April 2017

PURPOSE: To describe a case of bilateral endogenous cryptococcal endophthalmitis in an immunocompetent host and to review adjunctive ophthalmic imaging patterns and treatment. METHODS: A retrospective case report. RESULTS: A 45-year-old female patient with two distinct presentations of endogenous cryptococcal endophthalmitis in each eye presented initially with progressive blurred vision in the left eye, beginning more than 10 years after a craniotomy with ventriculoperitoneal shunt. Complete ophthalmic imaging was conducted and compared with data from previous literature. Administration of amphotericin-B had poorly responded; however, consolidation of fluconazole resulted in disease stabilization. CONCLUSIONS: Bilateral intraocular cryptococcal infection can present with two distinct patterns of posterior segment findings. A review of ophthalmic imaging patterns found consistency in some characteristics of A-scan ultrasonogram and fundus fluorescein angiogram. Besides conventional treatment, voriconazole is likely to play an important role in the management of cryptococcal endophthalmitis.

PURPOSE: To compare diagnostic performance and structure-function correlations of multifocal electroretinogram (mfERG), full-field flash ERG (ff-ERG) photopic negative response (PhNR) and transient pattern-reversal ERG (PERG) in a non-human primate (NHP) model of experimental glaucoma (EG). METHODS: At baseline and after induction of chronic unilateral IOP elevation, 43 NHP had alternating weekly recordings of retinal nerve fiber layer thickness (RNFLT) by spectral domain OCT (Spectralis) and retinal function by mfERG (7F slow-sequence stimulus, VERIS), ff-ERG (red 0.42 log cd-s/m(2) flashes on blue 30 scotopic cd/m(2) background, LKC UTAS-E3000), and PERG (0.8° checks, 99% contrast, 100 cd/m(2) mean, 5 reversals/s, VERIS). All NHP were followed at least until HRT-confirmed optic nerve head posterior deformation, most to later stages. mfERG responses were filtered into low- and high-frequency components (LFC, HFC, >75 Hz). Peak-to-trough amplitudes of LFC features (N1, P1, N2) and HFC RMS amplitudes were measured and ratios calculated for HFC:P1 and N2:P1. ff-ERG parameters included A-wave (at 10 ms), B-wave (trough-to-peak) and PhNR (baseline-to-trough) amplitudes as well as PhNR:B-wave ratio. PERG parameters included P50 and N95 amplitudes as well as N95:P50 ratio and N95 slope. Diagnostic performance of retinal function parameters was compared using the area under the receiver operating characteristic curve (A-ROC) to discriminate between EG and control eyes. Correlations to RNFLT were compared using Steiger's test. RESULTS: Study duration was 15 ± 8 months. At final follow-up, structural damage in EG eyes measured by RNFLT ranged from 9% above baseline (BL) to 58% below BL; 29/43 EG eyes (67%) and 0/43 of the fellow control eyes exhibited significant (>7%) loss of RNFLT from BL. Using raw parameter values, the largest A-ROC findings for mfERG were: HFC (0.82) and HFC:P1 (0.90); for ff-ERG: PhNR (0.90) and PhNR:B-wave (0.88) and for PERG: P50 (0.64) and N95 (0.61). A-ROC increased when data were expressed as % change from BL, but the pattern of results persisted. At 95% specificity, the diagnostic sensitivity of mfERG HFC:P1 ratio was best, followed by PhNR and PERG. The correlation to RNFLT was stronger for mfERG HFC (R = 0.65) than for PhNR (R = 0.59) or PERG N95 (R = 0.36), (p = 0.20, p = 0.0006, respectively). The PhNR flagged a few EG eyes at the final time point that had not been flagged by mfERG HFC or PERG. CONCLUSIONS: Diagnostic performance and structure-function correlation were strongest for mfERG HFC as compared with ff-ERG PhNR or PERG in NHP EG.

Posner-Schlossman syndrome (PSS) shares some clinical features with uveitis and open angle glaucoma. Cytokines and autoantibodies have been associated with uveitis and open angle glaucoma. However, the role of serum cytokines and autoantibodies in the pathogenesis of PSS remains unknown. This study aimed to evaluate the associations of type 1 T helper (Th1) and Th17 related cytokines and autoantibodies with PSS. Peripheral blood serum samples were collected from 81 patients with PSS and 97 gender- and age-matched healthy blood donors. Th1 and Th17 related cytokines, including interleukin-1β (IL-1β), IL-12, tumor necrosis factor-α (TNF-α), interferon- γ (IFN-γ), IL-6 and IL-17, and glucose-6-phosphate isomerase (GPI) were determined by double antibody sandwich ELISA. Anti-nuclear antibody (ANA), anti-keratin antibody (AKA) and anti-neutrophil cytoplasmic antibody (ANCA) were detected by indirect immunofluorescence assay. Anti-cardiolipin antibody (ACA)-IgG, ACA-IgM, ACA-IgA, anti-double stranded DNA (anti-dsDNA) and anti-cyclic citrullinated peptide antibody (anti-CCP) were detected by indirect ELISA. Serum levels of IL-1β, IL-12 and IL-6 in PSS patients were significantly lower than those in controls (P < 0.003), and these associations survived the Bonferroni correction (Pc < 0.018). There was no significant difference in serum levels of TNF-α, IFN-γ and IL-17 between the PSS and control groups (Pc > 0.12). Positive rate of serum anti-dsDNA in PSS patients was significantly higher than that in the control group (P = 0.002, Pc = 0.018), while positive rates of serum ANA, AKA, ANCA, ACA-IgG, ACA-IgM, ACA-IgA, GPI and anti-CCP in the PSS group were not significantly different from those in the control group (Pc > 0.09). These results suggest that anti-dsDNA may contribute to the pathogenesis of PSS, while Th1 and Th17 related cytokines and other autoantibodies may not be major contributors to PSS.

Whole-brain networks derived from diffusion tensor imaging (DTI) data require the identification of seed and target regions of interest (ROIs) to assess connectivity patterns. This study investigated how initiating tracts from gray matter (GM) or white matter (WM) seed ROIs impacts (1) structural networks constructed from DTI data from healthy elderly (control) and individuals with Alzheimer's disease (AD) and (2) between-group comparisons using these networks. DTI datasets were obtained from the Alzheimer's disease Neuroimaging Initiative database. Deterministic tractography was used to build two whole-brain networks for each subject; one in which tracts were initiated from WM ROIs and another in which they were initiated from GM ROIs. With respect to the first goal, in both groups, WM-seeded networks had approximately 400 more connections and stronger connections (as measured by number of streamlines per connection) than GM-seeded networks, but shared 94% of the connections found in the GM-seed networks. With respect to the second goal, between-group comparisons revealed a stronger subnetwork (as measured by number of streamlines per connection) in controls compared to AD using both WM-seeded and GM-seeded networks. The comparison using WM-seeded networks produced a larger (i.e., a greater number of connections) and more significant subnetwork in controls versus AD. Global, local, and nodal efficiency were greater in controls compared to AD, and between-group comparisons of these measures using WM-seeded networks had larger effect sizes than those using GM-seeded networks. These findings affirm that seed location significantly affects the ability to detect between-group differences in structural networks.

Human adenoviruses (HAdVs) shut down host cellular cap-dependent mRNA translation while initiating the translation of viral late mRNAs in a cap-independent manner. HAdV 5' untranslated regions (5'UTRs) are crucial for cap-independent initiation, and influence mRNA localization and stability. However, HAdV translational regulation remains relatively uncharacterized. The HAdV tripartite leader (TPL), composed of three introns (TPL 1-3), is critical to the translation of HAdV late mRNA. Herein, we annotated and analyzed 72 HAdV genotypes for the HAdV TPL and another previously described leader, the i-leader. Using HAdV species D, type 37 (HAdV-D37), we show by reverse transcription PCR and Sanger sequencing that mRNAs of the HAdV-D37 E3 transcription unit are spliced to the TPL. We also identified a polycistronic mRNA for RID-α and RID-β. Analysis of the i-leader revealed a potential open reading frame within the leader sequence and the termination of this potential protein in TPL3. A potential new leader embedded within the E3 region was also detected and tentatively named the j-leader. These results suggest an underappreciated complexity of post-transcriptional regulation, and the importance of HAdV 5'UTRs for precisely coordinated viral protein expression along the path from genotype to phenotype.

Transposons can be used to easily generate and label the location of mutations throughout bacterial and other genomes. Transposon insertion mutants may be screened for a phenotype as individual isolates, or by selection applied to a pool of thousands of mutants. Identifying the location of a transposon insertion is critical for connecting phenotype to the genetic lesion. In this unit, we present an easy and detailed approach for mapping transposon insertion sites using arbitrarily-primed PCR (AP-PCR). Two rounds of PCR are used to (1) amplify DNA spanning the transposon insertion junction, and (2) increase the specific yield of transposon insertion junction fragments for sequence analysis. The resulting sequence is mapped to a bacterial genome to identify the site of transposon insertion. In this protocol, AP-PCR as it is routinely used to map sites of transposon insertion within Staphylococcus aureus, is used to illustrate the principle. Guidelines are provided for adapting this protocol for mapping insertions in other bacterial genomes. Mapping transposon insertions using this method is typically achieved in 2 to 3 days if starting from a culture of the transposon insertion mutant. © 2017 by John Wiley & Sons, Inc.

Antigen-presenting cells (APCs) play an important role in transplant rejection and tolerance. In high-risk corneal transplantation, where the graft bed is inflamed and vascularized, immature APCs in the donor corneal stroma quickly mature and migrate to lymphoid tissues to sensitize host T cells. In this study, using a mouse model of corneal transplantation, we investigated whether enrichment of tolerogenic APCs (tolAPCs) in donor corneas can enhance graft survival in corneal allograft recipients with inflamed graft beds. Treatment of donor corneas with interleukin-10 (IL-10) and transforming growth factor-β1 (TGFβ1) altered the phenotype and function of tissue-residing APCs. Transplantation of these tolAPC-enriched corneas decreased frequencies of interferon gamma (IFNγ)(+) effector T cells (Teffs), as well as allosensitization in the hosts, diminished graft infiltration of CD45(+) and CD4(+) cells, and significantly improved corneal allograft survival compared to saline-injected controls. These data provide a novel approach for tolAPC-based immunotherapy in transplantation by direct cytokine conditioning of the donor tissue.

OBJECTIVE: To examine early performance on an eye surgery simulator and its relationship to subsequent live surgical performance in a single large residency program. DESIGN: Retrospective study. SETTING: Massachusetts Eye and Ear, Harvard Medical School, Department of Ophthalmology. METHODS: In a retrospective study, we compared performance of 30 first-year ophthalmology residents on an eye surgery simulator to their surgical skills as third-year residents. Variables collected from the eye surgery simulator included scores on the following modules of the simulator (Eyesi, VRmagic, Mannheim, Germany): antitremor training level 1, bimanual training level 1, capsulorhexis level 1 (configured), forceps training level 1, and navigation training level 1. Subsequent surgical performance was assessed using the total number of phacoemulsification cataract surgery cases for each resident, as well as the number performed as surgeon during residency and scores on global rating assessment of skills in intraocular surgery (GRASIS) scales during the third year of residency. Spearman correlation coefficients were calculated between each of the simulator performance and subsequent surgical performance variables. We also compared variables in a small group of residents who needed extra help in learning cataract surgery to the other residents in the study. MAIN OUTCOME MEASURES: Relationships between Eyesi scores early in residency and surgical performance measures in the final year of residency. RESULTS: A total of 30 residents had Eyesi data from their first year of residency and had already graduated so that all subsequent surgical performance data were available. There was a significant correlation between capsulorhexis task score on the simulator and total surgeries (r = 0.745, p = 0.008). There was a significant correlation between antitremor training level 1 (r = 0.554, p = 0.040), and forceps training level 1 (r = 0.622, p = 0.023) with primary surgery numbers. There was a significant correlation between forceps training level 1 (r = 0.811, p = 0.002), and navigation training level 1 (r = 0.692, p =0.013) with total GRASIS score. There was a significant inverse correlation between total GRASIS score and residents in need of extra help (r = -0.358, p =0.003). CONCLUSION: Module scores on an eye surgery simulator early in residency may predict a resident׳s future performance in the operating room. These scores may allow early identification of residents in need of supplemental training in cataract surgery.

The use of viral vectors for inner ear gene therapy is receiving increased attention for treatment of genetic hearing disorders. Most animal studies to date have injected viral suspensions into neonatal ears, via the round window membrane. Achieving transduction of hair cells, or sensory neurons, throughout the cochlea has proven difficult, and no studies have been able to efficiently transduce sensory cells in adult ears while maintaining normal cochlear function. Here, we show, for the first time, successful transduction of all inner hair cells and the majority of outer hair cells in an adult cochlea via virus injection into the posterior semicircular canal. We used a "designer" AAV, AAV2/Anc80L65, in which the main capsid proteins approximate the ancestral sequence state of AAV1, 2, 8, and 9. Our injections also transduced ~10% of spiral ganglion cells and a much larger fraction of their satellite cells. In the vestibular sensory epithelia, the virus transduced large numbers of hair cells and virtually all the supporting cells, along with close to half of the vestibular ganglion cells. We conclude that this viral vector and this delivery route hold great promise for gene therapy applications in both cochlear and vestibular sense organs.

Transplantation of cultured oral mucosal epithelial cells (OMECs) is a promising treatment strategy for limbal stem cell deficiency. In order to improve the culture method, we investigated the effects of four culture media and tissue harvesting sites on explant attachment, growth, and phenotype of OMECs cultured from Sprague-Dawley rats. Neither choice of media or harvesting site impacted the ability of the explants to attach to the culture well. Dulbecco's modified Eagle's medium/Ham's F12 (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI) supported the largest cellular outgrowth. Fold outgrowth was superior from LL explants compared to explants from the buccal mucosa (BM), HP, and transition zone of the lower lip (TZ) after six-day culture. Putative stem cell markers were detected in cultures grown in DMEM and RPMI. In DMEM, cells from TZ showed higher colony-forming efficiency than LL, BM, and HP. In contrast to RPMI, DMEM both expressed the putative stem cell marker Bmi-1 and yielded cell colonies. Our data suggest that OMECs from LL and TZ cultured in DMEM give rise to undifferentiated cells with high growth capacity, and hence are the most promising for treatment of limbal stem cell deficiency.

Verteporfin (VP) was first used in Photodynamic therapy, where a non-thermal laser light (689 nm) in the presence of oxygen activates the drug to produce highly reactive oxygen radicals, resulting in local cell and tissue damage. However, it has also been shown that Verteporfin can have non-photoactivated effects such as interference with the YAP-TEAD complex of the HIPPO pathway, resulting in growth inhibition of several neoplasias. More recently, it was proposed that, another non-light mediated effect of VP is the formation of cross-linked oligomers and high molecular weight protein complexes (HMWC) that are hypothesized to interfere with autophagy and cell growth. Here, in a series of experiments, using human uveal melanoma cells (MEL 270), human embryonic kidney cells (HEK) and breast cancer cells (MCF7) we showed that Verteporfin-induced HMWC require the presence of light. Furthermore, we showed that the mechanism of this cross-linking, which involves both singlet oxygen and radical generation, can occur very efficiently even after lysis of the cells, if the lysate is not protected from ambient light. This work offers a better understanding regarding VP's mechanisms of action and suggests caution when one studies the non-light mediated actions of this drug.

Regulation of biological processes occurs through complex, synergistic mechanisms. In this study, we discovered the synergistic orchestration of multiple mechanisms regulating the normal and diseased state (age related macular degeneration, AMD) in the retina. We uncovered gene networks with overlapping feedback loops that are modulated by nuclear hormone receptors (NHR), miRNAs, and epigenetic factors. We utilized a comprehensive filtering and pathway analysis strategy comparing miRNA and microarray data between three mouse models and human donor eyes (normal and AMD). The mouse models lack key NHRS (Nr2e3, RORA) or epigenetic (Ezh2) factors. Fifty-four total miRNAs were differentially expressed, potentially targeting over 150 genes in 18 major representative networks including angiogenesis, metabolism, and immunity. We identified sixty-eight genes and 5 miRNAS directly regulated by NR2E3 and/or RORA. After a comprehensive analysis, we discovered multimodal regulation by miRNA, NHRs, and epigenetic factors of three miRNAs (miR-466, miR1187, and miR-710) and two genes (Ell2 and Entpd1) that are also associated with AMD. These studies provide insight into the complex, dynamic modulation of gene networks as well as their impact on human disease, and provide novel data for the development of innovative and more effective therapeutics.

Motor, sensory, and integrative activities of the brain are coordinated by a series of midline-bridging neuronal commissures whose development is tightly regulated. Here we report a new human syndrome in which these commissures are widely disrupted, thus causing clinical manifestations of horizontal gaze palsy, scoliosis, and intellectual disability. Affected individuals were found to possess biallelic loss-of-function mutations in the gene encoding the axon-guidance receptor 'deleted in colorectal carcinoma' (DCC), which has been implicated in congenital mirror movements when it is mutated in the heterozygous state but whose biallelic loss-of-function human phenotype has not been reported. Structural MRI and diffusion tractography demonstrated broad disorganization of white-matter tracts throughout the human central nervous system (CNS), including loss of all commissural tracts at multiple levels of the neuraxis. Combined with data from animal models, these findings show that DCC is a master regulator of midline crossing and development of white-matter projections throughout the human CNS.