses when surface active reagents were added to solutions. Results: Surface selection of PM-IRRAS was demonstrated by suppression of water and phosphate signals in buffers with monolayers of oleic acid. Phosphate signals were shown to reflect relative concentrations. Absorption peaks attributable to phospholipids were detected by PM-IRRAS on the human tear film surface and were augmented by the addition of phospholipid. Conclusions: The data provide strong evidence that phospholipids are present at the surface of tears.

Pathologic Study of Supernumerary Orbital Band in Type I Duane Syndrome.

BACKGROUND/AIMS: Accessory orbital bands are relatively rare and very few reports detail histopathology. Cases in the literature describe the composition of the bands as muscular and/or fibrous. The composition of the supernumerary band lying deep in the medial rectus muscle in a patient with type I Duane syndrome was investigated. METHODS: Histochemical stains were used in conjunction with polarized light for differentiating compressed collagen from muscle. Immunohistochemistry was used for verification of the presence of muscle. RESULTS: Compressed collagen appeared red using Masson trichrome staining. Collagen was positively identified by illumination with polarized light on several stains including the underutilized Sirius red dye. CONCLUSIONS: The findings of dense collagen fibers in the fibrotic band with focal striated muscle correlated with the restrictive strabismus. In concert with other cases in the literature, it is proposed that the fibrous bands are generally associated with restrictive strabismus. Bands that are muscular may or may not be associated with strabismus. Special techniques are needed to positively identify compressed collagen.

Methods toward simplification of time resolved fluorescence anisotropy in proteins labeled with NBD (4-chloro-7-nitrobenzofurazan) adducts.

The analysis of time resolved fluorescence anisotropy for NBD tagged proteins is difficult when multiple exponential components arise from heterogeneous amino acid fluorescent adducts. Two approaches were taken toward simplification. First, N terminal selective labeling of tear lipocalin with NBD-Cl was attempted at pH 7.0. While lysines were predominantly labeled at pH 8.0, selective N terminal labeling was attained at neutral pH. Second, fluorescence anisotropic decay analysis was simplified to recover only the rotational correlation time of the protein not the side chain. The boundaries for analysis of anisotropic decays were limited to the longer lifetimes. A modified tail fit enabled fitting the anisotropic decay to a single exponential. The correlation time for tear lipocalin matched published values. Additionally, a method for normalization of acquisition times of vertically (VV) and horizontally (VH) polarized fluorescence emission decays is presented for time-resolved anisotropy. Here it is applied to Picoharp software (Picoquant, Berlin). Picoharp software is programmed with an automatic stop at unequal acquisition times if the fluorescent counts exceeds a default. The method adjusts the intensity decays to the same acquisition time and is applicable to all time-resolved anisotropic decay data collected with time-correlated photon counting. •NBD labeling at pH 7.0 was not selective for N terminus of LCN1.•Constraints for range simplifies fittings of anisotropic decays.•Different acquisition times for decays can be normalized to facilitate fitting in data obtained by Picoharp.

Ellipsometry of human tears.

PURPOSE: The outer surface layer of tears is presumably composed of lipid. The thickness of this layer is considered critical to retard evaporation. Prior thickness measurements differ widely. Advances in ellipsometry have availed more precise and accurate measurements for thin films. The range in thickness of the surface layer of tears was studied by ellipsometry to uncover the source of prior discrepancies. METHODS: Tear surface layers of normal and dry eye subjects were measured by in-vitro ellipsometry. Lateral and Z resolutions of ∼1 µm and 0.1 nm, were achieved respectively. Thicknesses were derived from matrices and a Levenberg-Marquardt multivariate regression algorithm to Fresnel equations for multi-layered films. RESULTS: Ellipsometric measurements of pooled and individual human tears in-vitro revealed a larger overall range (0-500 nm) of surface film thicknesses than previously reported by any one study. Each sample showed thin areas (0-2.6 nm) with interspersed thicker regions (∼200-500 nm). Repeat measurements of a single donor collected at weekly intervals showed a broad range of surface thicknesses within and between samples. Thickness measurements from a dry eye subject overlapped that of normal subjects. CONCLUSION: The data show that published disparity in surface film thickness may be attributable to limitations of prior methodologies. The range and overlap of surface film thicknesses challenge less rigorous methodologies that claim to segregate normal and dry eye.

Ligand binding complexes in lipocalins: Underestimation of the stoichiometry parameter (n).

The stoichiometry of a ligand binding reaction to a protein is given by a parameter (n). The value of this parameter may indicate the presence of protein monomer or dimers in the binding complex. Members of the lipocalin superfamily show variation in the stoichiometry of binding to ligands. In some cases the stoichiometry parameter (n) has been variously reported for the same protein as mono- and multimerization of the complex. Prime examples include retinol binding protein, ß lactoglobulin and tear lipocalin, also called lipocalin-1(LCN1). Recent work demonstrated the stoichiometric ratio for ceramide:tear lipocalin varied (range n = 0.3-0.75) by several different methods. The structure of ceramide raises the intriguing possibility of a lipocalin dimer complex with each lipocalin molecule attached to one of the two alkyl chains of ceramide. The stoichiometry of the ceramide-tear lipocalin binding complex was explored in detail using size exclusion chromatography and time resolved fluorescence anisotropy. Both methods showed consistent results that tear lipocalin remains monomeric when bound to ceramide. Delipidation experiments suggest the most likely explanation is that the low 'n' values result from prior occupancy of the binding sites by native ligands. Lipocalins such as tear lipocalin that have numerous binding partners are particularly prone to an underestimated apparent stoichiometry parameter.

Ligand binding studies by high speed centrifugal precipitation and linear spectral summation using ultraviolet-visible absorption spectroscopy.

In ligand-protein binding experiments the major challenge is to separate bound from free ligand. Equilibrium and gel filtration separation techniques are often hampered by competition for the ligand and non-specific binding. Biophysical assays have attempted to circumvent this problem using titration calorimetry and spectroscopic methods. However, insoluble ligands require solvents that can overwhelm the discernible enthalpic changes of the protein and ligands. Spectroscopic methods are effective but may suffer from insensitivity (NMR) or the need for a lipid analog e.g., fluorescence and electron paramagnetic resonance. Our purpose is to compare the standard fluorescence assay to a technique we call high speed centrifugal precipitation. High speed centrifugal precipitation is suited to ligands that are insoluble in aqueous. The method permits separation of insoluble free ligand from that bound to the protein. The concentration of the each fraction can be precisely measured by absorbance spectrophotometry. A second technique, linear spectral summation has been published for protein-ligand associations using fluorescence of labeled ligands [1]. Here, the method is altered for use with ultraviolet-visible (UV-Vis) absorption spectroscopy. If the ligand complex shows a shift in the peak absorption of >8 nm, the bound and free concentrations can be measured simultaneously. The composite spectra of the samples are fit by linearly scaling UV-Vis absorption spectra of pure bound and free components at each point. •Ligand- protein binding kinetics is accessible with an ordinary spectrophotometer.•Concentrations are accurately measured from molar extinction coefficients.•The methods are ideal for lipid ligands that show absorption spectral peaks shifts in the bound and free states and/or are insoluble.

Data on Orphan tear lipid analogs, synthesis and binding to tear lipocalin.

Data found in this article include the structures of the orphan tear lipids and their analogs that are binding candidates to tear lipocalin, the mass spectrum of products of collision induced dissociation of putative synthesized compounds of synthesized (O-oleoyl)-16 hydroxypalmitic acid. These data and analyses support the research article "Interaction of ceramides and tear lipocalin" Glasgow et al. (2018) [1].

Correlation of Immunocytochemistry of BRCA1-associated Protein-1 (BAP1) With Other Prognostic Markers in Uveal Melanoma.

PURPOSE: Prior studies have shown that nuclear reactivity for BRCA1-associated protein-1 (BAP1) yields prognostic information for paraffin-embedded uveal melanomas. Lacking are immunocytochemical studies of BAP1 on fine needle aspiration biopsies of uveal melanoma that correlate with prognosis or other markers of prognosis. Our purpose was to fill this gap. DESIGN: Experimental laboratory study. METHODS: Fine needle aspiration biopsies were performed prospectively on 113 patients with uveal melanomas, garnering limited subsets of cases for comparison. Agreement between immunocytochemistry for BAP1 nuclear staining vs chromosome 3 ploidy analysis and gene expression profiling was assessed by 2 × 2 contingency table analysis. RESULTS: The presence or absence of suppression of nuclear expression of BAP1 was strongly associated (73%, P = .000002) with monosomy and disomy chromosome 3, respectively. BAP1 nuclear expression was also correlated with gene expression profiling. Chromosome 3 ploidy analysis correlated with gene expression profiles. CONCLUSION: When adequate material is obtained, immunocytology using BAP1 is a potentially informative tool for prognostication of uveal melanoma.

Interaction of ceramides and tear lipocalin.

The distribution of lipids in tears is critical to their function. Lipids in human tears may retard evaporation by forming a surface barrier at the air interface. Lipids complexed with the major lipid binding protein in tears, tear lipocalin, reside in the bulk (aqueous) and may have functions unrelated to the surface. Many new lipids species have been revealed through recent mass spectrometric studies. Their association with lipid binding proteins has not been studied. Squalene, (O-acyl) omega-hydroxy fatty acids (OAHFA) and ceramides are examples. Even well-known lipids such as wax and cholesteryl esters are only presumed to be unbound because extracts of protein fractions of tears were devoid of these lipids. Our purpose was to determine by direct binding assays if the aforementioned lipids can bind tear lipocalin. Lipids were screened for ability to displace DAUDA from tear lipocalin in a fluorescence displacement assay. Di- and tri-glycerides, squalene, OAHFA, wax and cholesterol esters did not displace DAUDA from tear lipocalin. However, ceramides displaced DAUDA. Apparent dissociation constants for ceramide-tear lipocalin complexes using fluorescent analogs were measured consistently in the submicromolar range with 3 methods, linear spectral summation, high speed centrifugal precipitation and standard fluorescence assays. At the relatively small concentrations in tears, all ceramides were complexed to tear lipocalin. The lack of binding of di- and tri-glycerides, squalene, OAHFA, as well as wax and cholesterol esters to tear lipocalin is consonant with residence of these lipids near the air interface.