Jose D. Rios

Sjögren’s Syndrome-Like Ocular Surface Disease in Thrombospondin-1 Deficient Mice

Thrombospondin-1 (TSP-1) is a major activator of latent transforming growth factor-beta in vitro as well as in vivo. Mice deficient in TSP-1, despite appearing normal at birth, develop a chronic form of ocular surface disease that is marked by increased apoptosis and deterioration in the lacrimal gland, associated dysfunction, and development of inflammatory infiltrates that result in abnormal tears. The increase in CD4(+) T cells in the inflammatory infiltrates of the lacrimal gland, and the presence of anti-Sjögrens syndrome antigen A and anti-Sjögrens syndrome antigen B antibodies in the serum resemble autoimmune Sjögrens syndrome. These mice develop an ocular surface disorder dry eye that includes disruption of the corneal epithelial layer, corneal edema, and a significant decline in conjuctival goblet cells. Externally, several mice develop dry crusty eyes that eventually close. The inflammatory CD4(+) T cells detected in the lacrimal gland, as well as those in the periphery of older TSP-1 null mice, secrete interleukin-17A, a cytokine associated with chronic inflammatory diseases. Antigen-presenting cells, derived from TSP-1 null, but not from wild-type mice, activate T cells to promote the Th17 response. Together, these results indicate that TSP-1 deficiency results in a spontaneous form of chronic dry eye and aberrant histopathology associated with Sjögrens syndrome.

Dexamethasone intravitreal implant for treatment of uveitic persistent cystoid macular edema in vitrectomized patients.

Purpose: To evaluate the safety and efficacy of Ozurdex (dexamethasone intravitreal implant) 0.7 mg in the treatment of uveitic macular edema in vitrectomized eyes. Methods: Data from 13 patients (17 eyes) with persistent uveitic cystoid macular edema and a history of pars plana vitrectomy in the study eyes that were treated with intravitreal injection of 0.7-mg dexamethasone implant were reviewed retrospectively. Main outcome measures were changes in central retinal thickness measured by optical coherence tomography and changes in best-corrected visual acuity. Results: The median age of patients was 61 years (range, 19–81 years). The median duration of uveitic macular edema was 12 months (range, 2–72 months). The mean baseline central retinal thickness (95% confidence interval) was 461.6 &mgr;m (403.8–519.4), decreased to 277.2 &mgr;m (244.6–309.8) at 4 weeks (P < 0.01), remained low at 349.9 &mgr;m (281.8–418.0) at 3 months (P = 0.01), and then reached 394.1 &mgr;m (328.3–459.8) at 6 months (P = 0.14). After 3 months, there was a median improvement of 2 lines of best-corrected visual acuity, with 52.9% of eyes gaining 2 lines or more (P < 0.01). At 6 months, there were 5 eyes that maintained the 2 lines gain and none had lost >1 line from baseline (P = 0.03). In 8 eyes (47.1%), reinjection of the implant was performed at a mean of 6.5 months. Ocular hypertension (47.1%), hypotony (11.8%), anterior chamber displacement of the implant (5.9%), and glaucoma, which required filtration surgery (5.9%), were the most common adverse events. Mean follow-up was 9.6 months (range, 6–17 months). Conclusion: In this small case series of eyes with limited follow-up, treatment with dexamethasone intravitreal implant injection for uveitic macular edema in vitrectomized eyes was associated with favorable visual outcomes and had an acceptable safety profile.

OPC-12759 increases proliferation of cultured rat conjunctival goblet cells.

Purpose: To determine if the gastroprotective drug OPC-12759 increased proliferation of rat conjunctival goblet cells in culture. Methods: Cultured goblet cells were incubated with 10−12 to 10−8 M OPC-12759 for 1 to 7 days. Fetal bovine serum (FBS) was used as a positive control. Cell proliferation was determined by a MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay and by immunohistochemical staining with anti-Ki-67, a marker of cell division. Goblet cells were identified by double-labeling with anti-Ki-67, a marker of cell division, and Ulex europaeus agglutinin I lectin, anti-MUC5AC and anticytokeratin 7. Stratified squamous cells were identified by using Griffonia (Bandeiraea) simplicifolia lectin and anticytokeratin 4 antibody. Results: As determined by MTT conversion to formazan, OPC-12579 at 10−11 M induced an almost 2-fold increase in goblet cell proliferation on Days 1 and 3 of incubation but not on Days 5 and 7. The FBS at 10% increased cell proliferation by 2- to 3-fold at each time point. Daily replenishment of OPC-12579 for 3 consecutive days induced cell proliferation at all concentrations. Proliferation as determined by the number of Ki-67 positive cells increased by 4- and 3-fold at Days 1 and 3, respectively with addition of 10−11 M OPC-12579. The FBS at 10% induced a 10-fold increase in goblet cell proliferation on Days 1, 3, and 5. Colocalization of Ulex europaeus agglutinin I, MUC5AC and anticytokeratin 7 with Ki-67 indicated that proliferating cells were goblet cells. Proliferating cells were negative for the nongoblet cell markers Bandeiraea lectin and anticytokeratin 4. Conclusions: The OPC-12759 stimulates proliferation of conjunctival goblet cells in primary culture.

Role of mitogen-activated protein kinase in cholinergic stimulation of conjunctival goblet cell secretion.

Conjunctival goblet cells are a primary source for the mucous layer of the tear film. We showed previously that conjunctival goblet cell mucin secretion is under neural control as activation of afferent sensory nerves in the cornea stimulates mucin secretion.1 This neural reflex is mediated either by the parasympathetic and sympathetic nerves that surround the goblet cells, or by antidromic stimulation of the sensory nerves in the conjunctiva and collateral sensory nerves from the cornea. Exogenous addition of the parasympathetic agonist carbachol or the parasympathetic neuropeptide VIP stimulated conjunctival goblet cell secretion.2,3 Cholinergic agonists released from parasympathetic nerves bind to and activate G protein linked- M2 and M3 muscarinic receptors. In most tissues, activation of these receptors stimulates phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 binds to specific receptors on the endoplasmic reticulum to increase intracellular Ca2+. The increased intracellular Ca2+ either directly, or in combination with Ca2+/calmodulin-dependent protein kinases, activates mucin secretion. DAG in turn stimulates protein kinase C (PKC). In many tissues, cholinergic agonists also activate the mitogen-activated protein kinase (MAPK) signaling pathway used by growth factor receptors, which usually regulate cell growth and proliferation. Activation of the MAPK pathway by cholinergic agonists can occur through several mechanisms. One such mechanism is through transactivation of the EGF receptor in an EGF-independent manner.4 The activated recepter then serves as a scaffold for recruitment of Grb2-SoS complex via tyrosine phosphorylation of Shc.

Role of Ca 2+ and Protein Kinase C in Cholinergic, and α 1 -Adrenergic Agonists and EGF Stimulated Mitogen-Activated Protein Kinase Activity in Lacrimal Gland

The MAP kinase pathway plays a key regulatory role in a variety of cellular functions including cell growth and differentiation, and gene expression.1,2 This pathway is characterized by a family of protein-serine/threonine kinases known as mitogen-activated protein kinases or MAPKs. Activation of MAPK is mediated by several mechanisms including the stimulation of G-protein coupled receptors (GPCR), such as muscarinic and angiotensin II receptors, or the activation of growth factor receptors via a protein-tyrosine kinase pathway.3,4 Protein kinase C (PKC), induced by phorbol ester tumor promoters, can activate the MAPK pathway in a Ras-independent manner.5 In addition, Ca2+ and cAMP-dependent pathways intersect with the MAPK signaling pathway, either stimulating or inhibiting it in different types of cells.6 Mechanisms of transactivation of epidermal growth factor (EGF) receptors in an EGF-independent manner, can mediate GPCR activation of MAPK.7

INVERTED INTERNAL LIMITING MEMBRANE FLAP TECHNIQUE FOR MACULAR HOLES IN HIGH MYOPIA WITH AXIAL LENGTH ≥30 mm.

Purpose: To evaluate the closure rate of macular holes in highly myopic eyes treated with the inverted internal limiting membrane flap technique. Methods: Retrospective study in 33 consecutive patients (33 eyes) with a myopic macular hole (axial length ≥30 mm) and no associated macular retinoschisis, undergoing 23-gauge pars plana vitrectomy combined with the inverted internal limiting membrane flap technique. Results: Mean initial logarithm of the minimum angle of resolution best-corrected visual acuity was 0.59 (range, 0.22–1.8) (Snellen fraction, 20/80). At the 1-month postoperative control visit, the macular hole was closed in all patients. Reopening of the hole occurred in 2 patients. Visual acuity improved in 13 patients (39.4%): final mean ETDRS (Early Treatment Diabetic Retinopathy Study) improvement was +80 letters, and logarithm of the minimum angle of resolution was 0.4 (20/50). Staphyloma with macular involvement was present in all patients. Dissociated optic nerve fiber layer was observed in 25 patients (75.7%) and was absent in 2 (6.1%); in the remaining 6 patients, the layer could not be assessed. Gliosis was found in 14 patients (42.4%). Conclusion: Vitrectomy plus the inverted internal limiting membrane flap technique was effective for treating macular holes in eyes with axial length ≥30 mm and no associated retinoschisis.

New Clathrin-Based Nanoplatforms for Magnetic Resonance Imaging

Background Magnetic Resonance Imaging (MRI) has high spatial resolution, but low sensitivity for visualization of molecular targets in the central nervous system (CNS). Our goal was to develop a new MRI method with the potential for non-invasive molecular brain imaging. We herein introduce new bio-nanotechnology approaches for designing CNS contrast media based on the ubiquitous clathrin cell protein. Methodology/Principal Findings The first approach utilizes three-legged clathrin triskelia modified to carry 81 gadolinium chelates. The second approach uses clathrin cages self-assembled from triskelia and designed to carry 432 gadolinium chelates. Clathrin triskelia and cages were characterized by size, structure, protein concentration, and chelate and gadolinium contents. Relaxivity was evaluated at 0.47 T. A series of studies were conducted to ascertain whether fluorescent-tagged clathrin nanoplatforms could cross the blood brain barriers (BBB) unaided following intranasal, intravenous, and intraperitoneal routes of administration. Clathrin nanoparticles can be constituted as triskelia (18.5 nm in size), and as cages assembled from them (55 nm). The mean chelate: clathrin heavy chain molar ratio was 27.04±4.8: 1 for triskelia, and 4.2±1.04: 1 for cages. Triskelia had ionic relaxivity of 16 mM−1s−1, and molecular relaxivity of 1,166 mM−1s−1, while cages had ionic relaxivity of 81 mM−1s−1 and molecular relaxivity of 31,512 mM−1s−1. Thus, cages exhibited 20 times higher ionic relaxivity and 8,000-fold greater molecular relaxivity than gadopentetate dimeglumine. Clathrin nanoplatforms modified with fluorescent tags were able to cross or bypass the BBB without enhancements following intravenous, intraperitoneal and intranasal administration in rats. Conclusions/Significance Use of clathrin triskelia and cages as carriers of CNS contrast media represents a new approach. This new biocompatible protein-based nanotechnology demonstrated suitable physicochemical properties to warrant further in vivo imaging and drug delivery studies. Significantly, both nanotransporters crossed and/or bypassed the BBB without enhancers. Thus, clathrin nanoplatforms could be an appealing alternative to existing CNS bio-nanotechnologies.