Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.

PURPOSE: Amino-amide or amino-ester local anesthetics, which are currently used for topical ocular anesthesia, are short acting and may delay corneal healing with long-term use. In contrast, site 1 sodium channel blockers (S1SCBs) are potent local anesthetics with minimal adverse tissue reaction. In this study, we examined topical local anesthesia with two S1SCBs, tetrodotoxin (TTX) or saxitoxin (STX) individually or in combination with α2-adrenergic receptor agonists (dexmedetomidine or clonidine), and compared them with the amino-ester ocular anesthetic proparacaine. The effect of test solutions on corneal healing was also studied. METHODS: Solutions of TTX ± dexmedetomidine, TTX ± clonidine, STX ± dexmedetomidine, dexmedetomidine, or proparacaine were applied to the rat cornea. Tactile sensitivity was measured by recording the blink response to probing of the cornea with a Cochet-Bonnet esthesiometer. The duration of corneal anesthesia was calculated. Cytotoxicity from anesthetic solutions was measured in vitro. The effect on corneal healing was measured in vivo after corneal debridement followed by repeated drug administration. RESULTS: Addition of dexmedetomidine to TTX or STX significantly prolonged corneal anesthesia beyond that of either drug alone, whereas clonidine did not. Tetrodotoxin or STX coadministered with dexmedetomidine resulted in two to three times longer corneal anesthesia than did proparacaine. S1SCB-dexmedetomidine formulations were not cytotoxic. Corneal healing was not delayed significantly by any of the test solutions. CONCLUSIONS: Coadministration of S1SCBs with dexmedetomidine provided prolonged corneal anesthesia without delaying corneal wound healing. Such formulations may be useful for the management of acute surgical and nonsurgical corneal pain.

PURPOSE/AIMS: To assess the effect of storage temperature on the viability, phenotype, metabolism, and morphology of cultured human oral keratinocytes (HOK). MATERIALS AND METHODS: Cultured HOK cells were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium (MEM) at nine temperatures in approximately 4°C increments from 4°C to 37°C for seven days. Cells were characterized for viability by calcein fluorescence, phenotype retention by immunocytochemistry, metabolic parameters (pH, glucose, lactate, and O2) within the storage medium by blood gas analysis, and morphology by scanning electron microscopy and light microscopy. RESULTS: Relative to the cultured, but non-stored control cells, a high percentage of viable cells were retained only in the 12°C and 16°C storage groups (85%±13% and 68%±10%, respectively). Expression of ABCG2, Bmi1, C/EBPδ, PCNA, cytokeratin 18, and caspase-3 were preserved after storage in the 5 groups between 4°C and 20°C, compared to the non-stored control. Glucose, pH and pO2 in the storage medium declined, whereas lactate increased with increasing storage temperature. Morphology was best preserved following storage of the three groups between 12°C, 16°C, and 20°C. CONCLUSION: We conclude that storage temperatures of 12°C and 16°C were optimal for maintenance of cell viability, phenotype, and morphology of cultured HOK. The storage method described in the present study may be applicable for other cell types and tissues; thus its significance may extend beyond HOK and the field of ophthalmology.

OBJECTIVES: Novel therapeutics are an important part of ophthalmologists' armamentarium, and the risks and benefits of these therapies must be carefully evaluated. We sought to quantify the characteristics of the pivotal clinical trials supporting the regulatory approval of new ophthalmic drugs and medical devices. DESIGN: Retrospective observational study. SETTING AND DATA SOURCE: Medical review dossiers for new ophthalmic drug and high-risk device approvals released publicly by the US Food and Drug Administration (FDA). MAIN OUTCOME MEASURES: Proportion of pivotal trials with randomisation, masking, active or placebo controls and subgroup analyses; total and median number of trial enrollees; and the number of drugs and devices approved with required postapproval studies. RESULTS: From 2002 to 2012, the FDA approved 11 ophthalmic drugs and 25 devices. The pivotal trials underlying the approvals of ophthalmic drugs in our study cohort enrolled a median of 809 patients. Virtually all drug trials were randomised and masked (91%), of which 7 (70%) used a placebo control. Pivotal trials for ophthalmic devices enrolled 324 patients on average, and significantly fewer trials for ophthalmic devices versus drugs were randomised (16% vs 91%; p<0.001) or masked (12% vs 91%; p<0.001). 8 (32%) ophthalmic devices and 6 (55%) ophthalmic drugs were approved with required postapproval studies. CONCLUSIONS: Ophthalmic therapeutics were approved based on varying levels of evidence. Postapproval studies could be used to confirm or refute early indications of safety and effectiveness of these therapeutics, with the study results accessible to patients and clinicians who need to make informed treatment decisions.

The orbital branch of the infraorbital artery, a key vascular structure that is not universally noted in orbital textbooks and atlases, is clinically significant, since injury to it can result in perioperative hemorrhage. We conducted a cadaver dissection to document its presence, measure its location, and evaluate it histopathologically. It was present in 8 of 9 orbits and was a mean distance of 16.6 mm (range 10-23) from the inferior orbital rim. In half of the specimens, there were 2 separate structures seen. Histopathology confirmed these structures to be neurovascular bundles.

In the study presented by D. S. Kohane and co-workers on page 1159, fluorescein molecules are initially bound to collagen fibers through UV-sensitive bonds. Collagen fibers are exposed to NIR light, which is upconverted to UV light through second harmonic generation. The UV-sensitive bonds absorb the upconverted UV light and undergo an irreversible cleavage releasing the fluorescein molecules.

OBJECTIVE: To evaluate the ability of trained nonphysician retinal imagers to perform diabetic retinopathy (DR) evaluation at the time of ultrawide field retinal (UWF) imaging in a teleophthalmology program. RESEARCH DESIGN AND METHODS: Clinic patients with diabetes received Joslin Vision Network protocol retinal imaging as part of their standard medical care. Retinal imagers evaluated UWF images for referable DR at the time of image capture. Training of the imagers included 4 h of standardized didactic lectures and 12 h of guided image review. Real-time evaluations were compared with standard masked gradings performed at a centralized reading center. RESULTS: A total of 3,978 eyes of 1,989 consecutive patients were imaged and evaluated. By reading center evaluation, 3,769 eyes (94.7%) were gradable for DR, 1,376 (36.5%) had DR, and 580 (15.3%) had referable DR. Compared with the reading center, real-time image evaluation had a sensitivity and specificity for identifying more than minimal DR of 0.95 (95% CI 0.94-0.97) and 0.84 (0.82-0.85), respectively, and 0.99 (0.97-1.00) and 0.76 (0.75-0.78), respectively, for detecting referable DR. Only three patients with referable DR were not identified by imager evaluation. CONCLUSIONS: Point-of-care evaluation of UWF images by nonphysician imagers following standardized acquisition and evaluation protocols within an established teleophthalmology program had good sensitivity and specificity for detection of DR and for identification of referable retinal disease. With immediate image evaluation, <0.1% of patients with referable DR would be missed, reading center image grading burden would be reduced by 60%, and patient feedback would be expedited.

Second harmonic generation is a process through which nonlinear materials such as collagen can absorb two photons and scatter one with twice the energy. Collagen upconverts 730 nm (near-IR) to 365 nm (UV) through second harmonic generation, which cleaves a molecule bound to collagen via a UV-sensitive linker.

NR2E3 encodes the photoreceptor-specific nuclear hormone receptor that acts as a repressor of cone-specific gene expression in rod photoreceptors, and as an activator of several rod-specific genes. Recessive variants located in the ligand-binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive- (S-) cone syndrome (ESCS), a retinal degeneration characterized by an excess of S-cones and non-functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease-causing LBD missense variants by bioluminescence resonance energy transfer (BRET(2) ) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q, and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P, and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev-erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.

IMPORTANCE: Brain imaging and fluid biomarkers are characterized in children at risk for autosomal dominant Alzheimer disease (ADAD). OBJECTIVE: To characterize and compare structural magnetic resonance imaging (MRI), resting-state and task-dependent functional MRI, and plasma amyloid-β (Aβ) measurements in presenilin 1 (PSEN1) E280A mutation-carrying and noncarrying children with ADAD. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional measures of structural and functional MRI and plasma Aβ assays were assessed in 18 PSEN1 E280A carriers and 19 noncarriers aged 9 to 17 years from a Colombian kindred with ADAD. Recruitment and data collection for this study were conducted at the University of Antioquia and the Hospital Pablo Tobon Uribe in Medellín, Colombia, between August 2011 and June 2012. MAIN OUTCOMES AND MEASURES: All participants had blood sampling, structural MRI, and functional MRI during associative memory encoding and resting-state and cognitive assessments. Outcome measures included plasma Aβ1-42 concentrations and Aβ1-42:Aβ1-40 ratios, memory encoding-dependent activation changes, resting-state connectivity, and regional gray matter volumes. Structural and functional MRI data were compared using automated brain mapping algorithms and search regions related to AD. RESULTS: Similar to findings in adult mutation carriers, in the later preclinical and clinical stages of ADAD, mutation-carrying children were distinguished from control individuals by significantly higher plasma Aβ1-42 levels (mean [SD]: carriers, 18.8 [5.1] pg/mL and noncarriers, 13.1 [3.2] pg/mL; P < .001) and Aβ1-42:Aβ1-40 ratios (mean [SD]: carriers, 0.32 [0.06] and noncarriers, 0.21 [0.03]; P < .001), as well as less memory encoding task-related deactivation in parietal regions (eg, mean [SD] parameter estimates for the right precuneus were -0.590 [0.50] for noncarriers and -0.087 [0.38] for carriers; P < .005 uncorrected). Unlike carriers in the later stages, mutation-carrying children demonstrated increased functional connectivity of the posterior cingulate cortex with medial temporal lobe regions (mean [SD] parameter estimates were 0.038 [0.070] for noncarriers and 0.190 [0.057] for carriers), as well as greater gray matter volumes in temporal regions (eg, left parahippocampus; P < . 049, corrected for multiple comparisons). CONCLUSIONS AND RELEVANCE: Children at genetic risk for ADAD have functional and structural brain changes and abnormal levels of plasma Aβ1-42. The extent to which the underlying brain changes are either neurodegenerative or developmental remains to be determined. This study provides additional information about the earliest known biomarker changes associated with ADAD.