Peter A. W. J. F. Schellekens

Intraocular biomarker identification in uveitis associated with juvenile idiopathic arthritis.

PURPOSE To investigate the presence of biomarkers in aqueous humor (AH) from patients with uveitis associated with juvenile idiopathic arthritis (JIA). METHODS AH (N = 73) AND SERUM (N = 105) SAMPLES FROM 116 CHILDREN WERE ANALYZED USING SURFACE ENHANCED LASER DESORPTION/IONIZATION TIME OF FLIGHT MASS SPECTROMETRY (SELDI-TOF MS). THE SAMPLES WERE DIVIDED INTO THE FOLLOWING GROUPS JIA, silent chronic anterior uveitis (AU), other uveitis entities, and noninflammatory controls. Statistical biomarker identification was performed using the SELDI-ToF Biomarker Analysis Cluster Wizard followed by multivariate statistical analysis. Biochemical identification of biomarkers was performed by polyacrylamide gel protein separation, followed by liquid chromatography tandem mass spectrometry. ELISA was performed in a number of AH samples representing all four study groups. RESULTS In the JIA group, one AH protein peak at mass/charge (m/z) 13,762 had qualitative and quantitative differences in expression compared with the other uveitis entities and the controls, but not to the group of silent chronic AU. Its quantitative expression in AH of patients with JIA and other silent chronic AU was positively associated with uveitis activity. The protein at m/z 13,762 in AH was identified as transthyretin (TTR). The TTR concentration in AH differed significantly between the study groups (P = 0.006) with considerably higher TTR concentrations in JIA and silent chronic AU samples positive for m/z 13,762 than those of the other uveitis and control groups. CONCLUSIONS TTR is a potential intraocular biomarker of JIA- associated uveitis. Its role in the pathogenesis of silent chronic AU with and without arthritis needs further investigation.

Diagnostic pars plana vitrectomy and aqueous analyses in patients with uveitis of unknown cause

Purpose: To compare the yield of diagnostic pars plana vitrectomy (PPV) with the yield of aqueous analyses in patients with uveitis of unknown cause. Methods: Seventy-five consecutive patients (84 eyes) with uveitis involving posterior eye segment who undergo a diagnostic PPV from 2005 through 2009 were retrospectively reviewed. Vitreous specimens were simultaneously analyzed by microbiological culture, flow cytometry, and cytology as well as by polymerase chain reaction and for intraocular antibody production by Goldmann–Witmer coefficient. In 53 eyes, both aqueous and vitreous samples were assessed. The primary outcome measure was the comparison between vitreous and aqueous analyses. Results: Vitreous analysis was positive in 18 of 84 eyes (21%). Positive results indicated infectious uveitis in 12 of 18 cases (67%) and lymphoma in 6 of 18 (33%) cases. Of the 53 eyes with both aqueous and vitreous samples available, aqueous analysis revealed the diagnosis in 6 of 53 eyes and vitreous in 9 of 53 eyes. Unilateral uveitis (P = 0.022), panuveitis and uveitis posterior (P ⩽ 0.001), preoperative immunosuppressive therapy (P = 0.004), and increasing age (P = 0.018) were associated with an increased diagnostic yield of PPV. Overall, 1 year after PPV, median visual acuity improved from 20/200 to 20/80 (Snellen, P ⩽ 0.001). Of 18 patients who were on immunosuppressive treatment before PPV, 8 (44%) were able to stop immunosuppressive therapy during 1-year follow-up. The complications of PPV consisted predominantly of cataract development (33/65, 51%). Conclusion: Diagnostic PPV with the analysis of vitreous fluid by multiple laboratories for infectious and malignant disorders was useful in diagnosing uveitis of unknown cause. Previous aqueous analysis was especially valuable for the diagnosis of intraocular infections and may therefore decrease the number of patients who would otherwise undergo an invasive diagnostic PPV. Furthermore, PPV was associated with improved visual acuity and decreased use of immunosuppressive therapy.

Detection of choroid- and retina-antigen reactive CD8+ and CD4+ T lymphocytes in the vitreous fluid of patients with birdshot chorioretinopathy

Birdshot chorioretinopathy (BSCR), a progressive form of non-infectious uveitis, is the strongest HLA-associated disease described to date, with >95% of the patients displaying HLA-A29. Since indirect evidence indicates the involvement of T cells in the etiopathology of the disease, we now isolated, cultured and analyzed the vitreous fluid-infiltrating T cells from two BSCR patients with respect to their phenotype, cytokine profile, clonal distribution and antigen specificity. Phenotypic analyses revealed the predominant presence of both CD4(+) and CD8(+) T cells in vitreous fluid. Further analyses on short term expanded and cloned T cells suggested that eye-infiltrating T cells generally displayed a Th1 like cytokine profile with secretion of high levels of IFN-γ and TNF-α. In one patient an oligoclonal CD4(+) and CD8(+) T cell infiltration, with a moderate to strongly skewed TCR Vβ usage was suggestive for an antigen driven infiltration/expansion. Indeed, a number of intraocular CD4(+) and CD8(+) T cells responded to crude retinal and choroidal lysates. These results, which demonstrate for the first time the existence of eye-antigen-specific T cells in the vitreous fluid of BSCR patients, substantiate the current view on the role of eye-antigen specific T cells in the etiopathology of BSCR.

INCIDENCE, RISK FACTORS, AND CLINICAL CHARACTERISTICS OF UNEXPLAINED VISUAL LOSS AFTER INTRAOCULAR SILICONE OIL FOR MACULA-ON RETINAL DETACHMENT.

Purpose: To investigate the incidence, risk factors, and clinical characteristics of unexplained visual loss after macula-on rhegmatogenous retinal detachment (RRD). Methods: Retrospective cohort of patients with primary macula-on rhegmatogenous retinal detachment treated by vitrectomy with gas or silicone oil (SO) tamponade in 2011 and 2012. Outcome was unexplained visual loss (>2 Snellen lines) 2 months after the last vitrectomy. Results: Incidence of unexplained visual loss was 0.7% (1/151) in patients treated by gas and 29.7% (11/37) in patients treated by SO (P = 0.001). Visual loss occurred both during SO tamponade and after removal. Cases underwent optical coherence tomography, perimetry, microperimetry, fluorescein angiography, and visual evoked potentials. Patients with unexplained visual loss after SO tamponade showed a small scotoma within the central 2° on microperimetry. Duration of SO tamponade was the only statistically significant factor related to the incidence of unexplained visual loss (P = 0.001). Conclusion: Incidence of SO-related visual loss was 30% with duration of tamponade as the only risk factor. This study is the first to apply microperimetry in these patients, which showed a distinct pattern of a small central scotoma. Therefore, microperimetry can be of great value in the diagnostic workup of patients with unexplained visual loss after vitrectomy.

Correlation between measurement of IL-10 and IL-6 in paired aqueous humour and vitreous fluid in primary vitreoretinal lymphoma.

Editor, P rimary vitreoretinal lymphoma (PVRL) is a rare and aggressive malignancy almost always of large diffuse B-cell origin that predominantly arises in the retina and adjacent structures (Chan et al. 2011). PVRL often masquerades as uveitis and is easily misdiagnosed. As PVRL is potentially lethal, early and accurate diagnosis is crucial for adequate treatment and prognosis. The gold standard for diagnosing PVRL requires detection of malignant lymphoid cells in vitreous or retinochoroidal biopsies that can be supported by flowcytometric evidence of monoclonality of lymphocytes in vitreous fluid (VF) (Gonzales & Chan 2007). However, outcome of cytology is frequently false negative due to fragility of cells in obtained specimens. An enzyme-linked immunosorbent assay (ELISA) measured vitreal IL-10 to IL-6 ratio greater than 1.0 is currently considered to be typical for PVRL over non-infectious uveitis (Wolf et al. 2003). Here, we present a less invasive alternative using modern multicytokine analysis on aqueous humour (AqH) instead of VF. Paracentesis to obtain AqH of the anterior chamber is safe and less invasive compared to collection of VF. However, the AqH samples consist of smaller volumes compared to undiluted VF and potentially limit the evaluation of both IL-10 and IL-6 levels by ELISA. The emerging use of multiplex bead-based array platform (Luminex), however, allows the detection and evaluation of multiple cytokines in small sample volume (<25 ll) (de Jager et al. 2003). Exploiting this exciting technique, we measured IL-10 and IL-6 levels in paired AqH and VF samples of patients (n = 11) with proven PVRL between 2005 and 2014, to investigate whether the use of AqH could replace VF. The samples were collected during diagnostic vitrectomy to confirm the diagnosis PVRL, and the remainders of the samples were used to measure IL10 to IL-6 ratios with approval from the medical–ethical institutional review board. The diagnosis of PVRL was based upon cytology/immunocytology of vitreous cells, histopathology of retinal biopsy or proven CNS lymphoma. None of the patients was treated for PVRL with intra-ocular methotrexate, rituximab, systemic chemotherapy or irradiation of the eye during/before the diagnostic vitrectomy. The samples were preserved at minus 80°C degrees immediately after harvesting. IL-10 and IL-6 levels were measured in 25 ll of undiluted sample. Correlation of the biomarkers with AqH and VF was analysed by computing the nonparametric Spearman’s rank correlation coefficient. The female/male ratio was 4/7 and the mean age of developing of lymphoma was 68.8 years (range 63– 82 years). All patients suffered from PVRL and two patients had additional CNS involvement. The geometric mean of the levels of IL-10 in AqH was 863.6 (95% CI: 169.3–4405) and in VF was 2188 (95% CI: 607.6–7880). The mean of the levels of IL-6 in AqH was 81.9 (95% CI: 45.7–147.1) and in VF was 79.1 (95% CI: 40.7–153.6). The levels of IL-10 (r = 0.92; p = 0.0002) and the levels of IL-6 (r = 0.83, p = 0.003) in paired AqH and VF strongly correlated. Accordingly, the IL-10 to IL-6 ratio in the paired AqH and VF correlated (r = 0.73, p = 0.01) (Fig. 1). The individual levels and the ratio of IL-10 and IL-6, in paired AqH and VF of PVRL patients, strongly correlate and demonstrate that the relative proportion of IL-10 to IL-6 is similar for both AqH and VF. We emphasize that the general opinion is that definite diagnosis must be made by cytologic or histopathologic evaluation of vitreous or retinochoroidal specimens that can be supported by IL-10 to IL-6 ratio. The present results suggest that the AqH IL-10 to IL-6 ratio may be as useful as the previously suggested vitreal IL-10 to IL-6 ratio and, thus, provides a less invasive alternative for the evaluation of these cytokines to support histopathology and immunocytologic evidence, or follow-up after treatment, for PVRL.

Ocular fluid analysis in children reveals interleukin‐29/interferon‐λ1 as a biomarker for juvenile idiopathic arthritis associated uveitis

Childhood uveitis is a vision‐threatening inflammatory eye disease commonly attributed to juvenile idiopathic arthritis (JIA). The pathogenesis is poorly understood, which makes clinical management challenging. We analyzed soluble mediators in ocular fluid (aqueous humor [AqH]) and serum from children with JIA‐associated uveitis and common childhood uveitis to identify potential biomarkers and investigate the ocular microenvironment of this sight‐threatening eye disease.

Electrolyte composition of retro-oil fluid and silicone oil-related visual loss

Up to one‐third of patients with intra‐ocular silicone oil (SO) tamponade for complex macula‐on retinal detachment may experience an unexplained visual loss during or after SO tamponade. Although the underlying mechanism is unknown, previous studies suggested that accumulation of retinal potassium could be involved. Hence, this study tested the hypothesis that intra‐ocular potassium levels are elevated during SO tamponade.

An Ocular Protein Triad Can Classify Four Complex Retinal Diseases.

Retinal diseases generally are vision-threatening conditions that warrant appropriate clinical decision-making which currently solely dependents upon extensive clinical screening by specialized ophthalmologists. In the era where molecular assessment has improved dramatically, we aimed at the identification of biomarkers in 175 ocular fluids to classify four archetypical ocular conditions affecting the retina (age-related macular degeneration, idiopathic non-infectious uveitis, primary vitreoretinal lymphoma, and rhegmatogenous retinal detachment) with one single test. Unsupervised clustering of ocular proteins revealed a classification strikingly similar to the clinical phenotypes of each disease group studied. We developed and independently validated a parsimonious model based merely on three proteins; interleukin (IL)-10, IL-21, and angiotensin converting enzyme (ACE) that could correctly classify patients with an overall accuracy, sensitivity and specificity of respectively, 86.7%, 79.4% and 92.5%. Here, we provide proof-of-concept for molecular profiling as a diagnostic aid for ophthalmologists in the care for patients with retinal conditions.

GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay

Abstract Loss‐of‐function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain‐of‐function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys‐GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/−12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal—but submaximal—GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra‐high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys‐GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys‐GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.

Retinal sensitivity following intraocular silicone oil and gas tamponade for rhegmatogenous retinal detachment

To investigate whether intraocular silicone oil (SO) tamponade is associated with functional changes in patients with both macula‐on and macula‐off rhegmatogenous retinal detachments (RRDs).