Ocular surface APCs are necessary for autoreactive T cell-mediated experimental autoimmune lacrimal keratoconjunctivitis.

As specialized sentinels between the innate and adaptive immune response, APCs are essential for activation of Ag-specific lymphocytes, pathogen clearance, and generation of immunological memory. The process is tightly regulated; however, excessive or atypical stimuli may ignite activation of APCs in a way that allows self-Ag presentation to autoreactive T cells in the context of the necessary costimulatory signals, ultimately resulting in autoimmunity. Studies in both animal models and patients suggest that dry eye is a chronic CD4(+) T cell-mediated ocular surface autoimmune-based inflammatory disease. Using a desiccating stress-induced mouse model of dry eye, we establish the fundamental role of APCs for both the generation and maintenance of ocular-specific autoreactive CD4(+) T cells. Subconjunctival administration of liposome-encapsulated clodronate efficiently diminished resident ocular surface APCs, inhibited the generation of autoreactive CD4(+) T cells, and blocked their ability to cause disease. APC-dependent CD4(+) T cell activation required intact draining cervical lymph nodes, as cervical lymphadenectomy also inhibited CD4(+) T cell-mediated dry eye disease. In addition, local depletion of peripheral conjunctival APCs blocked the ability of dry eye-specific CD4(+) T cells to accumulate within the ocular surface tissues, suggesting that fully primed and targeted dry eye-specific CD4(+) T cells require secondary activation by resident ocular surface APCs for maintenance and effector function. These data demonstrate that APCs are necessary for the initiation and development of experimental dry eye and support the standing hypothesis that dry eye is a self-Ag-driven autoimmune disease.

Comparison of prostaglandin E2 receptor subtype 4 agonist and sulfasalazine in mouse colitis prevention and treatment.

Prodrugs of 5-aminosalicylic acid (5-ASA), such as sulfasalazine, have been the mainstay for the treatment and maintenance of inflammatory bowel disease (IBD) for decades, which is attributable to their antiadaptive immune activity. However, 5-ASA compromises regeneration of intestinal epithelia and induces apoptosis. The majority of patients eventually undergo colectomy. Agonists for the prostaglandin E(2) subtype 4 (EP4) receptor have been shown to protect epithelial barrier against colitis-inducing agents and could be valuable alternatives for sulfasalazine. Here, we compared sulfasalazine and a novel EP4 agonist for their abilities to prevent colitis induction and relieve symptoms of established colitis in a dextran sulfate sodium-indomethacin mouse model. The EP4 agonist dose-dependently alleviated weight loss in colitis mice. Compared with sulfasalazine at 100 mg/kg on the colitis induction model, the EP4 agonist at 0.2 mg/kg was superior in reducing colitis symptoms, preventing increase of innate immune cells, and ameliorating inflammation in colon. In mice with established colitis, sulfasalazine quickly reversed weight loss but with fading efficacy. The EP4 agonist, in contrast, had slow but sustained effects on body weight gain and was more efficacious in epithelial regeneration. Such temporal differences between sulfasalazine and the EP4 agonist actions seemingly led to no additive effect in combination therapy. In conclusion, the EP4 agonist would be more efficacious in the maintenance of remission because of both anti-innate immune responses and epithelial regeneration activity, whereas sulfasalazine would be more suitable for induction of remission because of its rapid onset of antiadaptive inflammation action.

Topical Brimonidine or Intravitreal BDNF, CNTF, or bFGF Protect Cones Against Phototoxicity.

PURPOSE: To develop a focal photoreceptor degeneration model by blue light-emitting diode (LED)-induced phototoxicity (LIP) and investigate the protective effects of topical brimonidine (BMD) or intravitreal brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor (bFGF). METHODS: In anesthetized, dark-adapted, adult female Swiss mice, the left eye was dilated and exposed to blue light (10 seconds, 200 lux). After LIP, full-field electroretinograms (ERG) and spectral-domain optical coherence tomography (SD-OCT) were obtained longitudinally, and reactive-Iba-1+monocytic cells, TUNEL+ cells and S-opsin+ cone outer segments were examined up to 7 days. Left eyes were treated topically with BMD (1%) or vehicle, before or right after LIP, or intravitreally with BDNF (2.5 µg), CNTF (0.2 µg), bFGF (0.5 µg), or corresponding vehicle right after LIP. At 7 days, S-opsin+ cone outer segments were counted within predetermined fixed-size areas (PFA) centered on the lesion in both flattened retinas. RESULTS: SD-OCT showed a circular region in the superior-temporal left retina with progressive thinning (207.9 ± 5.6 µm to 160.7 ± 6.8 µm [7 days], n = 8), increasing TUNEL+ cells (peak at 3 days), decreasing S-opsin+ cone outer segments, and strong microglia activation. ERGs were normal by 3 days. Total S-opsin+ cones in the PFA for LIP-treated and fellow-retinas were 2330 ± 262 and 5601 ± 583 (n = 8), respectively. All neuroprotectants (n = 7-11), including topical BMD pre- or post-LIP, or intravitreal BDNF, CNTF, and bFGF, showed significantly greater S-opsin+ cone survival than their corresponding vehicle-treated groups. CONCLUSIONS: LIP is a reliable, quantifiable focal photoreceptor degeneration model. Topical BMD or intravitreal BDNF, CNTF, or bFGF protect against LIP-induced cone-photoreceptor loss. TRANSLATIONAL RELEVANCE: Topical BMD or intravitreal BDNF, CNTF, or bFGF protect cones against phototoxicity.

Bimatoprost, prostamide activity, and conventional drainage.

PURPOSE: Despite structural similarity with prostaglandin F(2 alpha), the ocular hypotensive agent bimatoprost (Lumigan; Allergan, Inc., Irvine, CA) shows unique pharmacology in vitro and functional activity in vivo. Unfortunately, the precise mechanisms that underlie bimatoprost's distinctive impact on aqueous humor dynamics are unclear. The purpose of the present study was to investigate the effects of bimatoprost and a novel prostamide-selective antagonist AGN 211334 on human conventional drainage. METHODS: Two model systems were used to test the consequences of bimatoprost and/or AGN 211334 treatment on conventional drainage. Human anterior segments in organ culture were perfused at a constant flow rate of 2.5 microL/min while pressure was recorded continuously. After stable baseline facilities were established, segments were treated with drug(s), and pressure was monitored for an additional 3 days. In parallel, the drugs' effects on hydraulic conductivity of human trabecular meshwork (TM) cell monolayers were evaluated. Pharmacological properties of AGN 211334 were characterized in isolated feline iris preparations in organ culture and heterologously expressed G-protein-coupled receptors were examined in vitro. RESULTS: Bimatoprost increased outflow facility by an average of 40% +/- 10% within 48 hours of treatment (n = 10, P < 0.001). Preincubation or coincubation with AGN 211334 significantly blunted bimatoprost's effects by 95% or 43%, respectively. Similar results were obtained in cell culture experiments in which bimatoprost increased hydraulic conductivity of TM cell monolayers by 78% +/- 25%. Pretreatment with AGN 211334 completely blocked bimatoprost's effects, while coincubation decreased its effects on average by 74%. In both models, AGN 211334 alone significantly decreased fluid flux across trabecular tissues and cells. CONCLUSIONS: The findings indicate that bimatoprost interacts with a prostamide receptor in the trabecular meshwork to increase outflow facility.

Levels of bimatoprost acid in the aqueous humour after bimatoprost treatment of patients with cataract.

AIM: To determine the aqueous humour concentration of the acid hydrolysis products of bimatoprost and latanoprost after a single topical dose of bimatoprost 0.03% or latanoprost 0.005% in humans. METHODS: Randomised, controlled, double-masked, prospective study. 48 eyes of 48 patients scheduled for routine cataract surgery were randomised in an 8:2:2 ratio to treatment with a single 30 mul drop of bimatoprost 0.03%, latanoprost 0.005% or placebo at 1, 3, 6 or 12 h before the scheduled cataract surgery. Aqueous humour samples were withdrawn at the beginning of the surgical procedure and analysed using high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Bimatoprost acid (17-phenyl trinor prostaglandin F2alpha) was detected in aqueous samples at a mean concentration of 5.0 nM at hour 1, 6.7 nM at hour 3 and 1.9 nM at hour 6 after bimatoprost treatment. After latanoprost treatment, the mean concentration of latanoprost acid (13,14-dihydro-17-phenyl trinor prostaglandin F2alpha) in aqueous samples was 29.1 nM at hour 1, 41.3 nM at hour 3 and 2.5 nM at hour 6. Acid metabolites were below the limit of quantitation in all samples taken 12 h after dosing and in all samples from placebo-treated patients. None of the samples from latanoprost-treated patients contained quantifiable levels of non-metabolised latanoprost. Non-metabolised bimatoprost was detected in aqueous samples at a mean concentration of 6.6 nM at hour 1 and 2.4 nM at hour 3 after bimatoprost treatment. CONCLUSIONS: Low levels of bimatoprost acid were detected in aqueous humour samples from patients with cataract treated with a single dose of bimatoprost. Latanoprost acid concentrations in samples from patients treated with latanoprost were at least sixfold higher. These results suggest that bimatoprost acid in the aqueous humour does not sufficiently account for the ocular hypotensive efficacy of bimatoprost.

Neuroprotective effect of memantine in different retinal injury models in rats.

PURPOSE: To evaluate the neuroprotective effect of memantine, an NMDA receptor channel blocker, in two retinal ganglion cell (RGC) injury models in rats. METHODS: Neuroprotective effect of memantine was tested in partial optic nerve injury and chronic ocular hypertensive models. In the optic nerve injury model, memantine (0.1 - 30 mg/kg) was injected intraperitoneally immediately after injury. Two weeks later, optic nerve function was determined by measuring compound action potential and surviving RGC was determined by retrograde labeling with dextran tetramethyl rhodamine. Chronic ocular hypertension was attained by laser photocoagulation of episcleral and limbal veins. Memantine (5 or 10 mg/kg) was administered continuously each day with an osmotic pump, either immediately after or 10 days after first laser photocoagulation, for 3 weeks, after which RGC survival was determined. RESULTS: Two weeks after partial optic nerve injury, there was approximately 80% reduction in RGC number. Memantine (5 mg/kg) caused a twofold increase in compound action potential amplitude and a 1.7-fold increase in survival of RGCs, respectively. In the chronic ocular hypertension model there was 37% decrease in RGCs after 3 weeks of elevated intraocular pressure. Memantine (10 mg/kg daily) reduced ganglion cell loss to 12% when applied immediately after first laser photocoagulation, and prevented any further loss when applied 10 days after first laser photocoagulation. CONCLUSION: The protective effect of memantine suggests that excessive stimulation of NMDA receptors by glutamate is involved in causing cell damage in these RGC injury models.

A novel in vivo model of focal light emitting diode-induced cone-photoreceptor phototoxicity: neuroprotection afforded by brimonidine, BDNF, PEDF or bFGF.

We have investigated the effects of light-emitting diode (LED)-induced phototoxicity (LIP) on cone-photoreceptors and their protection with brimonidine (BMD), brain-derived neurotrophic factor (BDNF), pigment epithelium-derived factor (PEDF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF). In anesthetized, dark adapted, adult albino rats a blue (400 nm) LED was placed perpendicular to the cornea (10 sec, 200 lux) and the effects were investigated using Spectral Domain Optical Coherence Tomography (SD-OCT) and/or analysing the retina in oriented cross-sections or wholemounts immune-labelled for L- and S-opsin and counterstained with the nuclear stain DAPI. The effects of topical BMD (1%) or, intravitreally injected BDNF (5 µg), PEDF (2 µg), CNTF (0.4 µg) or bFGF (1 µg) after LIP were examined on wholemounts at 7 days. SD-OCT showed damage in a circular region of the superotemporal retina, whose diameter varied from 1,842.4±84.5 µm (at 24 hours) to 1,407.7±52.8 µm (at 7 days). This region had a progressive thickness diminution from 183.4±5 µm (at 12 h) to 114.6±6 µm (at 7 d). Oriented cross-sections showed within the light-damaged region of the retina massive loss of rods and cone-photoreceptors. Wholemounts documented a circular region containing lower numbers of L- and S-cones. Within a circular area (1 mm or 1.3 mm radius, respectively) in the left and in its corresponding region of the contralateral-fellow-retina, total L- or S-cones were 7,118±842 or 661±125 for the LED exposed retinas (n = 7) and 14,040±1,860 or 2,255±193 for the fellow retinas (n = 7), respectively. BMD, BDNF, PEDF and bFGF but not CNTF showed significant neuroprotective effects on L- or S-cones. We conclude that LIP results in rod and cone-photoreceptor loss, and is a reliable, quantifiable model to study cone-photoreceptor degeneration. Intravitreal BDNF, PEDF or bFGF, or topical BMD afford significant cone neuroprotection in this model.

Mitochondrial permeability transition pore in inflammatory apoptosis of human conjunctival epithelial cells and T cells: effect of cyclosporin A.

PURPOSE: To investigate the role of mitochondrial permeability transition pore (MPTP) and effect of cyclosporin A (CsA) on inflammatory apoptosis of human conjunctival epithelial cells (IOBA-NHC) and T cells. METHODS: IOBA-NHC and Jurkat cells were stimulated with IFNγ, TNFα, αFas, or PMA/αCD3, in the presence or absence of CsA. MPTP was determined using the calcein-cobalt technique. Mitochondrial membrane potential (ΔΨm) was measured with JC-1. Apoptosis was quantified by Annexin V/PI staining. Apoptosis mediators were evaluated by flow cytometry or Western blot. RESULTS: In IOBA-NHC, TNFα, and IFNγ induced MPTP opening, ΔΨm loss, and increased cell apoptosis. This was accompanied by upregulation of Fas/FasL; Bax; and caspase-3, -8, and -9 activation. Addition of CsA prevented IOBA-NHC from cell death by blocking MPTP opening, ΔΨm loss, Fas/FasL, and caspase activation. In PMA/αCD3-activated Jurkat T cells, MPTP opening and ΔΨm loss were increased along with cell apoptosis and upregulated Fas/FasL/caspase expressions. CsA further promoted T-cell apoptosis, ΔΨm loss, and upregulation of Fas/FasL/caspase. CONCLUSIONS: Inflammation induces aberrant MPTP opening, resulting in an increased apoptosis in conjunctival epithelial cells. CsA protected IOBA-NHC from cell death by blocking both intrinsic and extrinsic apoptosis pathways. CsA promoted T-cell apoptosis via upregulating Fas/FasL and caspase activities with a minimal effect on MPTP. The findings suggest that the differential effect of CsA on T cells versus ocular surface resident epithelial cells may contribute to its therapeutic efficacy in treating ocular inflammation such as dry eye disease.

Comparison of human ocular distribution of bimatoprost and latanoprost.

PURPOSE: This study investigated the ocular distribution of bimatoprost, latanoprost, and their acid hydrolysis products in the aqueous humor, cornea, sclera, iris, and ciliary body of patients treated with a single topical dose of 0.03% bimatoprost or 0.005% latanoprost for understanding concentration-activity relationships. METHODS: Thirty-one patients undergoing enucleation for an intraocular tumor not affecting the anterior part of the globe were randomized to treatment with bimatoprost or latanoprost at 1, 3, 6 or 12 h prior to surgery. Concentrations of bimatoprost, bimatoprost acid, latanoprost, and latanoprost acid in the human aqueous and ocular tissues were measured using liquid chromatography tandem mass spectrometry. RESULTS: Following topical administration, intact bimatoprost was distributed in human eyes with a rank order of cornea/sclera >iris/ciliary body >aqueous humor. Bimatoprost acid was also detected in these tissues, where its low levels in the cornea relative to that of latanoprost acid indicated that bimatoprost hydrolysis was limited. Latanoprost behaved as a prodrug that entered eyes predominantly via the corneal route. Levels of latanoprost acid were distributed as cornea >>aqueous humor>iris>sclera>ciliary body. CONCLUSIONS: Our study provided experimental evidence that levels of bimatoprost in relevant ocular tissues, and not only aqueous humor, are needed to understand the mechanisms by which bimatoprost lowers intraocular pressure (IOP) in human subjects. The data suggest that bimatoprost reached the target tissues favoring the conjunctival/scleral absorption route. Findings of intact bimatoprost in the target ciliary body indicated its direct involvement in reducing IOP. However, bimatoprost acid may have only a limited contribution on the basis that bimatoprost has greater/similar IOP-lowering efficacy than latanoprost, yet bimatoprost acid levels were a fraction of latanoprost acid levels in the aqueous humor and cornea and only sporadically detectable in the ciliary body. In this report, human ocular tissues were examined concurrently with aqueous humor for the in vivo distribution of bimatoprost, bimatoprost acid, latanoprost, and latanoprost acid.

Autoantibodies contribute to the immunopathogenesis of experimental dry eye disease.

PURPOSE: The purpose of this study was to determine if autoantibodies play a role in the immunopathogenesis of experimental dry eye disease. METHODS: Dry eye was induced by exposing female C57BL/6 wild-type mice or hen egg lysozyme B-cell receptor transgenic mice to desiccating stress (subcutaneous scopolamine [0.5 mg/0.2 mL] 3 times a day, humidity < 40%, and sustained airflow) for 3 weeks, allowing sufficient time for a humoral immune response. Serum or purified IgG isolated from dry-eye mice or untreated controls was passively transferred to nude recipient mice, which were evaluated for ocular surface inflammation 3 days after transfer. To determine if complement activation contributed to serum-induced dry eye disease, cobra venom factor was used to deplete complement activity. RESULTS: Autoantibodies against kallikrein 13 were identified in serum from dry-eye mice, but were undetectable in untreated controls. Autoantibody-containing serum or purified IgG from dry-eye mice was sufficient to mediate complement-dependent ocular surface inflammation. Serum or purified IgG caused marked inflammatory burden and tissue damage within the ocular surface tissues, including elevated Gr1+ neutrophil infiltration and proinflammatory cytokines/chemokines associated with goblet cell loss. Moreover, complement C3b deposition was found within the ocular surface tissues of mice receiving dry-eye serum, but not in recipients of control serum. Functionally, complement depletion attenuated the ability to transfer dry-eye-specific serum or IgG-mediated disease. CONCLUSIONS: These data demonstrate for the first time a complement-dependent pathogenic role of dry-eye-specific autoantibodies, and suggest autoantibody deposition within the ocular surface tissues contributes to the predominantly T-cell-mediated immunopathogenesis of dry eye disease.

Brimonidine prevents axonal and somatic degeneration of retinal ganglion cell neurons.

BACKGROUND: Brimonidine is a common drug for lowering ocular pressure and may directly protect retinal ganglion cells in glaucoma. The disease involves early loss of retinal ganglion cell transport to brain targets followed by axonal and somatic degeneration. We examined whether brimonidine preserves ganglion cell axonal transport and abates degeneration in rats with elevated ocular pressure induced by laser cauterization of the episcleral veins. RESULTS: Ocular pressure was elevated unilaterally by 90% for a period of 8 weeks post- cauterization. During this time, brimonidine (1mg/kg/day) or vehicle (phosphate-buffered saline) was delivered systemically and continuously via subcutaneous pump. Animals received bilateral intravitreal injections of fluorescent cholera toxin subunit ß (CTB) two days before sacrifice to assess anterograde transport. In retinas from the vehicle group, elevated pressure induced a 44% decrease in the fraction of ganglion cells with intact uptake of CTB and a 14-42% reduction in the number of immuno-labelled ganglion cell bodies, with the worst loss occurring nasally. Elevated pressure also caused a 33% loss of ganglion cell axons in vehicle optic nerves and a 70% decrease in CTB transport to the superior colliculus. Each of these components of ganglion cell degeneration was either prevented or significantly reduced in the brimonidine treatment group. CONCLUSIONS: Continuous and systemic treatment with brimonidine by subcutaneous injection significantly improved retinal ganglion cell survival with exposure to elevated ocular pressure. This effect was most striking in the nasal region of the retina. Brimonidine treatment also preserved ganglion cell axon morphology, sampling density and total number in the optic nerve with elevated pressure. Consistent with improved outcome in the optic projection, brimonidine also significantly reduced the deficits in axonal transport to the superior colliculus associated with elevated ocular pressure. As transport deficits to and from retinal ganglion cell projection targets in the brain are relevant to the progression of glaucoma, the ability of brimonidine to preserve optic nerve axons and active transport suggests its neuroprotective effects are relevant not only at the cell body, but throughout the entire optic projection.

EP4 agonist alleviates indomethacin-induced gastric lesions and promotes chronic gastric ulcer healing.

AIM: To investigate EP4-selective agonist effect on indomethacin-induced gastric lesions and on the spontaneous healing of chronic gastric ulcers. METHODS: In a mouse model of gastric bleeding with high dose of indomethacin (20 mg/kg), an EP4-selective agonist was administered orally. Stomach lesions and gastric mucous regeneration were monitored. In a mouse model of chronic gastric ulcer induced by acetic acid, EP4 agonist effect on the healing of chronic gastric ulcer was evaluated in the presence or absence of low dose indomethacin (3 mg/kg). In cultured human gastric mucous cells, EP4 agonist effect on indomethacin-induced apoptosis was assessed by flow cytometry. RESULTS: The EP4-selective agonist reduced high dose indomethacin-induced acute hemorrhagic damage and promoted mucous epithelial regeneration. Low-dose indomethacin aggravated ulcer bleeding and inflammation, and delayed the healing of the established chronic gastric ulcer. The EP4 agonist, when applied locally, not only offset indomethacin-induced gastric bleeding and inflammation, but also accelerated ulcer healing. In the absence of indomethacin, the EP4 agonist even accelerated chronic gastric ulcer healing and suppressed inflammatory cell infiltration in the granulation tissue. In vitro, the EP4 agonist protected human gastric mucous cells from indomethacin-induced apoptosis. CONCLUSION: EP4-selective agonist may prevent indomethacin-induced gastric lesions and promote healing of existing and indomethacin-aggravated gastric ulcers, via promoting proliferation and survival of mucous epithelial cells.

Bimatoprost-induced calcium signaling in human T-cells does not involve prostanoid FP or TP receptors.

PURPOSE: The prostamide bimatoprost and prostanoid FP receptor agonists are highly efficacious drugs for glaucoma treatment. The presence of both prostamide and prostanoid FP receptors in bimatoprost-sensitive preparations has made prostamide receptor classification difficult. This study investigated a novel bimatoprost-sensitive preparation. METHODS: Human peripheral blood T lymphoblasts (Molt-3) and human osteoblasts (hFOB) were cultured for intracellular calcium signaling studies and quantitative real-time PCR analysis of RNA. RESULTS: Bimatoprost stimulated concentration-related increases in [Ca(2 +)](i) in a human T-cell line that does not express human FP receptor/variants, according to PCR analysis. The calcium signal induced by bimatoprost was not antagonized by prostanoid FP receptor antagonist/partial agonist AL-8810 or selective TP receptor antagonist SQ 29548. Conversely, bimatoprost did not elevate [Ca(2 +)](i) in human osteoblasts, which were confirmed to contain RNA of human FP receptor/variants. CONCLUSIONS: Molt-3 cells have been identified as a bimatoprost-sensitive preparation in which the activity of bimatoprost is independent of prostanoid FP receptors.

Glaucoma alters the circadian timing system.

Glaucoma is a widespread ocular disease and major cause of blindness characterized by progressive, irreversible damage of the optic nerve. Although the degenerative loss of retinal ganglion cells (RGC) and visual deficits associated with glaucoma have been extensively studied, we hypothesize that glaucoma will also lead to alteration of the circadian timing system. Circadian and non-visual responses to light are mediated by a specialized subset of melanopsin expressing RGCs that provide photic input to mammalian endogenous clock in the suprachiasmatic nucleus (SCN). In order to explore the molecular, anatomical and functional consequences of glaucoma we used a rodent model of chronic ocular hypertension, a primary causal factor of the pathology. Quantitative analysis of retinal projections using sensitive anterograde tracing demonstrates a significant reduction (approximately 50-70%) of RGC axon terminals in all visual and non-visual structures and notably in the SCN. The capacity of glaucomatous rats to entrain to light was challenged by exposure to successive shifts of the light dark (LD) cycle associated with step-wise decreases in light intensity. Although glaucomatous rats are able to entrain their locomotor activity to the LD cycle at all light levels, they require more time to re-adjust to a shifted LD cycle and show significantly greater variability in activity onsets in comparison with normal rats. Quantitative PCR reveals the novel finding that melanopsin as well as rod and cone opsin mRNAs are significantly reduced in glaucomatous retinas. Our findings demonstrate that glaucoma impacts on all these aspects of the circadian timing system. In light of these results, the classical view of glaucoma as pathology unique to the visual system should be extended to include anatomical and functional alterations of the circadian timing system.

Bimatoprost: mechanism of ocular surface hyperemia associated with topical therapy.

Bimatoprost is a safe and well-tolerated intraocular pressure (IOP) lowering drug that was approved in the United States in 2001 for the treatment of glaucoma and ocular hypertension. It is highly efficacious and produces greater mean reductions in IOP than other currently available antiglaucoma drugs. Conjunctival hyperemia is a common side effect of bimatoprost, but the hyperemia is typically mild and transient. No association has been found between signs of inflammation and the presence of hyperemia in bimatoprost-treated patients. Preclinical studies have elucidated the pharmacological mechanism of bimatoprost-related hyperemia and have examined the possible involvement of inflammation. Bimatoprost, as well as the free acid of latanoprost, elicited endothelium-dependent vasorelaxation in the rabbit jugular vein preparation, a quantitative in vitro model for ocular surface hyperemia (OSH). The vasorelaxant responses to either bimatoprost or latanoprost free acid were significantly inhibited by L-NAME, a nitric oxide synthase inhibitor. Similarly, the in vivo OSH responses to topically applied bimatoprost or latanoprost in dog eyes were significantly inhibited by L-NAME. As predicted, prostaglandin E(2) (PGE(2))-induced conjunctival hyperemia was not inhibited by L-NAME, since PGE(2) has a direct relaxant effect on the vascular smooth muscle. In-life observations and histopathological assessment of ocular surface tissues following bimatoprost treatment were performed for multiple-dose one month, 6 month, or 12 month safety studies in rabbits, dogs, and non-human primates. Results of these studies showed no evidence of bimatoprost-related inflammation in the ocular surface tissues. In summary, OSH related to bimatoprost treatment in laboratory animals occurs by endothelial-derived nitric oxide-mediated vasodilatation and is not associated with inflammation. These studies suggest that conjunctival hyperemia, a side effect of bimatoprost treatment, results from non-inflammatory, pharmacologically based vasodilatation.

Bimatoprost and prostaglandin F(2 alpha) selectively stimulate intracellular calcium signaling in different cat iris sphincter cells.

Bimatoprost is a synthetic analog of prostaglandin F(2 alpha) ethanolamide (prostamide F(2 alpha)), and shares a pharmacological profile consistent with that of the prostamides. Like prostaglandin F(2 alpha) carboxylic acid, bimatoprost potently lowers intraocular pressure in dogs, primates and humans. In order to distinguish its mechanism of action from prostaglandin F(2 alpha), fluorescence confocal microscopy was used to examine the effects of bimatoprost, prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) on calcium signaling in resident cells of digested cat iris sphincter, a tissue which exhibits contractile responses to both agonists. Constant superfusion conditions obviated effective conversion of bimatoprost. Serial challenge with 100 nM bimatoprost and prostaglandin F(2 alpha) consistently evoked responses in different cells within the same tissue preparation, whereas prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) elicited signaling responses in the same cells. Bimatoprost-sensitive cells were consistently re-stimulated with bimatoprost only, and prostaglandin F(2 alpha) sensitive cells could only be re-stimulated with prostaglandin F(2 alpha). The selective stimulation of different cells in the same cat iris sphincter preparation by bimatoprost and prostaglandin F(2 alpha), along with the complete absence of observed instances in which the same cells respond to both agonists, strongly suggests the involvement of distinct receptors for prostaglandin F(2 alpha) and bimatoprost. Further, prostaglandin F(2 alpha) but not bimatoprost potently stimulated calcium signaling in isolated human embryonic kidney cells stably transfected with the feline- and human-prostaglandin F(2 alpha) FP-receptor and in human dermal fibroblast cells, and only prostaglandin F(2 alpha) competed with radioligand binding in HEK-feFP cells. These studies provide further evidence for the existence of a bimatoprost-sensitive receptor that is distinct from any of the known prostaglandin receptor types.

Immune-related mechanisms participating in resistance and susceptibility to glutamate toxicity.

Glutamate is an essential neurotransmitter in the CNS. However, at abnormally high concentrations it becomes cytotoxic. Recent studies in our laboratory showed that glutamate evokes T cell-mediated protective mechanisms. The aim of the present study was to examine the nature of the glutamate receptors and signalling pathways that participate in immune protection against glutamate toxicity. We show, using the mouse visual system, that glutamate-induced toxicity is strain dependent, not only with respect to the amount of neuronal loss it causes, but also in the pathways it activates. In strains that are genetically endowed with the ability to manifest a T cell-dependent neuroprotective response to glutamate insult, neuronal losses due to glutamate toxicity were relatively small, and treatment with NMDA-receptor antagonist worsened the outcome of exposure to glutamate. In contrast, in mice devoid of T cell-dependent endogenous protection, NMDA receptor antagonist reduced the glutamate-induced neuronal loss. In all strains, blockage of the AMPA/KA receptor was beneficial. Pharmacological (with alpha2-adrenoceptor agonist) or molecular intervention (using either mice overexpressing Bcl-2, or DAP-kinase knockout mice) protected retinal ganglion cells from glutamate toxicity but not from the toxicity of NMDA. The results suggest that glutamate-induced neuronal toxicity involves multiple glutamate receptors, the types and relative contributions of which, vary among strains. We suggest that a multifactorial protection, based on an immune mechanism independent of the specific pathway through which glutamate exerts its toxicity, is likely to be a safer, more comprehensive, and hence more effective strategy for neuroprotection. It might suggest that, because of individual differences, the pharmacological use of NMDA-antagonist for neuroprotective purposes might have an adverse effect, even if the affinity is low.

ICAM-1 expression predisposes ocular tissues to immune-based inflammation in dry eye patients and Sjögrens syndrome-like MRL/lpr mice.

PURPOSE: We previously reported that immune-based inflammation occurs on the ocular surface of humans as well as canines with keratoconjunctivitis sicca (KCS). Intercellular adhesion molecule-1 (ICAM-1) was found to be upregulated on lymphocytes and/or vascular endothelial cells resulting in lymphocytic diapedesis to the lacrimal and conjunctival tissues. The purpose of the current study was to demonstrate the role of ICAM-1 in (1) resident epithelial cell response during ocular inflammation, (2) local and/or peripheral lymphocyte activation or accumulation in the ocular tissues, and (3) whether anti-ICAM-1 is effective to attenuate immune-mediated ocular inflammation. METHODS: ICAM-1 levels in various ocular tissues of human with KCS and/or MRL/lpr mouse were evaluated by immunohistochemistry and in situ hybridization for protein and messenger RNA (mRNA) expression, respectively. Soluble ICAM-1 concentrations in MRL/lpr mouse plasma over the course of disease development were measured by ELISA. Cell proliferation within ocular tissues was assessed by bromodeoxyuridine (BrdU) incorporation and immunohistochemical detection. The level of T cell activation was determined by IL-2 receptor (CD25, a marker of T cell activation and proliferation) and CD69 (a marker of T cell activation) immunoreactivity using FACS analysis. To examine the effectiveness of anti-ICAM-1/LFA-1 in elimination of lacrimal gland inflammation, MRL/lpr mice were injected intraperitoneally (i.p.) with or without monoclonal antibodies against ICAM-1 and LFA-1 at three or eight weeks of age. RESULTS: Increased endogenous ICAM-1 expression at the level of protein and mRNA was detected in the epithelial cells present in the conjunctival and accessory lacrimal tissues in dry eye patients. In MRL/lpr mice, ICAM-1 expression by lacrimal acinar epithelial cells and conjunctival epithelial cells were detected in addition to inflammatory infiltrates and vascular endothelial cells at 16 weeks of age. Soluble ICAM-1 levels were markedly increased concomitantly with disease progression over time as compared with the controls. No significant lymphocytic proliferation (a lack of BrdU and CD25 immunoreactivities) was detected within lacrimal glands of MRL/lpr mice at the disease onset. However, a population of the infiltrated T cells were CD69 positive, indicating the activation stage of a T cell subset. Treatment using monoclonal antibodies against murine ICAM-1 and LFA-1 resulted in a decrease in the number of inflammatory infiltrates in MRL/lpr mice. CONCLUSIONS: Our findings suggest that ICAM-1 upregulation locally and systemically promote lymphocyte activation and migration to the ocular surface (OS). Ocular resident epithelium is an active component of ocular surface and is capable of interacting with invasive lymphocytes by ICAM-1 production in response to immune activation and inflammation. ICAM-1 synthesized by epithelial cells may serve as a signaling molecule for predisposition of ocular surface inflammation and facilitate potential antigen presentation by epithelial cells. Lymphocytic infiltrates in the lacrimal gland of the MRL/lpr mouse appeared to be the result of the accumulation, but not proliferation of circulating lymphocytes diapodesed from the vasculature that had migrated into the local ocular tissues. The potential use of anti-ICAM-1 therapy in treating immune-based inflammatory diseases such as dry eye deserves further investigation.