Molecular characterization of foveal versus peripheral human retina by single-cell RNA sequencing.

The human retina is a complex tissue responsible for detecting photons of light and converting information from these photons into the neurochemical signals interpreted as vision. Such visual signaling not only requires sophisticated interactions between multiple classes of neurons, but also spatially-dependent molecular specialization of individual cell types. In this study, we performed single-cell RNA sequencing on neural retina isolated from both the fovea and peripheral retina in three human donors. We recovered a total of 8,217 cells, with 3,578 cells originating from the fovea and 4,639 cells originating from the periphery. Expression profiles for all major retinal cell types were compiled, and differential expression analysis was performed between cells of foveal versus peripheral origin. Globally, mRNA for the serum iron binding protein transferrin (TF), which has been associated with age-related macular degeneration pathogenesis, was enriched in peripheral samples. Cone photoreceptor cells were of particular interest and formed two predominant clusters based on gene expression. One cone cluster had 96% of cells originating from foveal samples, while the second cone cluster consisted exclusively of peripherally isolated cells. A total of 148 genes were differentially expressed between cones from the fovea versus periphery. Interestingly, peripheral cones were enriched for the gene encoding Beta-Carotene Oxygenase 2 (BCO2). A relative deficiency of this enzyme may account for the accumulation of carotenoids responsible for yellow pigment deposition within the macula. Overall, this data set provides rich expression profiles of the major human retinal cell types and highlights transcriptomic features that distinguish foveal and peripheral cells.

CEP290 gene transfer rescues Leber congenital amaurosis cellular phenotype.

Mutations in CEP290 are the most common cause of Leber congenital amaurosis (LCA), a severe inherited retinal degenerative disease for which there is currently no cure. Autosomal recessive CEP290-associated LCA is a good candidate for gene replacement therapy, and cells derived from affected individuals give researchers the ability to study human disease and therapeutic gene correction in vitro. Here we report the development of lentiviral vectors carrying full-length CEP290 for the purpose of correcting the CEP290 disease-specific phenotype in human cells. A lentiviral vector containing CMV-driven human full-length CEP290 was constructed. Following transduction of patient-specific, iPSC-derived, photoreceptor precursor cells, reverse transcriptase-PCR analysis and western blotting revealed vector-derived expression. As CEP290 is important in ciliogenesis, the ability of fibroblast cultures from CEP290-associated LCA patients to form cilia was investigated. In cultures derived from these patients, fewer cells formed cilia compared with unaffected controls. Cilia that were formed were shorter in patient-derived cells than in cells from unaffected individuals. Importantly, lentiviral delivery of CEP290 rescued the ciliogenesis defect. The successful construction and viral transfer of full-length CEP290 brings us closer to the goal of providing gene- and cell-based therapies for patients affected with this common form of LCA.

External pneumatic compression and fibrinolysis in abdominal surgery.

INTRODUCTION: External pneumatic compression (EPC) devices prevent lower extremity deep venous thrombosis by increasing venous flow and thereby reducing stasis. Early studies suggested that they also enhance systemic fibrinolytic activity and thus prevent thrombus formation; more recent studies have been conflicting. The hypothesis of this study was that EPC devices enhance systemic fibrinolysis or reduce postoperative fibrinolytic impairment in patients undergoing abdominal surgical procedures. METHODS: Each of 48 patients (98% male; mean age, 67 years) undergoing major intra-abdominal surgical procedures (36 bowel procedures, 12 aortic reconstructions) was prospectively randomized to one of three treatments for deep venous thrombosis prophylaxis: subcutaneous heparin injections (HEP group), use of a thigh-length sequential EPC device (EPC group), or both (HEP + EPC group). Antecubital venous samples were collected for measurement of systemic fibrinolytic activity on the day before surgery, after induction of anesthesia but before prophylaxis was initiated, and on postoperative days 1, 3, and 5. Fibrinolysis was assessed through measurement of the activities of the rate limiting fibrinolytic activator, tissue plasminogen activator, and its inhibitor plasminogen activator inhibitor-1 with amidolytic methods. RESULTS: On the day before surgery, plasminogen activator inhibitor-1 activity was elevated in all groups in comparison with that in age-matched and sex-matched controls (20.3 +/- 0.6 AU/mL). In the HEP group, plasminogen activator inhibitor-1 activity was further elevated above the value for the day before surgery on postoperative day 1 (28.5 +/- 4.3 AU/mL; P =.04) and postoperative day 3 (25.1 +/- 1.9 AU/mL; P =.07). No significant decrease in plasminogen activator inhibitor-1 activity occurred in either group treated with EPC devices in comparison with the HEP group at any time. There were no changes in tissue plasminogen activator activity postoperatively in the HEP group and no significant increases in either EPC group at any point. CONCLUSIONS: Reduced systemic fibrinolytic activity ("fibrinolytic shutdown") occurred in these patients after abdominal surgery; it was manifested as increased plasminogen activator inhibitor-1 activity. EPC devices did not enhance systemic fibrinolysis or prevent postoperative shutdown either by decreasing plasminogen activator inhibitor-1 activity or by increasing tissue plasminogen activator activity. These data suggest that EPC devices do not prevent deep venous thrombosis by fibrinolytic enhancement; effective prophylaxis is achieved only when the devices are used in a manner that reduces lower extremity venous stasis.

A 5-year evaluation of the adenoclone test for the rapid diagnosis of adenovirus from conjunctival swabs.

The rapid diagnosis of adenoviral ocular infections affords the opportunity to limit the transmission of virus within the community and avoid expensive, unnecessary, and ineffective therapy. This study evaluated the results of a 5-year experience with the Adenoclone test (Cambridge Biotech, Worcester, MA), an enzyme immunoassay, applied directly to conjunctival swabs obtained from infected eyes. The sensitivity of this test was determined on 372 consecutive adenovirus culture-positive ocular specimens. A subset of 106 specimens was evaluated, including a retrospective chart review to determine the relationship between the Adenoclone result and the time to viral cytopathic effect (CPE) in A549 cell culture, ocular titers (90% tissue culture infectious dose; TCID90), serotype, and associated clinical parameters. Overall, the sensitivity for Adenoclone was 38% (142 of 372), which improved to 65% (129 of 199) for samples positive in culture during the first week. A positive Adenoclone test result was associated with a shorter time to CPE in cell culture (p = 0.0001). The mean ocular titers (log TCID90) associated with a positive test result were found to be at a significantly higher dilution than a negative result (-1.70 +/- 0.93 vs. -0.88 +/- 1.00, p < 0.0001). A positive Adenoclone outcome was independent of the serotype but directly associated with a recent visit to an ophthalmologist's office, follicular conjunctivitis, and conjunctival chemosis. For the rapid diagnosis of adenoviral ocular infections, the Adenoclone test remains useful, but a more sensitive test based on nonradioactive amplification is eagerly anticipated.

Biological activity of N-(4-hydroxyphenyl) retinamide-O-glucuronide in corneal and conjunctival cells of rabbits and humans.

Previous studies of topical retinoic acid for treatment of ocular surface disease met with limited success due to instability and irritancy of the retinoid and lack of efficacy in keratoconjunctivitis sicca. There has, however, been continued interest in the treatment of mucin deficiency and cicatrizing conjunctival diseases, such as ocular cicatricial pemphigoid (OCP), topically with retinoids. In this study the biological activity of stable, water-soluble, synthetic retinoid, N-(4-hydroxyphenyl) retinamide-O-glucuronide (4-HPROG) was investigated in vivo and in vitro using conjunctival and corneal epithelium and fibroblasts. Vitamin A-deficient rabbits with stage 3-4 corneal xerosis and squamous metaplasia confirmed by conjunctival impression cytology were treated with topical 0.1% 4-HPROG in an artificial tear vehicle for 3 weeks. Impression cytology was repeated at 2 and 3 weeks and at 3 weeks conjunctival biopsies were fixed for histology. Growth curves were generated using conjunctival fibroblasts of rabbits and humans (normals and patients with cicatrizing conjunctival disease including OCP and Stevens-Johnson syndrome) cultured in the 10(-8)-10(-6) M 4-HPROG. In vivo, corneal xerosis cleared in three days. A normal conjunctival epithelium was restored by 2 weeks and goblet cells were present by 3 wk, with no change in vehicle-treated controls. No ocular irritation occurred. In vitro, 10(-6) M 4-HPROG inhibits growth of rabbit conjunctival fibroblasts. The retinoid had no effect on proliferation of conjunctival fibroblasts from normal humans but the doubling time of cells from patients with OCP increased significantly, from 50.9 +/- 10.01 h (control) to 61.5 +/- 8.95 h (retinoid). Proliferation of conjunctival fibroblasts from a patient with Stevens-Johnson syndrome was also inhibited. N-(4-hydroxyphenyl) retinamide-O-glucuronide is biologically active and merits further study to determine its efficacy in controlling conjunctival fibrosis and treating ocular surface squamous metaplasia.

The multipotential cells of the limbus.

Ample evidence exists that there is a centripetal movement of cells from the periphery of the cornea toward the centre. While conjunctival cells have the capacity to transdifferentiate into corneal epithelial cells, the limbal region appears to act as a barrier between the conjunctival and corneal epithelia, even after large epithelial defects are created. The existence of limbal stem cells is suggested by the apparent role of the limbus in acting as a source of peripheral corneal cells. While specific staining of limbal cells has been reported in the rabbit, there is no positive identification of such stem cells in the human. However, in the human there is negative staining for both a keratin cytoskeleton antigen and a cell surface antigen in the limbal epithelial zone. Efforts positively to identify human limbal stem cells continue, as do efforts to culture and transplant such cells.

Standardization of formaldehyde-induced fluorescence and its measurement to quantify serotonin emission in pulmonary neuroendocrine cells.

We describe a modified and standardized quantitative FIF procedure for producing fluorophores and measuring emission intensity of serotonin-containing neuroepithelial bodies (NEB's) in the rabbit lung. This technique, using epifluorescence, was reproduced without significant differences between control groups. Important considerations for reproducibility were: using the same humidity (80% RH) and reaction time (2 h) during the vapor treatment, sectioning at constant relative humidity, avoiding unnecessary heating (sections should not be stretched over a hot plate) and avoiding exposure of sections to light. Optimal emission readings were obtained with sectioning and mounting at 40--50% RH. Readings were reduced by 25% when the mercury light source was switched from 200 W to 100 W. It was also important to let the instruments warm up long enough to avoid drift during quantitation. Each NEB should be subjected to the same duration of light exposure for alignment (30 s) before measuring fluorescence to avoid differences from photodecomposition.

Pulmonary neuroendocrine cells: decreased serotonin fluorescence and stable argyrophil-cell numbers in acute hypoxia.

We recorded serotonin-emission intensity using formalin induced fluorescence and counted the number of neuroendocrine cells (NEC's) and neuroepithelial bodies (NEB's) containing argyrophil granules. Comparison of serotonin-emission intensity of NEB's of acutely hypoxic neonates (520 mmHg for 2-2 1/2 h) and normoxic controls showed significantly lower fluorescence in hypoxic animals. Neuroendocrine cell numbers as shown wit Grimelius silver-nitrate stain did not change. This suggests either that the Grimelius stain reacts with compounds other than serotonin, or that the decrease in serotonin in acutely hypoxic rabbits is not sufficient to alter argyrophil stainability.