Optical coherence tomography biomarkers as outcome predictors to guide dexamethasone implant use in patients with iERM: a randomized controlled trial.

BACKGROUND: We aimed to investigate the anatomical features of optical coherence tomography (OCT) and vitreous cytokine levels as predictors of outcomes of combined phacovitrectomy with intravitreal dexamethasone (DEX) implants for idiopathic epiretinal membrane (iERM) treatment. METHODS: A prospective, single-masked, randomized, controlled clinical trial included 48 eyes. They were randomly assigned in a 1:1 ratio to undergo the DEX group (combined phacovitrectomy with ERM peeling and Ozurdex implantation) and control group (phacovitrectomy only). Best-corrected visual acuity (BCVA) and central macular thickness (CMT) were assessed at 1 d, 1 week, 1 month, and 3 months. The structural features of OCT before surgery were analysed for stratified analysis. Baseline soluble CD14 (sCD14) and sCD163 levels in the vitreous fluid were measured using ELISA. RESULTS: BCVA and CMT were not significantly different in the DEX and control groups. Eyes with hyperreflective foci (HRF) at baseline achieved better BCVA (Ptime*group=0.746; Pgroup=0.043, Wald χ²=7.869) and lower CMT (Ptime*group = 0.079; Pgroup = 0.001, Wald χ²=6.774) responses to DEX during follow-up. In all patients, the mean vitreous level of sCD163 in eyes with HRF was significantly higher than that in eyes without HRF (P = 0.036, Z=-2.093) at baseline. In the DEX group, higher sCD163 predicted greater reduction in CMT from baseline to 1 month (r = 0.470, P = 0.049). CONCLUSIONS: We found that intraoperative DEX implantation did not have beneficial effects on BCVA and CMT over a 3-month period in all patients with iERM, implying that the use of DEX for all iERM is not recommended. In contrast, for those with HRF on OCT responded better to DEX implants at the 3-month follow-up and thier vitreous fluid expressed higher levels of sCD163 at baseline. These data support the hypothesis that DEX implants may be particularly effective in treating cases where ERM is secondary to inflammation. TRIAL REGISTRATION: The trail has been registered at Chinese Clinical Trail Registry( https://www.chictr.org.cn ) on 2021/03/12 (ChiCTR2100044228). And all patients in the article were enrolled after registration.

Anterior Chamber Flare as a Non-Invasive Assessment of Intraocular Immune Status and Ocular Complications in Proliferative Diabetic Retinopathy.

Proliferative diabetic retinopathy (PDR) is a vision-threatening complication of diabetes mellitus (DM). Anterior chamber (AC) flare and intraocular cytokines are potent biomarkers reflecting the intraocular immune status in PDR. This study aimed to elucidate the complex interrelationship between AC flare and intraocular cytokines in PDR eyes. A retrospective observational study was conducted on 19 PDR eyes of 19 patients with type 2 DM, and on 19 eyes of 19 patients with idiopathic macular hole or epiretinal membrane as controls. AC flare was measured before pars plana vitrectomy (PPV). Aqueous humor (AH) and vitreous fluid (VF) samples were collected at the time of PPV, and the quantities of 27 cytokines in both intraocular fluids were analyzed. In the PDR and control groups, Spearman's rank correlation analysis revealed a positive correlation between AC flare and IL-8 level in both AH and VF. Additionally, IL-8 levels in AH correlated positively with IL-8 levels in VF. In the PDR group, receiver operating characteristic curve analysis identified IL-8 level in AH as a significant predictor for both diabetic macular edema (DME) and vitreous hemorrhage (VH) complications. The cut-off values of IL-8 were established at ≥26.6 pg/mL for DME and ≥7.96 pg/mL for VH. Given the positive correlation between AC flare and AH IL-8 level, the present findings suggest that AC flare value may potentially be a non-invasive biomarker for predicting DME.

Haemoglobin spherulosis: Fine needle aspiration biopsy of an unusual ocular lesion.

OBJECTIVE: Haemoglobin spherulosis (HS) is a rare lesion occurring post-vitreous haemorrhage that is absent from the cytopathology literature. METHODS: We present the fine needle aspiration (FNA) biopsy cytological features of a case of HS occurring in a 73-year-old woman. RESULTS: FNA smears of hemorrhagic vitreous fluid showed numerous variably sized smooth glassy spherules ranging from 3-20 µm. Occasional red cells and a rare lymphocyte were observed, but other cell types were absent. Immunohistochemical staining of a cell block showed diffuse positive staining of spherules with haemoglobin A. CONCLUSIONS: HS is a rare lesion that occurs post-subretinal haemorrhage and may be encountered by pathologists examining ocular cytological specimens.

Vitreous composition modification after transpalpebral electrical stimulation of the eye: Biochemical analysis.

Electrical stimulation (ES) of the eye represents a therapeutic approach in various clinical applications ranging from retinal dystrophies, age-related macular degeneration, retinal artery occlusion and nonarteritic ischemic optic neuropathy. In clinical practice, ES of the eye is mainly performed with a transcorneal or transpalpebral approach. These procedures are non-invasive and well-tolerated by the patients, reporting only minimal and transient adverse events, while serious adverse effects were not observed. Despite the growing literature on animal models, only clinical parameters have been investigated in humans and few data are available about biochemical changes induced by ES of the eye. The purpose of this study is to investigate the possible mechanism that regulates the beneficial effects of ES on retinal cells function and survival in humans. 28 patients undergoing pars plana vitrectomy (PPV) for idiopathic epiretinal membrane (iERM) were randomly divided in two groups: 13 patients were treated with transpalpebral ES before surgery and 15 underwent surgery with no prior treatment. Vitreous samples were collected for biochemical analysis during PPV. ES treatment leads to a reduction in the vitreous expression of both proinflammatory cytokines, namely IL-6 and IL-8, and proinflammatory lipid mediators, such as lysophosphatidylcholine. Indeed, we observed a 70% decrease of lysophosphatidylcholine 18:0, which has been proven to exert the greatest proinflammatory activities among the lysophosphatidylcholine class. The content of triglycerides is also affected and significantly decreased following ES application. The vitreous composition of patients undergoing PPV for iERM displays significant changes following ES treatment. Proinflammatory cytokines and bioactive lipid mediators expression decreases, suggesting an overall anti-inflammatory potential of ES. The investigation of the mechanism by which this treatment alters the retinal neurons leading to good outcomes is essential for supporting ES therapeutic application in various types of retinal diseases.

Evaluation of vitreous levels of advanced glycation end products and angiogenic factors as biomarkers for severity of diabetic retinopathy.

BACKGROUND/AIMS: Glyceraldehyde-derived advanced glycation end products (glycer-AGE; also called Toxic-AGE [TAGE]) play a crucial role in the pathogenesis of diabetic angiopathy. However, the relationships between vitreous glycer-AGE levels and diabetic retinopathy (DR) severity, and between glycer-AGE levels and the levels of other angiogenic factors remain unknown. We investigated the correlation between levels of vitreous biomarkers, including glycer-AGE and angiogenic factors (vascular endothelial growth factor [VEGF], interleukin [IL]-8, leptin, placental growth factor [PlGF], endoglin, and fibroblast growth factor [FGF]-2) in patients with DR, using three DR staging groups. METHODS: In this cross-sectional study, we examined 33 eyes from 33 patients with diabetes mellitus who underwent a vitrectomy (non-proliferative DR [NPDR, n = 8]; PDR with simple vitreous haemorrhage [VH, n = 17]; or PDR with a fibrovascular proliferative membrane [FVM, n = 8]). Vitreous levels of glycer-AGE and VEGF were evaluated using enzyme-linked immunosorbent assays. Vitreous levels of IL-8, leptin, PlGF, endoglin, and FGF-2 were evaluated using beaded assay methods. RESULTS: Vitreous levels of glycer-AGE in the FVM group were significantly higher than those in the NPDR and VH groups (all p < 0.05). Vitreous levels of VEGF (r = 0.85, p = 1.7 × 10-6) and leptin (r = 0.60, p = 5.0 × 10-3) were significantly correlated with levels of PlGF. CONCLUSION: The two systems (VEGF-PlGF-leptin and glycer-AGE) were represented in these measured biomarkers. High vitreous levels of both VEGF and glycer-AGE may be linked to more severe DR, suggesting that anti-VEGF and anti-TAGE therapy may be an important part of the therapeutic strategy for DR.

Relationships between vitreous levels of soluble receptor for advanced glycation end products (sRAGE) and renal function in patients with diabetic retinopathy.

PURPOSE: We investigated the relationship between vitreous levels of soluble receptor for advanced glycation end products (sRAGE) and vascular endothelial growth factor (VEGF) and renal function, and correlations between vitreous sRAGE levels and proliferative diabetic retinopathy (PDR) activity. METHODS: We examined 33 eyes from 33 patients with diabetes mellitus who underwent a vitrectomy (eight patients in the non-PDR [NPDR] group and 25 in the PDR group). Serum creatinine levels and estimated glomerular filtration rate (eGFR) were measured and classified according to the chronic kidney disease (CKD)-staging method. Enzyme-linked immunosorbent assay (ELISA) was performed to quantify vitreous sRAGE and VEGF levels. RESULTS: Vitreous sRAGE levels were significantly higher in PDR group compared to NPDR group (p = 0.00003). Vitreous sRAGE levels were significantly higher in patients with CKD stage 5 (end-stage renal failure or hemodialysis) than in patients with CKD stage 1 or 2 (p < 0.01) and 3 or 4 (p < 0.05), and were significantly correlated with eGFR (r = - 0.490, p = 0.007) and creatinine levels (r = 0.484, p = 0.006). Within the PDR group, patients with low (<27 pg/mL) sRAGE levels required repeat vitreous surgeries for early postoperative vitreous hemorrhage significantly more frequently than those with high (≥27 pg/mL) sRAGE levels (p = 0.0067). CONCLUSIONS: Vitreous sRAGE levels were significantly correlated with renal function, and low vitreous sRAGE levels in patients with PDR were associated with postoperative vitreous hemorrhage. Vitreous sRAGE may be a useful biomarker for renal dysfunction associated with diabetic retinopathy.

Serum autoantibodies against hexokinase 1 manifest secondary to diabetic macular edema onset.

AIMS: Autoantibodies against hexokinase 1 (HK1) were recently proposed to be associated with diabetic macular edema (DME). We hypothesized that anti-HK1 autoantibodies can be used as DME markers and to predict DME onset. MATERIALS AND METHODS: Serum from patients with 1) DME, 2) diabetes mellitus (DM), 3) allergies or autoimmunities, and 4) control subjects was tested for anti-HK1 and anti-hexokinase 2 (HK2) autoantibodies by immunoblotting. Patients with DM were prospectively followed for up to nine years, and the association of anti-HK1 antibodies with new-onset DME was evaluated. The vitreous humor was also tested for autoantibodies. RESULTS: Among patients with DME, 32 % were positive for anti-HK1 autoantibodies (42 % of those with underlying type 1 DM and 31 % of those with underlying type 2 DM), and 12 % were positive for anti-HK2 autoantibodies, with only partial overlap of these two groups of patients. Anti-HK1 positive were also 7 % of patients with DM, 6 % of patients with allergies and autoimmunities, and 3 % of control subjects. The latter three groups were anti-HK2 negative. Only one of seven patients with DM who were initially anti-HK1 positive developed DME. CONCLUSIONS: Anti-HK1 autoantibodies can be used as DME markers but fail to predict DME onset.

Identification of new pathogenic candidates for diabetic macular edema using fluorescence-based difference gel electrophoresis analysis.

BACKGROUND: Diabetic macular edema is the main cause of visual impairment in diabetic patients. The aim of the present study was to explore the differential proteomic pattern of the vitreous fluid from diabetic macular edema patients by means of fluorescence-based difference gel electrophoresis (DIGE). METHODS: Samples of vitreous from eight type 2 diabetic patients [four with diabetic macular edema without proliferative diabetic retinopathy and four with proliferative diabetic retinopathy without diabetic macular edema), and eight from non-diabetic subjects with idiopathic macular hole (control group) were selected from our vitreous bank for proteomic analysis. To further confirm the potential candidates identified by DIGE, 18 additional samples (six proliferative diabetic retinopathy, six diabetic macular edema and six macular hole, matched by age) were analysed by enzyme-linked immuno sorbent assay (ELISA). RESULTS: Selecting an abundance ratio of 1.5-fold, p < 0.05, as the threshold for the study, four proteins were specifically associated with diabetic macular edema. Hemopexin was significantly higher in the vitreous fluid of patients with diabetic macular edema in comparison with both control subjects and proliferative diabetic retinopathy patients. By contrast, clusterin, transthyretin and crystallin S were significantly decreased in the vitreous of patients with diabetic macular edema. The differential production of hemopexin, clusterin and transthyretin was further confirmed by ELISA. CONCLUSIONS: Proteomic analysis by DIGE was useful in identifying new potential candidates involved in the pathogenesis of diabetic macular edema. These results could open up new strategies in the treatment of diabetic macular edema.

Rapid diagnostic work-up of Streptococcus dysgalactiae endophthalmitis by a novel culture processing system.

The diagnosis of infective endophthalmitis is supported by microbiological work-up. Rapid work-up is critical to confirm clinical suspicion and appropriate antimicrobial therapy. We report the novel use of an automated liquid culture processing system (FAST system, Qvella, ON, Canada) in a vitreous fluid culture. A 59-year-old patient with post-operative endophthalmitis presented with acute right eye pain and blurred vision. Vitreous fluid collected for microbiology culture was of limited quantity and only inoculated to thioglycolate broth. The broth recovered beta-haemolytic, group G Streptococcus dysgalactiae susceptible to penicillin and vancomycin. Experimental application of the FAST system to purify the organism from broth culture yielded the same identification and susceptibility test results but 1 day sooner. Despite prompt treatment with appropriate antibiotics, including vancomycin and ceftazidime, disease progressed rapidly and required enucleation to achieve a stable therapeutic outcome. Use of automated processing of monomicrobial broth cultures has thus far focused on positive blood culture broths, but could potentially include other liquid-based cultures such as for sterile body fluids of critical nature.

Association between Systemic Factors and Vitreous Fluid Cytokines in Proliferative Diabetic Retinopathy.

Proliferative diabetic retinopathy (PDR) is a vision-threatening complication of diabetes mellitus (DM). Systemic and intraocular factors are intricately related to PDR, and vitreous fluid (VF) cytokines are representative intraocular biomarkers. However, the associations between systemic factors and VF cytokines and their influence on PDR pathology are unclear. This study aimed to examine the correlation between systemic factors and VF cytokines and analyze their contributions to the pathology of PDR using multivariate analyses. We conducted a retrospective observational study on 26 PDR eyes of 25 patients with type 2 DM, and 30 eyes of 30 patients with idiopathic macular hole or epiretinal membrane as controls. Fifteen systemic and laboratory tests including blood pressure (BP) and body mass index (BMI), and 27 cytokines in VF were analyzed. BP and BMI correlated positively with VF levels of IL-6 and IP-10 in PDR patients, while no significant correlation was found between systemic factors and VF cytokines in controls. MCP-1 and VEGF-A in VF separately clustered with different systemic factors in controls, but these cytokines lost the property similarity with systemic factors and acquired property similarity with each other in PDR. Systemic factors contributed to only 10.4%, whereas VF cytokines contributed to 42.3% out of 52.7% variance of the whole PDR dataset. Our results suggest that intraocular factors play a major role in the pathology of PDR, whereas systemic factors may have limited effects, and that BP and BMI control in PDR could be useful interventions to improve intraocular immune condition.

Correlations Between Different Angiogenic and Inflammatory Factors in Vitreous Fluid of Eyes With Proliferative Diabetic Retinopathy.

Purpose: To investigate the expression of various angiogenesis and inflammation mediators in the vitreous fluid of eyes with proliferative diabetic retinopathy (PDR). Methods: A total of 38 eyes with PDR and 37 control eyes were included. Vitreous fluid was collected during vitrectomy. Vitreous levels of colony stimulating factor-1 receptor (CSF-1R), syndecan-1, placental growth factor (PIGF), and angiopoietin-like protein 4 (ANGPTL-4) were measured by multiplex immunoassay. Vitreous levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule-1 (ICAM-1) were measured by cytometric beads array. Levels of these mediators were compared between the PDR and control eyes. Correlations between levels of different mediators and between these mediators and kidney function metrics in the PDR group were also analyzed. Results: Vitreous levels of syndecan-1, PIGF, ANGPTL-4, VEGF, and IL-8 were significantly higher in the PDR group compared to the control group (all p < 0.05). Levels of VEGF were significantly correlated with levels of syndecan-1, PIGF, and ANGPTL-4 (r = 0.370 to 0.497, all p < 0.05). Significant positive correlations were detected between levels of any two of the following mediators including syndecan-1, PIGF, ANGPTL-4, and IL-8 (r = 0.370 to 0.906, all p < 0.05). Apart from VEGF, levels of these mediators were positively correlated with serum creatinine and blood urea nitrogen (r = 0.328 to 0.638, all p < 0.05), and negatively correlated with fasting blood glucose and estimated glomerular filtration rate (r = -0.325 to -0.603, all p < 0.05). Conclusions: Correlations between different angiogenesis and inflammation mediators were observed in eyes with PDR, suggesting cross-talks of different angiogenesis and inflammation pathways in the pathogenesis of PDR. The levels of angiogenesis and inflammation in PDR are correlated with kidney damage, indicating possible common pathways in diabetic retinopathy and nephropathy.

Intraocular Viral Communities Associated With Post-fever Retinitis.

The virome of ocular fluids is naive. The results of this study highlight the virome in the vitreous fluid of the eye of individuals without any ocular infection and compare it with the virome of the vitreous fluid of individuals with retinitis. A total of 1,016,037 viral reads were generated from 25 vitreous fluid samples comprising control and post-fever retinitis (PFR) samples. The top 10 viral families in the vitreous fluids comprised of Myoviridae, Siphoviridae, Phycodnaviridae, Herpesviridae, Poxviridae, Iridoviridae, Podoviridae, Retroviridae, Baculoviridae, and Flaviviridae. Principal coordinate analysis and heat map analysis clearly discriminated the virome of the vitreous fluid of the controls from that of the PFR virome. The abundance of 10 viral genera increased significantly in the vitreous fluid virome of the post-fever retinitis group compared with the control group. Genus Lymphocryptovirus, comprising the human pathogen Epstein-Barr virus (EBV) that is also implicated in ocular infections was significantly abundant in eight out of the nine vitreous fluid viromes of post-fever retinitis group samples compared with the control viromes. Human viruses, such as Hepacivirus, Circovirus, and Kobuvirus, were also significantly increased in abundance in the vitreous fluid viromes of post-fever retinitis group samples compared with the control viromes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analysis and the network analysis depicted an increase in the immune response by the host in the post-fever retinitis group compared with the control group. All together, the results of the study indicate changes in the virome in the vitreous fluid of patients with the post-fever retinitis group compared to the control group.

Increased Levels of DKK1 in Vitreous Fluid of Patients with Pathological Myopia and the Correlation between DKK1 Levels and Axial Length.

Purpose: Dickkopf 1 (DKK1) functions as a natural antagonist of the canonical Wnt/ß-catenin pathway. The purpose of this study was to examine the expression of DKK1 in vitreous samples of patients with pathological myopia, in order to search for possible correlations between DKK1 and axial length.Materials and Methods: The expression of DKK1 and other cytokines in vitreous samples of 44 non-myopic eyes, 42 eyes with low-to-moderate myopia, and 51 eyes with pathological myopia were examined using multiplex cytokine detection technology. Ophthalmologic characteristics, including axial length and subfoveal choroidal thickness, were clinically measured for further analysis.Results: The intravitreous levels of DKK1 (P < .0001) were markedly higher in the pathological myopia group than in the control group. There were no differences of DKK1 levels in different vitreoretinal conditions. Additionally, we found that the DKK1 levels were positively correlated with HGF (ß = 0.268, P = .032), and TIMP-3 (ß = 0.209, P = .047) levels, as well as with axial length (ß = 0.714, P < .0001) in the pathological myopia group.Conclusions: Elevated levels of DKK1 were found in the eyes with elongated axial length.

Viral Loads in Ocular Fluids of Acute Retinal Necrosis Eyes Infected by Varicella-Zoster Virus Treated with Intravenous Acyclovir Treatment.

Acute retinal necrosis (ARN) is a rare viral endophthalmitis, and human herpesvirus is the principal pathogen. Early diagnosis and treatment are critical to avoid visual impairment by ARN, and pars plana vitrectomy (PPV) is required in advanced cases. In this study, we evaluated the transition of viral load in ocular fluids of ARN eyes with varicella-zoster virus (VZV) after intravenous acyclovir treatment. Fourteen eyes of 13 patients were analyzed retrospectively. All patients received intravenous acyclovir treatment, and eventually, all eyes underwent PPV. A polymerase chain reaction (PCR) test showed a 100% detection rate in all aqueous humor samples collected before the treatment (Pre-AH), as well as aqueous humor (Post-AH) and vitreous fluid samples (VF), collected during PPV conducted after the treatment. Within eight days or less of acyclovir treatment, viral loads both in AH and VF did not decrease significantly. Furthermore, the viral load of Pre-AH had a strong correlation with that of VH. These data suggest that in ARN eyes with VZV infection, the AH sample for the PCR test was reliable to confirm the pathogen. We propose that short-term treatment of intravenous acyclovir may be insufficient for reducing intraocular viral load, and the Pre-AH sample could be a predictor of viral activity in the eyes after acyclovir treatment.

Increased interleukin-26 expression in proliferative diabetic retinopathy.

AIM: To detect the possible role of interleukin (IL)-26 in diabetic retinopathy (DR) patients. METHODS: Subjects were divided into diabetes without retinopathy (DWR) group (n=20), non-proliferative diabetic retinopathy (NPDR) group (n=20), proliferative diabetic retinopathy (PDR) group (n=20) and normal control group (n=20). The protein expression of IL-26 in the serum and vitreous fluid were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA change of IL-26 in peripheral blood mononuclear cells (PBMCs) was assessed by real-time polymerase chain reaction. RESULTS: The serum expression of IL-26 in PDR group was significantly elevated compared with the normal control group, DWR group and NPDR group. The vitreous fluid concentration of IL-26 in PDR patients (without anti-VEGF therapy) was also higher compared to normal controls. However, no obvious significance was found concerning the expression of IL-26 in vitreous fluid between PDR after anti-VEGF therapy and normal controls. In PDR group, the mRNA level of IL-26 significantly increased compared with the normal controls and DWR patients in the PBMCs. CONCLUSION: Protein and mRNA expression of IL-26 are increased in serum, vitreous fluid and PBMCs in PDR patients, suggesting that IL-26 may be associated with the pathogenesis of PDR.

Role of Regulatory T Cells in Tubercular Uveitis.

PURPOSE: To study the role of regulatory T cells (Tregs) in patients with tubercular uveitis. METHODS: Frequencies of peripheral Tregs, Th1, Th17 cells, and intracellular cytokines were determined in 17 tubercular uveitis patients and 18 disease controls. Function of Tregs, Th1, and Th17 cells was assessed in vitro. Simultaneously, ocular levels of IFN-γ, IL-17A, IL-4, and IL-10 were also measured. RESULTS: Frequencies of peripheral Tregs in tubercular uveitis subjects were significantly lower compared with disease controls. Furthermore, expression of TGF-ß and IL-2Rα, but not CTLA4, was reduced in Tregs of the tubercular uveitis group. The tubercular uveitis group demonstrated heightened Th1, Th17 responses following in vitro stimulation with phorbol myristate acetate (PMA)/ionomycin. Interestingly, Treg suppression assay did not show a significant difference between the two groups. Ocular levels of IFN-γ, IL-17A, and IL-10 were also elevated in tubercular uveitis group. CONCLUSIONS: Low Treg frequency and hyporesponsive function contribute to proinflammatory responses manifesting at ocular level in tubercular uveitis.

Retinal pigment epithelium adenoma in vitreous fluid cytology.

Ocular cytology specimens are relatively uncommon, adding to the difficulty of their evaluation by cytopathologists. While melanomas account for a majority of primary intraocular pigmented lesions, other diagnostic considerations must be included in the differential. This brief report highlights a case of a pigmented ocular lesion in a 24-year-old man and key morphologic, immunohistochemical, and clinical differences between melanoma, melanocytoma, choroidal nevus, and retinal pigment epithelium (RPE) adenoma.

Deregulation of ocular nucleotide homeostasis in patients with diabetic retinopathy.

Clear signaling roles for ATP and adenosine have been established in all tissues, including the eye. The magnitude of signaling responses is governed by networks of enzymes; however, little is known about the regulatory mechanisms of purinergic signaling in the eye. By employing thin-layer chromatographic assays with 3H-labeled substrates, this study aimed to evaluate the role of nucleotide homeostasis in the pathogenesis of vitreoretinal diseases in humans. We have identified soluble enzymes ecto-5'-nucleotidase/CD73, adenylate kinase-1, and nucleoside diphosphate kinase in the vitreous fluid that control active cycling between pro-inflammatory ATP and anti-inflammatory adenosine. Strikingly, patients with proliferative form of diabetic retinopathy (DR) had higher adenylate kinase activity and ATP concentration, when compared to non-proliferative DR eyes and non-diabetic controls operated for rhegmatogenous retinal detachment, macular hole, and pucker. The non-parametric correlation analysis revealed positive correlations between intravitreal adenylate kinase and concentrations of ATP, ADP, and other angiogenic (angiopoietins-1 and -2), profibrotic (transforming growth factor-ß1), and proteolytic (matrix metalloproteinase-9) factors but not erythropoietin and VEGF. Immunohistochemical staining of postmortem human retina additionally revealed selective expression of ecto-5'-nucleotidase/CD73 on the rod-and-cone-containing photoreceptor cells. Collectively, these findings provide novel insights into the regulatory mechanisms that influence purinergic signaling in diseased eye and open up new possibilities in the development of enzyme-targeted therapeutic approaches for prevention and treatment of DR. KEY MESSAGE: Ecto-5'-nucleotidase/CD73 and adenylate kinase-1 circulate in human vitreous fluid. Adenylate kinase activity is high in diabetic eyes with proliferative retinopathy. Diabetic eyes display higher intravitreal ATP/ADP ratio than non-diabetic controls. Soluble adenylate kinase maintains resynthesis of inflammatory ATP in diabetic eyes.

CD18 expression in granulocytes infiltrating the vitreous fluid in patients with diabetic retinopathy.

AIM: To assess the levels of CD18 on the surface of granulocytes infiltrating the vitreous fluid in patients with diabetic retinopathy (DR). METHODS: Vitreous samples from twelve patients with non-proliferative DR with significant macula edema (group A), 33 patients with proliferative DR (grade 3 as group B, n=14, and, grade 4 as group C, n=19) were obtained during pars plana vitrectomy. Vitreous samples from 12 patients with macular hole as controls (group D) were analyzed together. The infiltrating of granulocytes and its surface level of CD18 were measured by flow cytometry. The level of CD18 was presented as the mean channel fluorescence (MCF) on a logarithmic scale. RESULTS: Granulocytes were detected in 6 of 12 vitreous samples from group A, 9 of 14 from group B, 15 of 19 from group C, and none of 12 from group D. MCF of CD18 on granulocytes from groups A, B, and C were 2.978±1.446, 3.201±0.692, and 4.072±0.837, respectively. The difference was significant (F=4.354, P=0.021). Subjects with more severe DR were more likely to have a higher level of CD18 MCF (trend test, χ (2)=7.351, P=0.007). CD18 MCF was significantly associated with the development of DR (r=0.46, P=0.005 and ß=0.147, P=0.035). CONCLUSION: Our results confirm the presence of granulocytes and the elevated levels of CD18 on the surface of them in the vitreous fluid from DR patients. These results may provide indirect evidence shown that granulocytes activation also has occurred in the retinal local compared to non-DR control.

Use of DNA microarray analysis in diagnosis of bacterial and fungal endophthalmitis.

BACKGROUND: To examine the utility of DNA microarray analysis for identifying causative microorganisms in endophthalmitis. METHODS: Thirteen samples of vitreous fluid (VF) were obtained from 13 patients during vitrectomy. Vitreous fluids from three patients with suspected endophthalmitis and ten controls without infection were subjected to testing for the presence of bacteria and fungi in culture tests, polymerase chain reaction (PCR) analysis, and DNA microarray analysis. RESULTS: No control sample was positive for bacteria or fungi in the culture test, PCR, or microarray analysis. Specimens from two patients (Cases 1 and 2) with suspected endophthalmitis were positive for bacteria in PCR, and a specimen from one patient (Case 3) was positive for fungi in PCR. Klebsiella pneumonia (Case 1), Streptococcus agalactiae (Case 2), and Candida parapsilosis (Case 3) in the PCR-positive specimens were identified by DNA microarray analysis within 24 hours. Culture results were also positive for K. pneumonia in Case 1, S. agalactiae in Case 2, and C. parapsilosis in Case 3, but required 3 to 4 days to obtain. CONCLUSIONS: Microarray analysis is complementary to routine cultures for identifying causative microorganisms and is likely to be a useful tool in patients with suspected endophthalmitis who require rapid diagnosis and early antibiotic treatment.