Whole-Exome Sequencing of Patients With Posterior Segment Uveitis.

OBJECTIVE: To elucidate molecular risk factors for posterior segment uveitis using a functional genomics approach. DESIGN: Genetic association cohort study. METHODS: Setting: Single-center study at an academic referral center. STUDY POPULATION: 164 patients with clinically diagnosed uveitis of the posterior segment. MAIN OUTCOME MEASURES: Exome sequencing was used to detect variants identified in 164 patients with posterior segment uveitis. A phenotype-driven analysis, protein structural modeling, and in silico calculations were then used to rank and predict the functional consequences of key variants. RESULTS: A total of 203 single nucleotide variants, in 23 genes across 164 patients, were included in this study. Both known and novel variants were identified in genes previously implicated in specific types of syndromic uveitis-such as NOD2 (Blau syndrome) and CAPN5 NIV (neovascular inflammatory vitreoretinopathy)-as well as variants in genes not previously linked to posterior segment uveitis. Based on a ranked list and protein-protein-interaction network, missense variants in NOD-like receptor family genes (NOD2, NLRC4, NLRP3, and NLRP1), CAPN5, and TYK2 were characterized via structural modeling and in silico calculations to predict how specific variants might alter protein structure and function. The majority of analyzed variants were notably different from wild type. CONCLUSIONS: This study implicates new pathways and immune signaling proteins that may be associated with posterior segment uveitis susceptibility. A larger cohort and functional studies will help validate the pathogenicity of the mutations identified. In specific cases, whole-exome sequencing can help diagnose nonsyndromic uveitis in patients harboring known variants for syndromic inflammatory diseases.

Possible involvement of NETosis in inflammatory processes in the eye: Evidence from a small cohort of patients.

Purpose: To evaluate whether NETosis is involved in cytokine-induced ocular inflammation and to track neutrophil extracellular traps (NET) complexes in patients with proliferative diabetic retinopathy (PDR). Methods: For the animal model, the eyes of C57BL/6J mice were intravitreally injected with interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), or saline. Histology and immunofluorescence staining for CD11b, neutrophil elastase (NE), myeloperoxidase (MPO), citrullinated histone 3 (H3Cit), and net-like structure were performed. Vitreous samples were collected from patients with PDR; the PDR1 group had no need for repeated surgical intervention, and the PDR2 group had repeated vitreous bleeding or other complication and controls. Levels of MPO, H3Cit-MPO, and NE-MPO complex were measured with enzyme-linked immunosorbent assay (ELISA). Results: Massive influx of CD11+ inflammatory cells, involving the anterior and posterior chambers, was observed in the murine eyes 24 h after the IL-8 or TNF-α injections. Cells excreted to their surroundings an extracellular net-like structure positive for NE, MPO, and H3Cit. H3Cit staining was abolished with the DNase I treatment, indicating the presence of extracellular DNA in the net-like structures. The vitreous samples of the patients with PDR2 contained statistically significantly higher levels of MPO (173±230) compared to those of the patients with PDR1 (12.0±33.0, p<0.05) or the controls (0.00, p<0.01). The levels of H3Cit-MPO and NE-MPO complexes were also statistically significantly higher in the patients with PDR2 (776.0±1274, 573.0±911.0, respectively) compared to those in the patients with PDR1 (0, p<0.05) and the controls (0, p<0.05). Conclusions: This study showed the existence of NETosis in cytokine-induced ocular inflammation in a mouse model and human samples. Furthermore, the extent of NET complex formation was higher in a subset of patients who exhibited more complicated PDR.

Distribution of topical ocular nepafenac and its active metabolite amfenac to the posterior segment of the eye.

Nepafenac ophthalmic suspensions, 0.1% (NEVANAC(®)) and 0.3% (ILEVRO™), are topical nonsteroidal anti-inflammatory drug (NSAID) products approved in the United States, Europe and various other countries to treat pain and inflammation associated with cataract surgery. NEVANAC is also approved in Europe for the reduction in the risk of postoperative macular edema (ME) associated with cataract surgery in diabetic patients. The efficacy against ME suggests that topical administration leads to distribution of nepafenac or its active metabolite amfenac to the posterior segment of the eye. This article evaluates the ocular distribution of nepafenac and amfenac and the extent of local delivery to the posterior segment of the eye, following topical ocular instillation in animal models. Nepafenac ophthalmic suspension was instilled unilaterally in New Zealand White rabbits as either a single dose (0.1%; one drop) or as multiple doses (0.3%, one drop, once-daily for 4 days, or 0.1% one drop, three-times daily for 3 days and one morning dose on day 4). Nepafenac (0.3%) was also instilled unilaterally in cynomolgus monkeys as multiple doses (one drop, three-times daily for 7 days). Nepafenac and amfenac concentrations in harvested ocular tissues were measured using high-performance liquid chromatography/mass spectrometry. Locally-distributed compound concentrations were determined as the difference in levels between dosed and undosed eyes. In single-dosed rabbit eyes, peak concentrations of locally-distributed nepafenac and amfenac showed a trend of sclera > choroid > retina. Nepafenac peak levels in sub-samples posterior to the eye equator and inclusive of the posterior pole (E-PP) were 55.1, 4.03 and 2.72 nM, respectively, at 0.25 or 0.50 h, with corresponding amfenac peak levels of 41.9, 3.10 and 0.705 nM at 1 or 4 h. By comparison, peak levels in sclera, choroid and retina sub-samples in a band between the ora serrata and the equator (OS-E) were 13- to 40-fold (nepafenac) or 11- to 23-fold (amfenac) higher, indicating an anterior-to-posterior directional concentration gradient. In multiple-dosed rabbit eyes, with 0.3% nepafenac instilled once-daily or 0.1% nepafenac instilled three-times daily, cumulative 24-h locally-distributed levels of nepafenac in E-PP retina were similar between these groups, whereas exposure to amfenac once-daily dosing nepafenac 0.3% was 51% of that achieved with three-times daily dosing of 0.1%. In single-dosed monkey eyes, concentration gradients showed similar directionality as observed in rabbit eyes. Peak concentrations of locally-distributed nepafenac were 1580, 386, 292 and 13.8 nM in E-PP sclera, choroid and retina, vitreous humor, respectively, at 1 or 2 h after drug instillation. Corresponding amfenac concentrations were 21.3, 11.8, 2.58 and 2.82 nM, observed 1 or 2 h post-instillation. The data indicate that topically administered nepafenac and its metabolite amfenac reach pharmacologically relevant concentrations in the posterior eye segment (choroid and retina) via local distribution, following an anterior-to-posterior concentration gradient. The proposed pathway involves a choroidal/suprachoroidal or periocular route, along with an inward movement of drug through the sclera, choroid and retina, with negligible vitreal compartment involvement. Sustained high nepafenac concentrations in posterior segment tissues may be a reservoir for hydrolysis to amfenac.

Characterization of a New Epitope of IRBP That Induces Moderate to Severe Uveoretinitis in Mice With H-2b Haplotype.

PURPOSE: Experimental autoimmune uveitis (EAU) induced in mice using the retinal antigen interphotoreceptor retinoid binding protein (IRBP) is an animal model for posterior uveitis in humans. However, EAU induced by native IRBP protein or its widely used epitope amino acid residues 1 to 20 of human IRBP (hIRBP1-20) is inconsistent, often showing low scores and incidence. We found an urgent need to identify a better pathogenic epitope for the C57BL/6 strain. METHODS: Mice were immunized with uveitogenic peptides or with native bovine IRBP. Clinical and histological disease and associated immunological responses were evaluated. Truncated and substituted peptides, as well as bioinformatic analyses, were used to identify critical major histocompatibility complex (MHC)/T cell receptor (TCR) contact residues and the minimal core epitope. RESULTS: The new uveitogenic epitope of IRBP, amino acid residues 651 to 670 of human IRBP (LAQGAYRTAVDLESLASQLT [hIRBP651-670]) is uveitogenic for mice of the H-2b haplotype and elicits EAU with a higher severity and incidence in C57BL/6 mice than the previously characterized hIRBP1-20 epitope. Using truncated and substituted peptides, as well as bioinformatic analysis, we identified the critical contact residues with MHC/TCR and defined the minimal core epitope. This made it possible to design MHC tetramers and use them to detect epitope-specific T cells in the uveitic eye and in lymphoid organs of hIRBP651-670-immunized mice. CONCLUSIONS: Data suggest that hIRBP651-670 is an epitope naturally processed from a conserved region of native IRBP, potentially explaining its relatively high uveitogenicity. This epitope should be useful for basic and preclinical studies of uveitis in the C57BL/6 model and gives access to genetically engineered mice available on this background.

Development and evaluation of dexamethasone nanomicelles with potential for treating posterior uveitis after topical application.

PURPOSE: This study aims at the development and preliminary evaluation of dexamethasone nanomicelles for treating posterior uveitis. Nanomicelles were formulated using polyoxyl 40 stearate (P40S) and polysorbate 80 (P80), which are approved by the FDA for ocular use. METHODS: Dexamethasone nanomicelles were prepared and characterized for critical micellar concentration, solubility of dexamethasone, particle size, surface charge, morphology, in vitro drug release, clarity, stability, filtration efficiency, and sterility. Ocular tolerance and the tissue drug distribution of dexamethasone were assessed in rabbits after single and multiple topical administration. RESULTS: Dexamethasone nanomicelles (0.1% w/v) were successfully developed and characterized with an optimized composition of P40S/P80=7/3 by weight. The mean diameter of blank and drug-loaded nanomicelles was 13.3±0.4 and 14.5±0.4 nm, respectively. Transmission electron microscopy images revealed the spherical structure of nanomicelles. Nanomicelles were found to be stable with respect to clarity, size and drug content at 4°C and 25°C for up to 6 months. No irritation or redness was observed in the treated eyes as compared with the untreated control rabbit eyes. Therapeutic concentrations of dexamethasone were observed in the retina and choroid after single and multiple topical application in rabbits. CONCLUSION: In conclusion, the nanomicelles of P40S and P80 could efficiently solubilize 0.1% dexamethasone in their cores. The results also indicate that mixed nanomicelles could be utilized as a potential delivery system for delivering dexamethasone to treat the back of the eye diseases such as posterior uveitis after topical application.

Beyond the cherry-red spot: Ocular manifestations of sphingolipid-mediated neurodegenerative and inflammatory disorders.

Sphingolipids are a ubiquitous membrane lipid present in every cell and found most abundantly in neural tissues. Disorders such as Tay-Sachs or Niemann-Pick disease are the most familiar examples of dysfunction in sphingolipid metabolism and are typically associated with neurodegeneration and ocular findings such as blindness. More recently, the role of bioactive sphingolipids has been established in a multitude of cellular events, including cell survival, growth, senescence and apoptosis, inflammation, and neovascularization. We discuss our current knowledge and understanding of sphingolipid metabolism and signaling in the pathogenesis of ocular diseases.

Laser-induced intrachoroidal dexamethasone drug delivery system to posterior eye segment.

PURPOSE: To investigate the feasibility of laser-induced intrachoroidal dexamethasone (DEX) delivery as a potentially useful therapy for adjusting the most effective drug level to the posterior segment eye diseases. METHODS: An implant was prepared by dissolving poly(DL-lactide) and DEX. In vitro release of DEX was evaluated at 7, 14, and 28 days by ELISA. In vivo, a DEX implant was inserted into a rabbit choroid, and 10, 50, or 200 burns of photocoagulation were applied at the implant lesion. After treatment, the vitreous humor was immediately aspirated and the DEX level was measured by liquid chromatography/mass spectrometry/mass spectrometry. Furthermore, the vitreous DEX level was measured at 1, 7, 14, and 28 days after implantation and 50 burns of photocoagulation. The toxicity of the laser-induced DEX implant was evaluated by ophthalmoscopy and light microscopy. Endotoxin-induced uveitis (EIU) was induced after DEX implantation and photocoagulation, and anti-inflammatory activities were evaluated by grading clinical signs, protein concentrations, and histopathologic studies. RESULTS: Photocoagulation significantly increased the DEX release from the implant at 7 days in vitro. In vivo, the DEX implant exposed to 10, 50, and 200 burns of photocoagulation increased the vitreous DEX levels in a dose-dependent manner. The vitreous DEX level in the DEX implant applied to 50 burns of photocoagulation peaked 1 day after treatment. The laser-induced DEX implant showed no retinal abnormalities except the implantation site, and significantly inhibited the EIU. CONCLUSIONS: Laser-induced intrachoroidal DEX delivery controls the DEX level in the vitreous humor and effectively prevents the experimental uveitis.

Fundus autofluorescence imaging in posterior uveitis.

Although the phenomenon of fundus autofluorescence has been known for decades, it has only recently been recognized as a measure of retinal pigment epithelial function and health. Characteristic fundus autofluorescence patterns have been described in eyes affected by inflammation of the posterior segment, and these patterns have provided insights into the pathogenesis of posterior uveitis entities. In addition, preliminary data indicate that fundus autofluorescence characteristics may serve as markers of disease activity, allow prediction of visual prognosis, and may help determine the adequacy of therapy. We provide an overview of the current state of fundus autofluorescence imaging technology and review our current knowledge of fundus autoflourescence findings and their clinical use in the posterior uveitis entities.

Constitutive neuronal expression of the immune regulator, programmed death 1 (PD-1), identified during experimental autoimmune uveitis.

PURPOSE: Programmed death-1 (PD-1) ligation downregulates active lymphocyte responses. The authors tested whether PD-1 or its ligands are expressed in the posterior segment during active intraocular inflammation. METHODS: Experimental autoimmune uveitis (EAU) was induced using interphotoreceptor retinoid-binding protein (IRBP 161-180). Ocular inflammation was evaluated by histology and expression of PD-1 ligand tested by immunohistochemistry. PD-1 expression was evaluated by immunohistochemistry, RT-PCR, and Western immunoblotting. RESULTS: Using immunohistochemistry, PD-1, but not its ligands, was constitutively expressed in retinal neurons of naive mouse retina. Both PD-1 and its ligands were observed, as expected, in sites of active inflammation. CONCLUSIONS: PD-1 and its ligands were expressed in sites of active inflammation, in accordance with many other models of inflammatory disease. Surprisingly, PD-1, not previously described outside the immune system, was constitutively expressed in retinal neurons, raising the possibility that PD-1 signaling may be important for neuronal function in the absence of an inflammatory insult.

Local ocular immunomodulation resulting from electrotransfer of plasmid encoding soluble TNF receptors in the ciliary muscle.

PURPOSE: Plasmid electrotransfer in the ciliary muscle allows the sustained release of therapeutic proteins within the eye. The aim of this study was to evaluate whether the ocular production of TNF-alpha soluble receptor, using this nonviral gene therapy method, could have a beneficial local effect in a model of experimental autoimmune uveoretinitis (EAU). METHODS: Injection of a plasmid encoding a TNF-alpha p55 receptor (30 microg) in the ciliary muscle, combined with electrotransfer (200 V/cm), was carried out in Lewis rat eyes 4 days before the induction of EAU by S-antigen. Control eyes received naked plasmid electrotransfer or simple injection of the therapeutic plasmid. The disease was evaluated clinically and histologically. Cytokines and chemokines were analyzed in the ocular media by multiplex assay performed 15 and 21 days after immunization. RESULTS: Ocular TNF-alpha blockade, resulting from the local secretion of soluble receptors, was associated with delayed and significantly less severe uveitis, together with a reduction of the retinal damages. Compared with the controls, treated eyes showed significantly lower levels of IL-1beta and MCP1, higher levels of IL-13 and IL-4, and reduced NOS-2 expression in infiltrating cells. Treatment did not influence TNF-alpha levels in inguinal lymph nodes. CONCLUSIONS: Taken together, these results indicate that local immunomodulation was achieved and that no systemic adverse effects of TNF-alpha blockade observed after systemic injection of TNF-alpha inhibitors should be expected.

Neuroprotective effect of an antioxidant, lutein, during retinal inflammation.

PURPOSE: Lutein has been the focus of recent study as a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate, with the use of a mouse endotoxin-induced uveitis (EIU) model, the neuroprotective effects of lutein against retinal neural damage caused by inflammation. METHODS: EIU was induced by intraperitoneal injection of lipopolysaccharide (LPS). Each animal was given a subcutaneous injection of lutein or vehicle three times: concurrently with and 3 hours before and after the LPS injection. Analysis was carried out 24 hours after EIU induction. Levels of rhodopsin protein and STAT3 activation were analyzed by immunoblotting. Lengths of the outer segments of the photoreceptor cells were measured. Dark-adapted full-field electroretinograms were recorded. Oxidative stress in the retina was analyzed by dihydroethidium and fluorescent probe. Expression of glial fibrillary acidic protein (GFAP) was shown immunohistochemically. RESULTS: The EIU-induced decrease in rhodopsin expression followed by shortening of the outer segments and reduction in a-wave amplitude were prevented by lutein treatment. Levels of STAT3 activation, downstream of inflammatory cytokine signals, and reactive oxygen species (ROS), which are both upregulated during EIU, were reduced by lutein. Pathologic change of Müller glial cells, represented by GFAP expression, was also prevented by lutein. CONCLUSIONS: The present data revealed that the antioxidant lutein was neuroprotective during EIU, suggesting a potential approach for suppressing retinal neural damage during inflammation.

Effects of heme oxygenase-1 inducer and inhibitor on experimental autoimmune uveoretinitis.

PURPOSE: Experimental autoimmune uveoretinitis (EAU) is an animal model of posterior uveitis and heme oxygenase-1 (HO-1) is a well-known anti-oxidant factor. However, there is no report a protective role of HO-1 on EAU in vivo. To verify that HO-1 is induced in EAU by interphotoreceptor retinoid-binding protein (IRBP), that an HO-1 inducers ameliorates the associated inflammation, and that an HO-1 inhibitor exacerbates this inflammation. METHODS: Forty four Lewis rats were given either 40 mol/kg hemin or 40 mol/kg SnPP (tin protoporphyrin IX) by intraperitoneal injection and twenty two uveitis control rats were injected with 0.5 mL of saline once daily 5-20 days after IRBP immunization inducing EAU. Three normal control rats were used for Western blotting and ELISA assay of HO-1. The clinical uveitis signs of inflammation were scored in the three groups from 0 to 4 on alternate three days. To confirm the clinical results, histological and immunohistochemical stain of HO-1 were performed on the day of peak inflammation and Western blotting and ELISA assay of HO-1 were performed on 6th, 12th and 18th day after IRBP immunization. RESULTS: Hemin, an inducer of HO-1, ameliorated the clinical signs of EAU. In contrast, SnPP-treated rats show that the severity of the clinical sign were exacerbated at the peak period of the disease. These results are roughly compatible with histological, immunoblotting, and immunohistochemical evaluations and an ELISA assay of HO-1. CONCLUSIONS: We suggest that HO-1 plays an important protective role in EAU.

Post-translational modification of crystallins in vitreous body from experimental autoimmune uveitis of rats.

Experimental autoimmune uveitis (EAU) is a well-known animal model of posterior uveitis that is one of the major causes of blindness. EAU could be induced in susceptible animals (i.e., Lewis rat) by immune reactions using evolutionarily conserved retinal proteins, such as interphoto-receptor retinoid binding protein (IRBP), or epitaphs of the protein. First, we prepared the following four test groups that subsequently increased or decreased inflammation. (1) Normal control group, (2) IRBP-induced uveitis group, (3) Hemin-treated uveitis group, and (4) Sn(IV) protoporphyrin IX dichloride (SnPP)-treated uveitis group. Second, in the vitreous bodies of Lewis rats, the infiltrated proteins were analyzed using two-dimensional electrophoresis (2-DE), MALDI-TOF/MS, and Micro LC/LC-MS/MS analysis. Finally, Western blotting was applied to confirm the relative amount of crystallins and phosphorylation sites of alphaB-crystallin. Thirty spots were identified in vitreous bodies, and 27 of these spots were members of the crystallin family. Unlike betaA4- and B2-crystallins (that were significantly increased without truncation), alphaA- and B-crystallins were only truncated in EAU vitreous body. Taken as a whole, in the rat EAU model, we suggest that post-translational truncations of alphaA- and alphaB-crystallins, phosphorylation of alphaB-crystallin, and new production of betaA4- and betaB2-crystallins are intercorrelated with uveitis progression and inflammatory responses.

Long-term follow-up of tacrolimus treatment in immune posterior uveitis.

PURPOSE: To study the efficacy of tacrolimus in immune posterior uveitis. METHODS: Twenty-one eyes of 11 patients with immune posterior uveitis under tacrolimus treatment were prospectively followed for 1 to 5 years. Tacrolimus dosage was adjusted to maintain blood levels in the range of 7 to 10 ng/mL. Systemic and ophthalmic evaluations were performed at baseline and during follow-up. RESULTS: After a mean follow-up of 45 months, no treatment other than tacrolimus was necessary to control the inflammation in 6 cases (54.5%). The number of annual recurrences decreased from 3.2 to 1.29 during tacrolimus treatment (p=0.021). In four patients, tacrolimus was suspended after a treatment period of 27+/-3.5 months and a follow-up period of 12 months free of uveitis relapses. All four were free from relapses following tacrolimus withdrawal. Visual acuity remained unchanged in 16/21 (76%) eyes, deteriorated in 4/21 (19%), and improved in 1/21 (5%). Renal function transiently deteriorated in four patients from basal serum creatinine levels of 0.84, 1.1, 0.88, and 0.78 mg/dL to maximum levels of 1.33, 2.48, 1.38, and 1.39 mg/dL, respectively. This deterioration was directly related with elevated tacrolimus serum levels, returning to normal when doses were reduced. During the overall controlled evolution period, a slight increase of serum creatinine from a basal value of 0.89+/-0.2 mg/dL to a final of 1+/-0.19 mg/dL was detected, which was not statistically significant. All secondary effects were mild, transient, and did not require interruption of long-term treatment to be controlled. CONCLUSIONS: Tacrolimus was well tolerated and useful in controlling posterior immune uveitis. Tacrolimus could be considered a real alternative to cyclosporine, and not only in cases of cyclosporine resistance or toxicity.

Analysis of immune privilege in eyes with Mycobacteria tuberculosa adjuvant-induced uveitis.

PURPOSE: To examine the effects of intravitreal Mycobacteria tuberculosa adjuvant (MTA) on ocular immune privilege. METHODS: MTA was injected into the vitreous cavity of BALB/c mouse eyes to induce anterior uveitis. The inflamed eyes were then examined for their capacity to afford immune privilege to injected allogeneic tumor cells and to promote anterior chamber-associated immune deviation (ACAID). Aqueous humor (AqH) was tested for IL-12 content and for its ability to inhibit T-cell activation. RESULTS: AqH removed from MTA-inflamed eyes at 6 and 12 h contained high levels of IL-12, which then fell almost to baseline at 24 h. This is relevant to the finding that the inflamed eye failed to support ACAID induction at an early time period and then regained the ACAID-induction capability at a later time. Nonetheless, AqH removed from MTA-inflamed eyes retained its capacity to suppress T-cell activation, and MTA-inflamed eyes afforded extended survival to alloantigenic tumor cells implanted into the anterior chamber. CONCLUSION: Intraocular inflammation evoked by MTA causes the local accumulation of IL-12 and simultaneously robs the eye of its capacity to promote systemic immune tolerance to eye-derived antigens. However, MTA-inflamed eyes retain immune privilege, as indicated by their support of the progressive growth of allogeneic tumor cells.

Contribution of TNF-alpha to leukocyte adhesion, vascular leakage, and apoptotic cell death in endotoxin-induced uveitis in vivo.

PURPOSE: To investigate the effect of TNF-alpha on leukocyte adhesion, vascular leakage, and apoptotic cell death in endotoxin-induced uveitis (EIU) in the rat. METHODS: EIU was induced in Long-Evans rats by a single footpad injection of lipopolysaccharide (LPS; 350 microg/kg) from Salmonella typhimurium. A single injection of recombinant TNF receptor P75 (etanercept) was given subcutaneously 24 hours before the administration of LPS. Twenty-four hours after administration of LPS, leukocyte adhesion was evaluated in vivo with SLO-acridine orange angiography and ex vivo with concanavalin A lectin staining of retinal flatmounts. Neutrophil activation was quantified by a myeloperoxidase activity assay. Vascular leakage was assessed by Evans blue extravasation. Retinal cell death was assessed with TUNEL staining and quantified with a modified ELISA protocol. Involvement of caspase-3 and -8 was determined by M30 antibody staining, Western blot analysis, and a test for enzymatic activity. RESULTS: Twenty-four hours after the LPS injection, significant increases in leukocyte rolling, adhesion, and activation were observed. In addition, increased levels of apoptosis in the vascular endothelium and the ganglion cell and inner nuclear layers and activation of caspase-8 and -3 were observed. After administration of the TNF-alpha inhibitor, significant reduction in the leukocyte rolling, adhesion, activation, and apoptosis in all the affected layers was observed. The quantitative analysis of vascular leakage revealed a significant decrease after treatment with etanercept. Retinal cell death quantification showed a significant decrease after treatment with the TNF-alpha inhibitor. CONCLUSIONS: Anti-TNF-alpha treatment reduces the LPS-induced increases in leukocyte rolling, adhesion, and vascular leakage in this rat model of inflammatory uveitis. These results suggest the involvement of TNF-alpha in inflammatory uveitis and its potential use as a therapeutic agent in the reduction of ocular inflammation.

Chemokine and chemokine receptor expression during experimental autoimmune uveoretinitis in mice.

BACKGROUND: Chemokines act as chemoattractants and activators of specific leukocytes at the site of inflammation. In this study, we investigated serial expression of chemokines and chemokine receptors in the eye with experimental autoimmune uveoretinitis (EAU) using RNAse protection assay, and confirmed their expression by immunohistochemical staining. METHODS: B10.A mice were immunized with 50 micro g of interphotoreceptor retinoid binding protein (IRBP) emulsified in complete Freund's adjuvant in order to induce EAU. The eyes were enucleated 0, 7, 14 and 21 days after IRBP immunization to analyze mRNA expression of chemokines and chemokine receptors in the posterior segment. In addition, expression of IP-10 and CXCR3 was analyzed by immunohistochemistry. RESULTS: The gene expression of RANTES, IP-10, and MCP-1 was upregulated on day 14 after immunization (early stage of EAU). The expression of chemokine receptors (CCR2 and CCR5) associated with Th1-type T cells correlated with their appropriate ligands. Furthermore, immunohistochemical study showed that IP-10 and CXCR3, the receptor for IP-10, were strongly expressed in the posterior segment of the eyes from mice with EAU. CONCLUSION: These results suggest that RANTES, IP-10 and MCP-1 may contribute to the recruitment of Th1-type T cells into the eye during the development of EAU in mice.

Leukocyte trafficking in experimental autoimmune uveitis: breakdown of blood-retinal barrier and upregulation of cellular adhesion molecules.

PURPOSE: To clarify the order of events occurring in the breakdown of the blood-retinal barrier (BRB) in experimental autoimmune uveoretinitis (EAU) and in particular to study the relationships between increased vascular permeability, upregulation of endothelial cell adhesion molecules, and leukocyte adhesion and infiltration during EAU. METHODS: B10.RIII mice were immunized with human interphotoreceptor retinoid binding protein (IRBP) peptide 161-180. Changes in the retinal microvasculature were examined on days 3, 6, 7, 8, 9, 10, 16, and 21 postimmunization (pi). Evans blue dye was administered intravenously to assess vascular permeability. Expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, P-selectin, E-selectin, and platelet endothelial cell adhesion molecule (PECAM)-1 was evaluated by in vivo administration of antibody and subsequent immunostaining of retinal wholemounts. Lymphocytes from inguinal lymph nodes of normal and chicken ovalbumin (OVA)- or IRBP peptide-immunized mice at day 5, 6, 7, 8, and 15 pi were labeled in vitro with calcein-AM (C-AM) and infused intravenously into syngeneic recipient mice, which had been immunized with peptide at the same corresponding time point. Wholemount preparations of retinas were observed 24 hours later by confocal microscopy to determine the adhesion and infiltration of lymphocytes. RESULTS: The first observation of an increase in vascular permeability occurred at day 7 pi and was restricted to focal areas of the retinal postcapillary venules of the inner vascular plexus. This progressively extended to the outer vascular plexus at day 9 pi. Specific adhesion of leukocytes to the endothelium of retinal venules of the inner vascular plexus was first observed at day 6 pi. Leukocyte extravasation into the retinal parenchyma from these vessels began at day 8 pi and extended to the outer vascular plexus at day 9 pi. The expression of adhesion molecules increased progressively during the development of EAU. In particular, the adhesion molecules ICAM-1, P-selectin, and E-selectin were expressed predominately in retinal venules, the sites of BRB breakdown, cell adhesion, and extravasation, from day 7 pi. The increases in expression of ICAM-1 and P-selectin were associated both spatially and temporally with breakdown of the BRB, cell adhesion, and extravasation. No increase in expression of P-selectin and ICAM-1 was observed in either the mesenteric vessels of EAU mice or the retinal vessels of OVA-immunized mice. CONCLUSIONS: The sequence of events in EAU appears to be focal adhesion of leukocytes to discrete sites on postcapillary venules, followed by upregulation of adhesion molecules, especially ICAM-1 and P-selectin, and breakdown of the BRB, leading to transendothelial migration of leukocytes and recruitment of large numbers of cells to the retinal parenchyma. These changes occur over a short period of 6 to 9 days pi and initiate the process of tissue damage during the following 2 to 3 weeks.

Apoptosis in patients with posterior uveitis.

BACKGROUND: Apoptosis plays a part in the pathogenesis of autoimmune diseases. OBJECTIVE: To investigate the expression of apoptotic markers in the eyes of patients with uveitis. METHODS: With the use of immunohistochemical and in situ apoptotic detection techniques, apoptotic molecules (Fas or Fas ligand [FasL]) and nuclear DNA fragmentation were examined in 8 enucleated eyes with Behçet's disease (1), sarcoidosis (1), subretinal fibrosis and uveitis (1), sympathetic ophthalmia (4), and the Vogt-Koyanagi-Harada syndrome (1); in 5 chorioretinal biopsy specimens with acute retinal necrosis (2), multifocal choroiditis (1), sarcoidosis (1), and subretinal fibrosis and uveitis (1); and in 3 normal control eyes. RESULTS: Fas and FasL were constitutively expressed in the normal human retina, but they were expressed much less in the choroid. Increased expression of Fas and FasL was found in the retina, chorioretinal scar, and choroidal granulomas in uveitic eyes. However, Fas and FasL expression was absent in the biopsy specimens with acute retinal necrosis, and little Fas or FasL was noted on infiltrating lymphocytes. DNA fragmentation was also identified in eyes with chorioretinal scar and gliosis. CONCLUSIONS: Apoptosis occurs in uveitic eyes and may play a regulatory role in limiting ocular inflammation. In uveitic eyes, a dysregulation of the Fas-FasL apoptotic pathway may lead to gliosis and fibrosis.

Sensitivity of laser flare photometry to monitor inflammation in uveitis of the posterior segment.

PURPOSE: Laser flare photometry is a new quantitative method for evaluating aqueous flare, making flare the only inflammatory parameter that can be evaluated precisely and objectively. The validity of the method already has been demonstrated in anterior segment inflammation. The aim of this study is to assess the validity and limitations of the method to quantify and monitor inflammation in uveitis with predominant involvement of the posterior segment. METHODS: Five well-defined conditions with uveitis predominant in the posterior segment were analyzed in this study: Behçet uveitis, pars planitis, posterior sarcoidosis, posterior pole toxoplasmosis, and birdshot chorioretinopathy. (1) Mean initial (pretreatment) flare was determined; (2) in the patients needing systemic steroid therapy, introduction of therapy was correlated with evoluting laser flare photometry; and (3) in patients with quiescent disease, the predictive value of a defined subclinical photometry-detected flare rise for disease recrudescence was analyzed. RESULTS: Initial pretreatment flare was 331.8 +/- 47.7 photon counts per millisecond (ph/msecond) (mean +/- standard error of the mean) for Behçet uveitis, 15.6 +/- 1.3 ph/msecond for pars planitis, 26.9 +/- 4.6 ph/msecond for posterior sarcoidosis, 7.5 +/- 1.0 ph/msecond for posterior pole toxoplasmosis, 5.8 +/- 0.7 ph/msecond for birdshot chorioretinopathy, and 4.7 +/- 0.1 ph/msecond for a group of 88 control eyes. A significant flare reduction after start of steroid therapy was seen in Behçet uveitis (78% reduction), sarcoidosis (44.8%), and pars planitis (51%), but not in toxoplasmosis or in birdshot. A small flare rise had a predictive value for disease recrudescence in 27/35 patients (predictive value, 0.77; sensitivity rate, 100%). The level of associated blood-aqueous barrier disruption for reliable follow-up of posterior uveitis was empirically determined to be 13 to 15 ph/msecond. CONCLUSION: Laser flare photometry was found to be very sensitive to monitor inflammation in uveitis of the posterior segment as long as a sufficient level of associated blood-aqueous barrier disruption (flare) was present.