Non-Occlusive Retinal Vascular Inflammation and Role of Red Blood Cell Deformability in Birdshot Chorioretinopathy.

Purpose: To investigate differences in red blood cell (RBC) deformability between birdshot chorioretinopathy (BCR) subjects and matched controls, and to postulate its relationship with lack of vascular occlusion in BCR. Methods: In a single center, prospective, non-randomized mechanistic study, blood samples were collected from eight healthy controls and nine BCR patients, and subjected to biochemical and hematological tests, as well as RBC indices assessment using dual-beam optical tweezers. Results: The mean age of the controls was 52.37 ± 10.70 years and BCR patients was 53.44 ± 12.39 years. Initial cell size (Io) for the controls was 8.48 ± 0.25 µm and 8.87 ± 0.31 µm for BCR RBCs (p = 0.014). The deformability index (DI) for the controls was 0.066 ± 0.02 and that for BCR RBCs was 0.063 ± 0.03 (p = 0.441). Conclusion: There was no statistically significant difference in DI between RBCs from BCR and healthy controls. This may explain the rare occurrence of retinal vascular occlusion despite the underlying vasculitic pathophysiology of BCR.

Circulating Endothelial Markers in Retinal Vasculopathy With Cerebral Leukoencephalopathy and Systemic Manifestations.

BACKGROUND AND PURPOSE: Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is a monogenic small vessel disease, caused by C-terminal truncating TREX1 mutations, that can be considered a model for stroke and vascular dementia. The pathophysiology of RVCL-S is largely unknown, but systemic endothelial involvement has been suggested, leading to pathology in the brain and other highly vascularized organs. Here, we investigated circulating endothelial markers to confirm endothelial involvement and identify biomarkers for disease activity. METHODS: We measured circulating levels of von Willebrand factor (VWF) antigen, VWF propeptide, and angiopoietin-2 in members of 3 Dutch RVCL-S families and matched unrelated healthy controls. Stratified analyses based on symptomatology and age were performed. RESULTS: We found elevated levels of VWF antigen, VWF propeptide, and angiopoietin-2 in TREX1 mutation carriers (n=31) compared with family members without a TREX1 mutation (n=33) and unrelated healthy controls (n=31; Kruskal-Wallis test P<0.001 for all comparisons). Effects were most pronounced in mutation carriers with clinical manifestations aged ≥40 years (Mann-Whitney U test P<0.001 for all comparisons). Compared with healthy controls, levels of VWF antigen (P=0.02) and angiopoietin-2 (P=0.04) were also elevated in mutation carriers aged <40 years. All 3 markers showed moderate correlations with markers of kidney and liver disease and inflammation (ie, systemic symptoms of RVCL-S). CONCLUSIONS: Our results confirm an important role of the endothelium in RVCL-S pathophysiology. VWF antigen, VWF propeptide, and angiopoietin-2 might serve as early biomarkers of disease activity. Our findings might also help to understand the pathophysiology of common neurovascular disorders, such as stroke.

The predictive role of markers of Inflammation and endothelial dysfunction on the course of diabetic retinopathy in type 1 diabetes.

AIMS: This study was undertaken to determine whether levels of inflammation and endothelial dysfunction biomarkers in serum samples collected at baseline in the Diabetes Control and Complications Trial (DCCT) cohort could predict the development of retinopathy. METHODS: Levels of clotting/fibrinolysis, inflammation and endothelial dysfunction biomarkers were measured in 1391 subjects with type 1 diabetes to determine whether their levels predicted increased risk to develop or accelerate progression of retinopathy during 16years of follow-up. RESULTS: Using regression models adjusted for DCCT treatment group, duration of diabetes, baseline retinopathy scores, HbA1c and albumin excretion rate, the baseline levels of sE-selectin and PAI-1 (active) were significantly associated with increased risk of a 3-step progression in retinopathy score in the primary prevention cohort (PPC). After adjusting for additional covariates (e.g., ACE/ARB and statin therapy), this association persisted. Levels of active and total PAI-1 in the same group were also significantly associated, after similar adjustments, with the time to progress to severe non-proliferative retinopathy during the follow-up period (54 and 29%, respectively of increased risk). No associations were observed in the secondary intervention cohort for any of the outcomes. CONCLUSIONS: High levels of sE-selectin and PAI-1 are associated with the development of retinopathy in patients with uncomplicated type 1 diabetes.

Increased homocysteine, homocysteine-thiolactone, protein homocysteinylation and oxidative stress in the circulation of patients with Eales' disease.

BACKGROUND: Eales' disease (ED) is an idiopathic retinal vascular disorder. It presents with inflammation and neovascularization in the retina. Adult men, aged between 15 and 40 years are more susceptible than women. Homocysteine has been implicated in other ocular diseases including age-related macular degeneration (ARMD), central retinal vein occlusion (CRVO) and optic neuropathy. The present study investigates the role of homocysteine in ED. METHODS: Forty male subjects, 20 with ED and 20 healthy controls, were recruited to the study. Their blood samples were used to measure thiobarbituric acid reactive substances (TBARS), glutathione (GSH), homocysteine, homocysteine-thiolactone, extent of homocysteine conjugation with proteins and plasma copper concentration. RESULTS: In the ED group, plasma homocysteine (18.6 ± 1.77 µmol/L, P < 0.001) and homocysteine-thiolactone (45.3 ± 6.8 nmol/L, P < 0.0001) concentrations were significantly higher compared to homocysteine (11.2 ± 0.64 µmol/L) and homocysteine-thiolactone (7.1 ± 0.94 nmol/L) concentrations in control subjects. TBARS (P < 0.011) and protein homocysteinylation (P < 0.030) were higher in the ED group while GSH (5.9 ± 0.44 µmol/L, P < 0.01) and copper (6.6 ± 0.42 µmol/L, P < 0.001) were lower compared to GSH (8.1 ± 0.41 µmol/L) and copper (15.4 ± 0.73 µmol/L) concentrations in control subjects. CONCLUSIONS: Increased homocysteine, and its metabolite thiolactone, is associated with the functional impairment of protein due to homocysteinylation in ED.

Interleukin-1ß-regulating antibody XOMA 052 (gevokizumab) in the treatment of acute exacerbations of resistant uveitis of Behcet's disease: an open-label pilot study.

OBJECTIVE: Uveitis and retinal vasculitis are sight-threatening manifestations of Behçet's disease with limited treatment options. This pilot study aimed to evaluate the safety, pharmacokinetics and clinical activity of XOMA 052 (gevokizumab), a recombinant humanised anti-interleukin 1ß antibody, in Behçet's disease patients with uveitis. METHODS: Patients with acute posterior or panuveitis, and/or retinal vasculitis, resistant to azathioprine and/or ciclosporin, and receiving 10 mg/day or less of prednisolone, were enrolled into the 98-day study. Immunosuppressive agents were discontinued at baseline. Patients received a single infusion of XOMA 052 (0.3 mg/kg). The safety and uveitis status and pharmacokinetics of XOMA 052 were evaluated. RESULTS: Seven patients enrolled and completed the study. No treatment-related adverse event was observed. XOMA 052 treatment was associated with rapid and durable clinical response in all patients. Complete resolution of intraocular inflammation was achieved in 4-21 days (median 14 days), with a median duration of response of 49 days (range 21-97 days); one patient remained exacerbation free throughout the study. CONCLUSIONS: Well tolerated, XOMA 052 resulted in a rapid onset and sustained reduction in intraocular inflammation in patients with resistant uveitis and retinal vasculitis. Moreover, the effect was observed despite discontinuation of immunosuppressive agents and without the need to increase corticosteroid dosages.

Elevated tumor necrosis factor in serum is associated with increased retinal ischemia in proliferative eales' disease.

OBJECTIVE: Tumor necrosis factor (TNF) was evaluated in the serum of patients with proliferative stage of Eales' disease to study its relation with the area of retinal capillary non-perfusion (ischemic retina). METHODS: Quantification of the levels of TNF was done using sandwich ELISA in 52 cases with proliferative Eales' disease and in 32 healthy controls. Seven 50° photographs of different fields of the fundus were taken on fluorescein angiography. The area of retinal capillary non-perfusion denoting retinal cell death was assessed in terms of optic disc areas. RESULTS: TNF levels were found to be significantly increased in the proliferative stage of the disease (mean 23.64 ± 3.7 pg/ml) as compared to controls (mean 12.49 ± 2.9 pg/ml; p < 0.001). Higher levels of TNF were found to be associated with an increased area of retinal capillary non-perfusion on fluorescein angiography. TNF levels of 20-31 pg/ml were observed in cases with neovascularization at the disc (n = 33) as compared to 17-21 pg/ml in cases with neovascularization elsewhere (n = 19). CONCLUSIONS: An increased level of TNF is associated with an increased area of the ischemic retina. It is hypothesized that retinal cell death signaling in proliferative Eales' disease is related to an increased TNF level.

Total antioxidant capacity in Eales' disease, uveitis & cataract.

BACKGROUND & OBJECTIVES: The human system possesses antioxidants that act harmoniously to neutralize the harmful oxidants. This study was aimed to evaluate the serum total antioxidant capacity (TAC) as a single parameter in Eales' disease (ED) and in an acute inflammatory condition such as uveitis and in cataract which is chronic, compared to healthy controls. METHODS: The TAC assay was done spectrophotometrically in the serum of Eales' disease cases (n=20) as well as in other ocular pathologies involving oxidative stress namely, uveitis and cataract (n=20 each). The oxidative stress measured in terms of TBARS, was correlated with the TAC. Individual antioxidants namely vitamin C, E and glutathione were also estimated and correlated with TAC. RESULTS: TAC was found to be significantly lower in Eales' disease with active vasculitis (0.28 ± 0.09 mM, P<0.001), Eales' disease with healed vasculitis (0.67 ± 0.09 mM), uveitis (0.46 ± 0.09 mM, P<0.001) and cataract (0.53 ± 0.1 mM, P=0.001) compared to the healthy controls, with a TAC level of 0.77 ± 0.09 mM. The TAC was found to correlate positively with vitamin E levels (P=0.05), GSH (P=0.02) but not with vitamin C, as seen in ED cases. In ED cases supplemented with vitamin E and C, there was a significant increase in the TAC level (P=0.02). INTERPRETATION & CONCLUSIONS: The TAC measurement provided a comprehensive assay for establishing a link between the antioxidant capacity and the risk of disease as well as monitoring antioxidant therapy. This method is a good substitute for assay of individual antioxidants as it clearly gives the status of the oxidative stress in the disease process.

Mannose-binding lectin as part of the complement pathway: characterization in non-inflamed and inflamed human eyes.

BACKGROUND: Mannose-binding lectin plays a central effector role in the lectin pathway of complement activation. Frequently occurring MBL2 polymorphisms result in mannose-binding lectin deficiency, which increases susceptibility to infection. We characterized mannose-binding lectin levels and function in non-inflamed and inflamed human eyes, and evaluated its relationship to blood mannose-binding lectin levels and function. DESIGN: Prospective, observational clinical study with controls and cases. PARTICIPANTS: Twenty-seven patients with paired blood and ocular samples (aqueous and/or vitreous) including 15 controls (non-inflamed) and 12 cases (inflamed). METHODS: Blood and ocular samples were collected from controls (n = 15) with quiet eyes during elective cataract surgery and cases with inflamed eyes including proven/suspected endophthalmitis (n = 11) and herpetic retinal vasculitis (n = 1). Mannan-binding and C4 deposition enzyme-linked quantify mannose-binding lectin levels and function. MAIN OUTCOME MEASURES: Blood and ocular mannose-binding lectin levels and function. RESULTS: Of 27 patients, 10 (37%) were mannose-binding lectin-deficient (defined as blood mannose-binding lectin levels <500 ng/mL). Blood mannose-binding lectin levels (P= 0.16) or function (P= 0.43) were not significantly different between controls and cases. As expected, there was a high correlation between blood mannose-binding lectin levels and function (r(2) = 0.74). However, there was significantly more mannose-binding lectin in inflamed eyes than non-inflamed eyes measured as level (P < 0.01) or C4 deposition function (P < 0.01). CONCLUSIONS: Our study demonstrated that mannose-binding lectin is significantly elevated in inflamed human eyes but virtually undetectable in non-inflamed control eyes, suggesting a role in sight-threatening ocular inflammation.

Assessment of gelatinase and tumor necrosis factor-α level in the vitreous and serum of patients with Eales disease: role of inflammation-mediated angiogenesis in the pathogenesis of Eales disease.

BACKGROUND: Eales disease (ED) is an idiopathic, inflammatory, venoocclusive disorder of peripheral retina resulting in retinal angiogenesis and vitreous hemorrhage. The objective of the present study is to investigate the expression and activation of gelatinase associated with the retinal neovascularization in ED and the relation between the levels of gelatinase and the cytokine tumor necrosis factor-α, known to upregulate matrix metalloproteinase (MMP) expression on various cells. METHODS: Vitreous and serum samples from 19 patients with ED who underwent retinal surgery were estimated for levels of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, and tumor necrosis factor-α by enzyme-linked immunosorbent assay method. Matrix metalloproteinase-2 and MMP-9 activities in serum and vitreous samples were evaluated by gelatin zymography method. Vitreous samples from 16 patients with macular hole undergoing vitrectomy were used as controls. RESULTS: Among the 2 gelatinase examined in vitreous and serum samples, only level and activity of MMP-9 were significantly higher in serum (P = 0.0001) and vitreous (P = 0.0002) samples of patients with ED than those of control subjects. Simultaneously, a positive correlation was found between intraocular tumor necrosis factor-α and MMP-9 concentration (Spearman correlation coefficient, r = 0.7040, P = 0.0023) in patients with ED. CONCLUSION: Increase in MMP-9 activity and its concentration in serum and vitreous of patients with ED compared with that of control subjects and correlation between intraocular levels of MMP-9 and tumor necrosis factor-α in patients with ED seem to provide a plausible explanation for inflammation-mediated angiogenesis during the development of this condition.

Interleukin-1 and tumor necrosis factor-alpha: novel targets for immunotherapy in Eales disease.

BACKGROUND: Eales disease is an idiopathic obliterative vasculopathy that primarily affects the peripheral retina of young adults. The authors evaluated interleukin 1 beta (IL-1beta), interleukin-6 (IL-6), Interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-alpha) in the serum of patients with Eales disease stages for the first time. METHODS: The study group consisted of 45 consecutive patients of Eales disease [inflammatory stage (n = 15) and proliferative stage (n = 30)] and 28 healthy controls. Immunoassays for the quantification of the levels of four cytokines including IL-1beta, IL-6, IL-10, and TNF-alpha in the serum samples were performed using ELISA kits. RESULTS: IL-1beta, IL-6, IL-10, and TNF-alpha levels were found to be increased significantly in the inflammatory stage of Eales disease as compared to controls (p < .001). IL-1beta levels decreased significantly during the proliferative stage of the disease as compared to the inflammatory stage (p = .03). TNF-alpha levels increased significantly during the proliferative stage as compared to the inflammatory stage (p = .02). CONCLUSIONS: Raised levels of IL-1beta and TNF-alpha were observed in the inflammatory stage and persisted in the proliferative stage of the disease. The IL-1 system and TNF-alpha represent novel target for immunotherapy for controlling inflammatory activity and/or the associated long-term sequelae related to angiogenesis in Eales disease.

Interferon-beta and adhesion molecules (E-selectin and s-intracellular adhesion molecule-1) are detected in sera from patients with retinal vasculitis and are induced in retinal vascular endothelial cells by Toll-like receptor 3 signalling.

Retinal vasculitis is a major component of ocular inflammation that plays a role in retinal tissue damage in patients with idiopathic uveitis and Behçet's disease. Here we show that type 1 interferons (IFN alpha/beta) were not detected in sera from normal individuals but were identified in up to 46% of the sera from retinal vasculitis patients. The predominant form of IFN observed was IFN-beta, which was detected in 39% of Behçet's disease patients and 47% of idiopathic uveitis patients. Seven patients whose sera contained IFN-beta were monitored prospectively. IFN-beta was shown to be present for 6-12 months in all seven of the sera samples tested. Furthermore, the adhesion molecule profile identified in this study was strikingly different when Behçet's and uveitis patient sera were compared to sera from normal controls. Sera from Behçet's disease patients contained significantly elevated levels of the soluble adhesion molecules, sE-selectin and s-intracellular adhesion molecule-1 (sICAM-1), whereas sera from patients with idiopathic uveitis contained significantly increased sE-selectin. In vitro studies evaluating the cell source of these cytokines revealed that polyriboinosinic polyribocytidylic acid (poly I:C) activated retinal vascular endothelial cells produce sE-selectin, sICAM-1 and IFN-beta. Production of these molecules was inhibited by pretreatment with anti-Toll-like receptor 3 (TLR-3) antibody. In conclusion, IFN-beta, sE-selectin and sICAM-1 are elevated in patients with retinal vasculitis and are induced in retinal vascular endothelial cells in vitro by activating the innate immune system through TLR-3. Further analysis of innate immune signalling may prove to be a novel target for future studies on pathogenic mechanisms and therapeutic approaches in retinal vasculitis.

Decreased platelet membrane fluidity in retinal periphlebitis in Eales' disease.

PURPOSE: Oxidative damage to cellular membranes plays an important role in the pathobiology of tissue injury. Free radical-induced peroxidation of membrane lipid and protein is associated with alterations in cellular, morphological, biochemical, and physical dynamics, which are related to the mobility of lipid molecules. Retinal photoreceptors and platelets have been shown to be an easy target of oxidants because of their high proportion of polyunsaturated fatty acids. This study was undertaken, for the first time, to investigate membrane fluidity in the platelets of patients with Eales' disease. METHODS: Assays of malonaldialdehyde levels and the enzymes superoxide dismutase and catalase and fluorescence polarization, for estimating membrane fluidity, were carried out on platelets from 20 patients with Eales' disease (stage 1 characterized by periphlebitis of small (1a) and large (1b) caliber vessels with superficial retinal hemorrhages) and 15 healthy controls. RESULTS: A significant increase was observed in the malonaldialdehyde levels. A significant decrease in the activity of superoxide dismutase and catalase was also observed. Platelet fluorescence polarization was significantly higher in the patients, indicating decreased membrane fluidity compared to controls (p<0.01). CONCLUSION: A decrease in platelet membrane fluidity occurs as a result of oxidative stress in retinal periphlebitis in Eales' disease. The decreased membrane fluidity suggests alterations in the physiological events, which may result in alterations in the functioning of retinal photoreceptors.

Response time and safety profile of pulsed oral methotrexate therapy in idiopathic retinal periphlebitis.

PURPOSE: To evaluate the response time and safety profile of low-dose oral methotrexate pulsed therapy in idiopathic retinal periphlebitis (Eales' disease). METHODS: A tertiary care center-based prospective interventional study, based on visual acuity grading, was undertaken. Twenty-one consecutive patients with idiopathic retinal periphlebitis were administered 12.5 mg methotrexate as a single oral dose, once per week for 12 weeks (cumulative dose = 150 mg). Each patient was assessed for change in visual acuity grades. Time of first therapeutic response was also noted. Drug safety was monitored by laboratory tests that included twice-weekly white blood cells and differential counts, twice-weekly platelet counts, and monthly liver function tests. RESULTS: Twenty-one eyes were assessed. Mean follow-up period was 6 months. All showed improvement in visual acuity grades. An excellent visual outcome (6/6 or better) was achieved in 18 (69%) eyes. Time of first therapeutic response varied from 2 to 6 weeks with a majority of eyes (80%) showing response by 4 weeks (median = 3 weeks). All the side effects of methotrexate were mild or moderate in severity and rapidly reversible on dose reduction or discontinuation. No patient had any constitutional symptoms severe enough to necessitate cessation of therapy. CONCLUSIONS: Low dose oral methotrexate pulse therapy (at a dose of 12.5 mg/week) is clinically effective within 4 weeks, and is associated with an acceptable safety profile.

Serum levels of chemokines correlate with disease activity in patients with retinal vasculitis.

Retinal vasculitis (RV) is characterised pathologically by migration of leucocytes across the blood-retinal barrier leading to oedema and photoreceptor cell dysfunction. Chemokines are a family of small molecules involved in leucocyte migration. In this study, levels of chemokines were measured in serum from patients with RV and correlated with disease activity and drug treatment. Serum samples (n= 100; 25 active, 75 inactive) were obtained from 50 patients with RV, and levels of the chemokines MIP-1alpha, macrophage inflammatory protein-1beta (MIP-1beta) and monocyte chemoattractant protein-1 (MCP-1) were measured by ELISA. For longitudinal analysis levels of the same chemokines were measured in six consecutive serum samples from 10 of the above patients. Chemokine levels were correlated with disease activity and current drug treatment for each sample. Sera from 20 healthy individuals were used as control samples. Serum levels of MIP-1beta were significantly raised in patients with RV, whether active or not, compared to healthy controls (P= 0.04). Levels of MIP-1beta and MCP-1 correlated with disease activity in some patients and with prednisolone levels in patients on this treatment alone. MIP-1alpha levels were not detectable in all samples but were present in significantly more samples from patients with active or inactive RV compared with healthy controls. Serum levels of the chemokines MIP-1beta and MIP-1alpha, but not MCP-1 were raised in patients with retinal vasculitis. Longitudinal analysis suggested that MIP-1beta and MCP-1 levels were controlled by drug treatment, particularly prednisolone. These data demonstrate that chemokines are involved in the pathogenesis of RV and may act as novel therapeutic targets.

Presence of a 88 kDa Eales protein in uveitis, tuberculosis, leprosy and rheumatoid arthritis.

BACKGROUND: Eales disease (ED) is an idiopathic retinal vasculitis affecting young adult males. We have earlier reported the identification, purification and partial characterization of a novel 88 kDa protein found in the serum of patients with ED. The aim of the present study was to look for the 88 kDa protein in serum samples obtained from cases of retinal vasculitis mimicking ED and in other systemic inflammatory diseases. MATERIAL/METHODS: Serum samples from healthy volunteers and from patients with ED, uveitis, parsplanitis ocular sarcoidosis, toxoplasmosis, leprosy, diabetic retinopathy, viral hepatitis, and rheumatoid arthritis were analyzed for the presence of the 88 kDa protein by polyacralymide gel electrophoresis (PAGE). The immunological identity of the 88 kDa protein found in ED and in other diseases was investigated by Western blot. Immunohistochemistry was performed on epiretinal membranes (ERM) obtained from ED patients to localize the 88 kDa protein. RESULTS: 88 kDa protein were detected in serum samples obtained from patients with posterior uveitis, tuberculosis, leprosy and rheumatoid arthritis. The 88 kDa protein found in serum from patients with ED is immunologically identical to that found in other systemic inflammatory conditions. 88 kDa protein was localized in inflammatory cells and in nonvascular endothelium in ERMs obtained from patients with ED. CONCLUSIONS: We have identified a novel acute phase reactant, which is elaborated in ocular and systemic inflammatory conditions other than Eales disease. Further work is necessary to decipher the precise role of the 88 kDa protein in the pathophysiology of these inflammatory diseases.