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Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter.

Fri, 2018-12-28 02:03
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Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter.

Microvasc Res. 2018 Dec 17;:

Authors: Giacalone JC, Miller MJ, Workalemahu G, Reutzel AJ, Ochoa D, Scott Whitmore S, Stone EM, Tucker BA, Mullins RF

Abstract
Age-related macular degeneration (AMD) is a common cause of blindness worldwide. While recent studies have revealed that the loss of choroidal endothelial cells (ChECs) is critical to the disease pathogenesis of dry AMD, in vitro studies are needed to fully elucidate the disease mechanism. However, these studies remain hindered due to the lack of publically available human ChEC lines. To address this need, ChECs were harvested form donor tissue and enriched for by using magnetic cell separation using anti-CD31 conjugated microbeads. Next, lenti-viral vectors with endothelial-specific promoters driving genes necessary for immortalization, CDH5p-hTERT and CDH5p TAg, were generated. Stable integration of both gene cassettes allowed cells to maintain their proliferative state and yielded an immortalized cell line (iChEC-1). Immunocytochemical analysis of iChEC-1 confirmed the expression of important ChEC markers such as CA4, a marker of choriocapillaris endothelial cells, CDH5, and CD34, pan-endothelial cell markers. qRT-PCR analysis of expanded clones from iChEC-1 further showed that the line maintained expression of other important endothelial markers, vWF, PECAM1, and PLVAP, similar to primary cells. Functional responses were characterized by tube-forming assays and repopulation of decellularized choroid with the immortalized cell line. In conclusion, the iChEC-1 line presents a suitable immortalized human ChEC line for future in vitro studies of AMD.

PMID: 30571950 [PubMed - as supplied by publisher]

Correlation of Optical Coherence Tomography and Retinal Histology in Normal and Pro23His Retinal Degeneration Pig.

Thu, 2018-12-13 21:28
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Correlation of Optical Coherence Tomography and Retinal Histology in Normal and Pro23His Retinal Degeneration Pig.

Transl Vis Sci Technol. 2018 Nov;7(6):18

Authors: Cheng J, Sohn EH, Jiao C, Adler KL, Kaalberg EE, Russell SR, Mullins RF, Stone EM, Tucker BA, Han IC

Abstract
Purpose: We correlate optical coherence tomography (OCT) retinal layer thickness measurements with histology in wild-type and retinal degenerative pigs.
Methods: OCT scans were obtained using the Bioptigen Envisu R2200. In normal pigs, three eyes were imaged in vivo, and three eyes were imaged after enucleation. In the Pro23His retinal degeneration pigs (P23H), one eye was imaged in vivo and four eyes were imaged after enucleation. All eyes were fixed in 4% paraformaldehyde and processed for histology. Corresponding retinal locations on OCT and histology were identified using anatomic landmarks (optic nerve, retinal vessels, visual streak). Individual retinal layer thicknesses were measured by two independent, masked graders, and intraclass correlation coefficients were used to determine agreement. OCT and histologic retinal thickness measurements were averaged and compared.
Results: OCT and histologic measurements correlated highly in normal and diseased eyes (R 2 = 0.91 and 0.92, respectively), and scans performed in vivo and ex vivo did not differ significantly. Despite good overall correlation, certain individual retinal layers (e.g., retinal nerve fiber layer [NFL], inner [INL] and outer [ONL] nuclear layers) appeared thicker on OCT compared to histology, while other layers (e.g., retinal pigment epithelium) appeared thinner. No statistically significant difference was found between OCT and histology for any retinal layer thickness measurement.
Conclusions: Retinal layer thickness measurements correlate well with histology in pig eyes, but differences in individual retinal layers may be seen.
Translational Relevance: OCT may be used in pigs to measure retinal thicknesses with good overall correlation to histologic measurements.

PMID: 30519502 [PubMed]

The ARMS2 A69S Polymorphism Is Associated with Delayed Rod-Mediated Dark Adaptation in Eyes at Risk for Incident Age-Related Macular Degeneration.

Thu, 2018-11-08 13:21
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The ARMS2 A69S Polymorphism Is Associated with Delayed Rod-Mediated Dark Adaptation in Eyes at Risk for Incident Age-Related Macular Degeneration.

Ophthalmology. 2018 Oct 30;:

Authors: Mullins RF, McGwin G, Searcey K, Clark ME, Kennedy EL, Curcio CA, Stone EM, Owsley C

Abstract
OBJECTIVE: To examine the association between sequence variants in genetic risk factors for age-related macular degeneration (AMD), and delayed rod-mediated dark adaptation (RMDA), the first functional biomarker for incident AMD, in older adults with normal macular health and early AMD.
DESIGN: Cross-sectional SUBJECTS: Older adults aged ≥60 years in normal macular health (defined as both eyes at step 1 on the Age-Related Eye Disease 9-step AMD classification system) and those with AMD in one or both eyes (defined as steps 2-9).
METHODS: Single nucleotide polymorphisms were genotyped in the CFH and ARMS2 genes using a Taqman assay. RMDA was assessed in one eye after photobleach with targets centered at 5° on the inferior vertical meridian. Rate of dark adaptation was defined by rod intercept time (RIT), duration (minutes) required for sensitivity to reach a criterion sensitivity level in the latter half of the second component of rod recovery. Associations between CFH and ARMS2 polymorphisms and RMDA were adjusted for age and smoking.
MAIN OUTCOME MEASURE: RIT.
RESULTS: The sample consisted of 543 participants having both genotype and RIT determination; 408 were in normal macular health and 135 had AMD, most having early AMD (124 of 135). For the combined sample, higher RIT (slower RMDA) was observed for both the A69S variant in ARMS2 and the Y402H variant in CFH (adjusted p=0.0001 and p=0.0023 respectively). For normal subjects the A69S variant in ARMS2 was associated with higher RIT (adjusted p=0.0011), whereas CFH Y402H was not (adjusted p=0.2175). For AMD cases, the A69S variant of ARMS2 and CFH Y402H were associated with higher RIT (adjusted p=0.0182 and p=0.0222 respectively). Those with a greater number of high-risk ARMS2 and CFH alleles had higher RIT, in both normal and AMD groups (adjusted p=0.0002 and p<0.0001 respectively).
CONCLUSIONS: We report a novel association wherein older adults with high risk ARMS2 and CFH genotypes are more likely to have delayed RMDA, the first functional biomarker for incident early AMD. Before the AMD clinical phenotype is present, those in normal macular health with the ARMS2 A69S allele have delayed RMDA. Understanding ARMS2 function is a research priority.

PMID: 30389424 [PubMed - as supplied by publisher]

Autoimmune retinopathy and optic neuropathy associated with enolase-positive renal oncocytoma.

Wed, 2018-09-26 22:42
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Autoimmune retinopathy and optic neuropathy associated with enolase-positive renal oncocytoma.

Am J Ophthalmol Case Rep. 2018 Dec;12:55-60

Authors: Cheng JL, Beebe JD, Nepple KG, Zakharia Y, Mullins RF, Flamme-Wiese MJ, Thurtell MJ, Han IC

Abstract
Purpose: To report a case of autoimmune retinopathy and optic neuropathy associated with an enolase-positive renal oncocytoma.
Observations: A 41-year-old man presented with subacute, painless, bilateral vision loss. On initial examination, visual acuity measured 20/125 OD and 20/1250 OS, and telangiectatic vessels were noted on the optic nerves and in the maculae. Goldmann perimetry showed bilateral, cecocentral scotomas, and electroretinography demonstrated reduced photopic and scotopic signals, concerning for autoimmune retinopathy. Serum testing showed multiple positive anti-optic nerve and anti-retinal antibodies, including to alpha-enolase. Extensive systemic workup was negative except for a large, exophytic, right renal mass. Biopsy was consistent with a benign oncocytoma, and immunohistochemical staining showed diffusely positive alpha-enolase staining. The patient was treated with a five-day course of intravenous methylprednisolone and plasmapheresis with minimal improvement. Surgical excision of the oncocytoma was performed. At 9-months post-operatively, visual acuity had improved to 20/40 OU, with corresponding improvement on visual field and electroretinography testing.
Conclusions and importance: To our knowledge, this is the first report of autoimmune retinopathy and optic neuropathy associated with a renal oncocytoma. The case highlights the importance of a thorough systemic workup in cases of suspected autoimmune retinopathy and reminds clinicians that even tumors considered benign can have distal effects on other organs.

PMID: 30229140 [PubMed]