December 2019

OBJECTIVE:  To evaluate to what extent indicators of placenta insufficiency are associated with low concentrations of insulin-like growth factor 1 (IGF-1) and IGF-1-binding protein-1 (IGFBP-1) in neonatal blood, and to what extent the concentrations of these growth factors are associated with concentrations of proteins with inflammatory, neurotrophic, or angiogenic properties. STUDY DESIGN:  Using multiplex immunoassays, we measured the concentrations of IGF-1 and IGFBP-1, as well as 25 other proteins in blood spots collected weekly from ≥ 880 infants born before the 28th week of gestation, and sought correlates of concentrations in the top and bottom quartiles for gestational age and day the specimen was collected. RESULTS:  Medically indicated delivery and severe fetal growth restriction (sFGR) were associated with low concentrations of IGF-1 on the first postnatal day and with high concentrations of IGFBP-1 on almost all days. Elevated concentrations of IGF-1 and IGFBP-1 were accompanied by elevated concentrations of many other proteins with inflammatory, neurotrophic, or angiogenic properties. CONCLUSION:  Disorders associated with impaired placenta implantation and sFGR appear to account for a relative paucity of IGF-1 on the first postnatal day. Elevated concentrations of IGF-1 and especially IGFBP-1 were associated with same-day elevated concentrations of inflammatory, neurotrophic, and angiogenic proteins.

Epithelial to mesenchymal transition (EMT) plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). We aimed to demonstrate the role of mouse double minute 2 (MDM2) in transforming growth factor-beta 2 (TGF-β2)-induced EMT in human retinal pigment epithelial cells (RPEs). Immunofluorescence was used to assess MDM2 expression in epiretinal membranes (ERMs) from patients with PVR. A single guide (sg)RNA targeting the second promoter of MDM2 was cloned into a mutant lentiviral Clustered Regularly Interspaced Short Palindromic Repeats (lentiCRISPR) v2 (D10A and H840A) vector for expressing nuclease dead Cas9 (dCas9)/MDM2-sgRNA in RPEs. In addition, MDM2-sgRNA was also cloned into a pLV-sgRNA-dCas9-Kruppel associated box (KRAB) vector for expressing dCas9 fused with a transcriptional repressor KRAB/MDM2-sgRNA. TGF-β2-induced expression of MDM2 and EMT biomarkers were assessed by quantitative polymerase chain reaction (q-PCR), western blot, or immunofluorescence. Wound-healing and proliferation assays were used to evaluate the role of MDM2 in TGF-β2-induced responses in RPEs. As a result, we found that MDM2 was expressed obviously in ERMs, and that TGF-β2-induced expression of MDM2 and EMT biomarkers Fibronectin, N-cadherin and Vimentin in RPEs. Importantly, we discovered that the dCas9/MDM2-sgRNA blocked TGF-β2-induced expression of MDM2 and the EMT biomarkers without affecting their basal expression, whereas the dCas9-KRAB/MDM2-sgRNA suppressed basal MDM2 expression in RPEs. These cells could not be maintained continuously because their viability was greatly reduced. Next, we found that Nutlin-3, a small molecule blocking the interaction of MDM2 with p53, inhibited TGF-β2-induced expression of Fibronectin and N-cadherin but not Vimentin in RPEs, indicating that MDM2 functions in both p53-dependent and -independent pathways. Finally, our experimental data demonstrated that dCas9/MDM2-sgRNA suppressed TGF-β2-dependent cell proliferation and migration without disturbing the unstimulated basal activity. In conclusion, the CRISPR/dCas9 capability for blocking TGF-β2-induced expression of MDM2 and EMT biomarkers can be exploited for a therapeutic approach to PVR.

Rapid and flexible learning during behavioral choices is critical to our daily endeavors and constitutes a hallmark of dynamic reasoning. An important paradigm to examine flexible behavior involves learning new arbitrary associations mapping visual inputs to motor outputs. We conjectured that visuomotor rules are instantiated by translating visual signals into actions through dynamic interactions between visual, frontal and motor cortex. We evaluated the neural representation of such visuomotor rules by performing intracranial field potential recordings in epilepsy subjects during a rule-learning delayed match-to-behavior task. Learning new visuomotor mappings led to the emergence of specific responses associating visual signals with motor outputs in 3 anatomical clusters in frontal, anteroventral temporal and posterior parietal cortex. After learning, mapping selective signals during the delay period showed interactions with visual and motor signals. These observations provide initial steps towards elucidating the dynamic circuits underlying flexible behavior and how communication between subregions of frontal, temporal, and parietal cortex leads to rapid learning of task-relevant choices.

Cell-cell communication plays a fundamental role in mediating corneal wound healing following injury or infection. Depending on the severity of the wound, regeneration of the cornea and the propensity for scar development are influenced by the acute resolution of the pro-fibrotic response mediated by closure of the wound via cellular and tissue contraction. Damage of the corneal epithelium, basement membrane, and anterior stroma following a superficial keratectomy is known to lead to significant provisional matrix deposition, including secretion of fibronectin and thrombospondin-1, as well as development of a corneal scar. In addition, corneal wounding has previously been shown to promote release of extracellular vesicles from the corneal epithelium, which, in addition to soluble factors, may play a role in promoting tissue regeneration. In this study, we report the development and characterization of a co-culture system of human corneal epithelial cells and corneal stromal fibroblasts cultured for 4 weeks to allow extracellular matrix deposition and tissue maturation. The secretion of provisional matrix components, as well as small and large extracellular vesicles, was apparent within the constructs, suggesting cell-cell communication between epithelial and stromal cell populations. Laminin-1β was highly expressed by the corneal epithelial layer with the presence of notable patches of basement membrane identified by transmission electron microscopy. Interestingly, we identified expression of collagen type III, fibronectin, and thrombospondin-1 along the epithelial-stromal interface similar to observations seen in vivo following a keratectomy, as well as expression of the myofibroblast marker, α-smooth muscle actin, within the stroma. Our results suggest that this corneal epithelial-stromal model may be useful in the study of the biochemical phenomena that occur during corneal wound healing.

Pathogenic MAGEL2 variants result in the phenotypes of Chitayat-Hall syndrome (CHS), Schaaf-Yang syndrome (SYS) and Prader-Willi syndrome (PWS). We present five patients with mutations in MAGEL2, including the first patient reported with a missense variant, adding to the limited literature. Further, we performed a systematic review of the CHS and SYS literature, assess the overlap between CHS, SYS and PWS, and analyze genotype-phenotype correlations among them. We conclude that there is neither a clinical nor etiological difference between CHS and SYS, and propose that the two syndromes simply be referred to as MAGEL2-related disorders.

PURPOSE: We observed four individuals in two unrelated but consanguineous families from Portugal and Brazil affected by early-onset retinal degeneration, sensorineural hearing loss, microcephaly, intellectual disability, and skeletal dysplasia with scoliosis and short stature. The phenotype precisely matched that of an individual of Azorean descent published in 1986 by Liberfarb and coworkers. METHODS: Patients underwent specialized clinical examinations (including ophthalmological, audiological, orthopedic, radiological, and developmental assessment). Exome and targeted sequencing was performed on selected individuals. Minigene constructs were assessed by quantitative polymerase chain reaction (qPCR) and Sanger sequencing. RESULTS: Affected individuals shared a 3.36-Mb region of autozygosity on chromosome 22q12.2, including a 10-bp deletion (NM_014338.3:c.904-12_904-3delCTATCACCAC), immediately upstream of the last exon of the PISD (phosphatidylserine decarboxylase) gene. Sequencing of PISD from paraffin-embedded tissue from the 1986 case revealed the identical homozygous variant. In HEK293T cells, this variant led to aberrant splicing of PISD transcripts. CONCLUSION: We have identified the genetic etiology of the Liberfarb syndrome, affecting brain, eye, ear, bone, and connective tissue. Our work documents the migration of a rare Portuguese founder variant to two continents and highlights the link between phospholipid metabolism and bone formation, sensory defects, and cerebral development, while raising the possibility of therapeutic phospholipid replacement.

: We evaluated visual acuity (VA) over 5 years in a subspecialty noninfectious uveitis population.: Retrospective data from 5,530 noninfectious uveitis patients with anterior, intermediate, posterior or panuveitis were abstracted by expert reviewers. Mean VA was calculated using inverse probability of censoring weighting to account for losses to follow-up.: Patients were a median of 41 years old, 65% female, and 73% white. Initial mean VA was worse among panuveitis (20/84) than posterior (20/64), intermediate (20/47), and anterior (20/37) uveitides. On average, mean VA improved by 0.62, 0.51, 0.37, and 0.26 logMAR-equivalent lines over 2 years, respectively (each < .001), then remained stable, except posterior uveitis mean VA worsened to initial levels.: Mean VA of uveitic eyes improved and, typically, improvement was sustained under uveitis subspecialty care. Because VA tends to improve under tertiary care, mean VA change appears a better outcome for clinical studies than time-to-loss of VA.

The MIRAgel (hydrogel) scleral buckle, introduced in the 1980s, was a novel material to repair retinal detachments. It was later discontinued due to the frequency of long-term complications related to buckle hydrolysis and expansion. These complications included pain, limited extraocular motility, and more serious complications such as infection or scleral perforation, which ultimately necessitated surgical extraction as late as 20-30 years after placement. Prompt and proper diagnosis and treatment is often delayed as these buckle-associated complications frequently mimic other orbital pathologies such as tumors or infections. The hydrolyzed MIRAgel buckle exhibits distinct radiographic features that are helpful in arriving at the correct diagnosis, particularly in cases of ambiguous clinical presentation or history. Here, we expand on the previously described radiographic features of hydrolyzed MIRAgel and compare them to features of common, mimicking orbital pathology.

Blindness due to corneal diseases is a common pathology affecting up to 23 million individuals worldwide. The tissue-engineered anterior human cornea, which is currently being tested in a Phase I/II clinical trial to treat severe corneal trophic ulcers with preliminary good feasibility and safety results. This bioartificial cornea is based on a nanostructured fibrin-agarose biomaterial containing human allogeneic stromal keratocytes and cornea epithelial cells, mimicking the human native anterior cornea in terms of optical, mechanical, and biological behavior. This product is manufactured as a clinical-grade tissue engineering product, fulfilling European requirements and regulations. The clinical translation process included several phases: an initial in vitro and in vivo preclinical research plan, including preclinical advice from the Spanish Medicines Agency followed by additional preclinical development, the adaptation of the biofabrication protocols to a good manufacturing practice manufacturing process, including all quality controls required, and the design of an advanced therapy clinical trial. The experimental development and successful translation of advanced therapy medicinal products for clinical application has to overcome many obstacles, especially when undertaken by academia or SMEs. We expect that our experience and research strategy may help future researchers to efficiently transfer their preclinical results into the clinical settings.

Non-arteritic anterior ischemic optic neuropathy (NAON) is the second most common optic neuropathy in adults. Despite extensive study, the etiology of NAION is not definitively known. The best evidence suggests that NAION is caused by an infarction in the region of the optic nerve head (ONH), which is perfused by paraoptic short posterior ciliary arteries (sPCAs) and their branches. To examine the gaps in knowledge that defies our understanding of NAION, a historical review was performed both of anatomical investigations of the ONH and its relevant blood vessels and the evolution of clinical understanding of NAION. Notably, almost all of the in vitro vascular research was performed prior our current understanding of NAION, which has largely precluded a hypothesis-based laboratory approach to study the etiological conundrum of NAION. More recent investigative techniques, like fluorescein angiography, have provided valuable insight into vascular physiology, but such light-based techniques have not been able to image blood vessels located within or behind the dense connective tissue of the sclera and laminar cribrosa, sites that are likely culpable in NAION. The lingering gaps in knowledge clarify investigative paths that might be taken to uncover the pathogenesis of NAION and possibly glaucoma, the most common optic neuropathy for which evidence of a vascular pathology also exists.

BACKGROUND/AIMS: Microperimetry is a technique that is increasingly used to assess visual function in age-related macular degeneration (AMD). In this study, we aimed to evaluate the relationship between retinal sensitivity measured with macular integrity assessment (MAIA) microperimetry and optical coherence tomography (OCT)-based macular morphology in AMD. METHODS: Prospective, cross-sectional study. All participants were imaged with colour fundus photographs used for AMD staging (Age-Related Eye Disease Study scale), spectral-domain OCT (Spectralis, Heidelberg, Germany) and swept-source OCT (Topcon, Japan). Threshold retinal sensitivity of the central 10° diameter circle was assessed with the full-threshold, 37-point protocol of the MAIA microperimetry device (Centervue, Italy). Univariable and multivariable multilevel mixed-effect linear regression models were used for analysis. RESULTS: We included 102 eyes with AMD and 46 control eyes. Multivariable analysis revealed that older age (p<0.0001), advanced AMD stage (p<0.0001) and reduced retinal thickness (p<0.0001) were associated with decreased mean retinal sensitivity. No associations were found between choroidal thickness and retinal sensitivity within the macula. Within the 10° diameter circle of the macula, the presence of ellipsoid disruption, subretinal fluid, atrophy and fibrosis, and outer retinal tubulation on OCT images was also associated with decreased retinal sensitivity (all p<0.05). CONCLUSIONS: There is an association between TRS as determined by MAIA microperimetry and several OCT structural parameters across various stages of AMD. This study highlights the relevance of microperimetry as a functional outcome measure for AMD.

AIM: To determine the association between dementia and age-related macular degeneration (AMD) using meta-analysis. METHODS: We searched in the MEDLINE, EMBASE, Web of Knowledge, PsycInfo and Cochrane database of systematic reviews for studies published from March 1959 to March 2018. We included cross-sectional, case-control and cohort studies that evaluated the association of dementia/Alzheimer's disease (AD) with AMD (as outcome) and the association of AMD with dementia/AD (as outcome). Studies that compared cognitive functions between AMD and controls were also included. The summary outcomes, namely odds ratio (OR), relative risk, mean differences and corresponding 95% CIs, were estimated using random effects models. We performed sensitivity analysis based on study quality and individual study effect to control for potential biases. RESULTS: Among 2159 citation records, we identified 21 studies consisting of 7 876 499 study subjects for meta-analysis. Patients with dementia (p≤0.017, OR≥1.24, I≤9%) or AD (p=0.001, OR=2.22, I=50%) were at risk for AMD, particularly for late AMD (p<0.001, OR=1.37, I=0). AMD was also significantly associated with increased risk of AD/cognitive impairment (p=0.037, OR=2.42, I=38%). Moreover, patients with AMD had poorer cognitive functions when compared with controls, including Mini-Mental State Examination (p<0.001, I≤79%) and Trail Making Test A (p<0.001, I=0). Sensitivity analysis and Egger's test indicated our results were less likely biased. CONCLUSIONS: A significant association between dementia/AD and AMD calls for greater clinical awareness. The cost-effectiveness of routine screening for the other condition in patients with primary diagnosis of dementia/AD or AMD requires further study.

BACKGROUND: Non-infectious uveitis (NIU) is an immune-mediated disease with clinical symptoms such as eye pain, redness, floaters, and light sensitivity. NIU is one of the leading causes of preventable blindness. OBJECTIVE: This review describes current and emerging therapies for NIU. METHODS: PubMed searches were conducted using the terms uveitis, therapy, corticosteroids, immunomodulators, biologics, intravitreal injections, intraocular implants, and adverse events deemed relevant if they presented data relating to prevalence, diagnosis, and treatment of uveitis. RESULTS: Diagnosis and management of NIU may require collaboration among different healthcare providers, including ophthalmologists and rheumatologists. Although many patients with NIU respond to corticosteroid (CS) therapy, long-term CS use can be associated with potentially severe adverse events. Localized CS therapies have been developed to reduce adverse events; however, some intravitreal injections and intraocular implants were linked to elevated intraocular pressure and cataracts. CS-sparing therapies such as biologics have demonstrated efficacy and safety while reducing CS burden. Biologics targeting tumor necrosis factor provide CS-sparing options for patients with NIU. Additional studies are needed to address long-term efficacy and safety of biologics targeting IL-6 and inhibitors of JAK/STAT. CONCLUSION: Biologics, JAK/STAT inhibitors, and improved localized therapies may provide additional options for patients with NIU.

The purpose of this review was to identify areas of consensus and disagreement among experts for the definition of success following strabismus surgery using the Delphi process. Three rounds of electronic questionnaires were sent to a panel of 28 strabismus experts. Throughout the process, members of the panel were masked to one another's identities to minimize the possibility of influence among members. Prior to data collection, we defined consensus as an 85% agreement on the answer to each question. Questions for which there was no consensus were reworded, and the resultant new questions were used in each subsequent round of questioning. We arrived at consensus for 23 of the 36 questions (64%). Consensus was obtained for recommending unique criteria for the definition of success for certain specific strabismus conditions. In addition, it was considered important that stereopsis and the range of single binocular vision be included in the definition of success for certain types of strabismus.

PURPOSE: To describe the long-term effect of a treatment protocol for ocular involvement in acute Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), including focused ocular examination and pathology-appropriate use of lubrication, topical corticosteroids, topical antibiotics, and amniotic membrane transplantation (AMT). DESIGN: Retrospective, comparative case series. METHODS: A total of 48 patients (96 eyes) were included in this study. Nine of 48 patients (18 eyes) had acute SJS/TEN from 2000 to 2007 and did not receive protocol care (Group I). Thirty-nine of 48 patients (78 eyes) had acute SJS/TEN from 2008 to 2017 and received protocol care (Group II). The main outcome measures were best-corrected visual acuity (BCVA) at final follow-up visit and incidence of complications in the chronic phase. RESULTS: No eyes in Group I received AMT for SJS/TEN, compared to 87% of qualifying eyes in Group II (P < .0001) There was a significant difference in the proportion of eyes with BCVA ≥20/40 at last follow-up between Group I and Group II (33% vs 92%, P < .001). The proportion of eyes with vision-threatening complications in the chronic phase was significantly higher in Group I versus Group II (67% vs 17%, P = .002), with most complications occurring in the first 2 years after disease onset in both groups. CONCLUSIONS: A specific protocol for acute ocular care in SJS/TEN, including aggressive use of AMT, was highly successful in reducing corneal blindness and severe vision-threatening complications of the disorder.

PURPOSE: To evaluate the finding of anomalous superior oblique muscles in congenital fibrosis of the extraocular muscles (CFEOM), a feature not previously emphasized in this condition. METHODS: The medical records of all patients clinically or genetically diagnosed with CFEOM at Boston Children's Hospital between 2010 and 2018 were reviewed retrospectively. Those who underwent strabismus surgery during the study period were included in the analysis. Baseline patient characteristics, type of CFEOM, results of genetic testing, and intraoperative features of the superior oblique muscle or tendon were recorded. RESULTS: Of 24 patients identified (age range, 1 month to 62 years), 10 (42%) had genetically confirmed CFEOM, and 22 underwent strabismus surgery, 14 (64%) involving the superior oblique muscle. Of these, 7 (50%) had anomalously inserted tendons (most commonly attached nasal to the superior rectus muscle), whereas 7 (50%) had increased superior oblique muscle tension. CONCLUSIONS: Half of CFEOM patients who underwent superior oblique surgery had abnormally inserted superior oblique tendons, and 50% had tight muscles or abnormally thin tendons, findings that have not been well-characterized in this condition. The findings suggest that abnormal insertion of the superior oblique muscles and tendons are additional features of the disease process in CFEOM that have not been described previously. These features may contribute to the severe upgaze limitation in CFEOM and highlight the importance of superior oblique tenotomy in surgical management.

Adeno-associated viruses (AAVs) have been employed successfully as gene therapy vectors in treating various genetic diseases for almost two decades. However, transgene packaging is usually imperfect, and developing a rapid and accurate method for measuring the proportion of DNA encapsidation is an important step for improving the downstream process of large scale vector production. In this study, we used two-dimensional class averages and three-dimensional classes, intermediate outputs in the single particle cryo-electron microscopy (cryo-EM) image reconstruction pipeline, to determine the proportion of DNA-packaged and empty capsid populations. Two different preparations of AAV3 were analyzed to estimate the minimum number of particles required to be sampled by cryo-EM in order for robust calculation of the proportion of the full versus empty capsids in any given sample. Cost analysis applied to the minimum amount of data required for a valid ratio suggests that cryo-EM is an effective approach to analyze vector preparations.