Adalimumab in refractory cystoid macular edema associated with birdshot chorioretinopathy

Purpose To report the clinical outcomes of adalimumab therapy in cases of birdshot chorioretinitis (BCR) with cystoid macular edema (CME) refractory to conventional immunotherapy. Methods This is a retrospective case series of three BCR patients treated with adalimumab for refractory CME. The main outcome measure was central subfield thickness (CST) on optical coherence tomography. Any patients treated with local steroids and/or receiving systemic steroids higher than 40 mg prednisolone daily during adalimumab therapy were excluded. Results At baseline, all patients were receiving systemic corticosteroids and two second-line immunosuppressive agents. The mean duration of treatment with adalimumab was 31.2 months (range 17.2–52). The mean CST was 327 ± 112.7 μm (mean ± SD) at baseline and 256.2 ± 39.7 μm at 6 months and 235.5 ± 32.5 μm at 12 months. Adalimumab permitted cessation or reduction in the daily dose of oral prednisolone plus withdrawal of a second-line agent in all patients. Conclusions In these patients, adalimumab was effective in the treatment of refractory CME

CytoBinning: Immunological insights from multi-dimensional data

New cytometric techniques continue to push the boundaries of multi-parameter quantitative data acquisition at the single-cell level particularly in immunology and medicine. Sophisticated analysis methods for such ever higher dimensional datasets are rapidly emerging, with advanced data representations and dimensional reduction approaches. However, these are not yet standardized and clinical scientists and cell biologists are not yet experienced in their interpretation. More fundamentally their range of statistical validity is not yet fully established. We therefore propose a new method for the automated and unbiased analysis of high-dimensional single cell datasets that is simple and robust, with the goal of reducing this complex information into a familiar 2D scatter plot representation that is of immediate utility to a range of biomedical and clinical settings. Using publicly available flow cytometry and mass cytometry datasets we demonstrate that this method (termed CytoBinning), recapitulates the results of traditional manual cytometric analyses and leads to new and testable hypotheses

Ex Vivo T Cell Cytokine Expression Predicts Survival in Patients with Severe Alcoholic Hepatitis

Alcoholic hepatitis (AH) is an acute inflammatory liver condition with high early mortality rate. Steroids improve shortterm survival but nonresponders have the worst outcomes. There is a clinical need to identify these high-risk individuals at the time of presentation. T cells are implicated in AH and steroid responsiveness. We measured ex vivo T cell cytokine expression as a candidate biomarker of outcomes in patients with AH. Consecutive patients (bilirubin >80 µmol/L and ratio of aspartate aminotransferase to alanine aminotransferase >1.5 who were heavy alcohol consumers with discriminant function [DF] ≥32), were recruited from University Hospitals Plymouth NHS Trust. T cells were obtained and stimulated ex vivo. Cytokine expression levels were determined by flow cytometry and protein multiplex analysis. Twenty-three patients were recruited (10 male; median age 51 years; baseline DF 67; 30% 90-day mortality). Compared to T cells from nonsurvivors at day 90, T cells from survivors had higher baseline baseline intracellular interleukin (IL)-10:IL-17A ratio (0.43 vs 1.20, p=0.02). Multiplex protein analysis identified interferon γ (IFNγ) and tumor necrosis factor-α (TNF-α) as independent predictors of 90-day mortality (p=0.04, p=0.01, respectively). The ratio of IFNγ to TNF-α was predictive of 90-day mortality (1.4 vs 0.2, p=0.03). These data demonstrate the potential utility of T cell cytokine release assays performed on pretreatment blood samples as biomarkers of survival in patients with severe AH. Our key findings were that intracellular IL10:IL-17A and IFNγ:TNF-α in culture supernatants were predictors of 90-day mortality. This offers the promise of developing T cell-based diagnostic tools for risk stratificatio

Glucocorticoid treatment in patients with newly diagnosed immune thrombocytopenia switches CD14(++)CD16(+) intermediate monocytes from a pro-inflammatory to an anti-inflammatory phenotype

Immune thrombocytopenia (ITP) is thought to result from an aberrant adaptive autoimmune response, involving autoantibodies, B and T lymphocytes, directed at platelets and megakaryocytes. Previous reports have demonstrated skewed CD4+ T-helper subset distribution and enhanced production of pro-inflammatory cytokines such as interleukin 17A and interferon gamma. The role of monocytes (MCs) in ITP is less widely described, but innate immune cells have a role in shaping CD4+ T-cell phenotypes. Glucocorticoids (GCs) are commonly used for first-line ITP treatment and modulate a broad range of immune cells including T cells and MCs. Using multiparameter flow cytometry analysis, we demonstrate the expansion of intermediate MCs (CD14++CD16+ ) in untreated patients with newly diagnosed ITP, with these cells displaying a pro-inflammatory phenotype, characterised by enhanced expression of CD64 and CD80. After 2 weeks of prednisolone treatment (1 mg/kg daily), the proportion of intermediate MCs reduced, with enhanced expression of the anti-inflammatory markers CD206 and CD163. Healthy control MCs were distinctly different than MCs from patients with ITP before and after GC treatment. Furthermore, the GC-induced phenotype was not observed in patients with chronic ITP receiving thrombopoietin receptor agonists. These data suggest a role of MCs in ITP pathogenesis and clinical response to GC therapy

Ex Vivo T Cell Cytokine Expression Predicts Survival in Patients with Severe Alcoholic Hepatitis

Alcoholic hepatitis (AH) is an acute inflammatory liver condition with high early mortality rate. Steroids improve shortterm survival but nonresponders have the worst outcomes. There is a clinical need to identify these high-risk individuals at the time of presentation. T cells are implicated in AH and steroid responsiveness. We measured ex vivo T cell cytokine expression as a candidate biomarker of outcomes in patients with AH. Consecutive patients (bilirubin >80 µmol/L and ratio of aspartate aminotransferase to alanine aminotransferase >1.5 who were heavy alcohol consumers with discriminant function [DF] ≥32), were recruited from University Hospitals Plymouth NHS Trust. T cells were obtained and stimulated ex vivo. Cytokine expression levels were determined by flow cytometry and protein multiplex analysis. Twenty-three patients were recruited (10 male; median age 51 years; baseline DF 67; 30% 90-day mortality). Compared to T cells from nonsurvivors at day 90, T cells from survivors had higher baseline baseline intracellular interleukin (IL)-10:IL-17A ratio (0.43 vs 1.20, p=0.02). Multiplex protein analysis identified interferon γ (IFNγ) and tumor necrosis factor-α (TNF-α) as independent predictors of 90-day mortality (p=0.04, p=0.01, respectively). The ratio of IFNγ to TNF-α was predictive of 90-day mortality (1.4 vs 0.2, p=0.03). These data demonstrate the potential utility of T cell cytokine release assays performed on pretreatment blood samples as biomarkers of survival in patients with severe AH. Our key findings were that intracellular IL10:IL-17A and IFNγ:TNF-α in culture supernatants were predictors of 90-day mortality. This offers the promise of developing T cell-based diagnostic tools for risk stratificatio

Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina

It is not currently possible to reliably visualise and track immune cells in the human central nervous system or eye. Previous work demonstrated that indocyanine green (ICG) dye could label immune cells and be imaged after a delay during disease in the mouse retina. We report a pilot study investigating if ICG can similarly label immune cells within the human retina. Twelve adult participants receiving ICG angiography as part of routine standard of care were recruited. Baseline retinal images were obtained prior to ICG administration then repeated over a period ranging from 2 hours to 9 days. Matched peripheral blood samples were obtained to examine systemic immune cell labelling and activation from ICG by flow cytometry with human macrophage cultures as positive controls. Differences between the delayed near infrared ICG imaging and 488 nm autofluorescence was observed across pathologies, likely arising from the retinal pigment epithelium (RPE). Only one subject demonstrated ICG signal on peripheral blood myeloid cells and only three distinct cell-sized signals appeared over time within the retina of three participants. No significant increase in immune cell activation markers were detected after ICG administration. ICG accumulated in the endosomes of macrophage cultures and was detectable above a minimum concentration, suggesting cell labelling is possible. ICG can label RPE and may be used as an additional biomarker for RPE health across a range of retinal disorders. Standard clinical doses of intravenous ICG do not lead to robust immune cell labelling in human blood or retina and further optimisation in dose and route are required

The DNA Methylation Inhibitor Zebularine Controls CD4(+) T Cell Mediated Intraocular Inflammation

CD4+ T cell mediated uveitis is conventionally treated with systemic immunosuppressive agents, including corticosteroids and biologics targeting key inflammatory cytokines. However, their long-term utility is limited due to various side effects. Here, we investigated whether DNA methylation inhibitor zebularine can target CD4+ T cells and control intraocular inflammation. Our results showed that zebularine restrained the expression of inflammatory cytokines IFN-γ and IL-17 in both human and murine CD4+ T cells in vitro. Importantly, it also significantly alleviated intraocular inflammation and retinal tissue damage in the murine experimental autoimmune uveitis (EAU) model in vivo, suggesting that the DNA methylation inhibitor zebularine is a candidate new therapeutic agent for uveitis

Application of OCT-angiography to characterise the evolution of chorioretinal lesions in acute posterior multifocal placoid pigment epitheliopathy

Purpose: The aim of this study was to determine a sequence of structural changes in acute posterior multifocal placoid pigment epitheliopathy (APMPPE) using optical coherence tomography-angiography (OCT-A) and comparing with other imaging modalities.Patients and methodsPatients with a new diagnosis of acute-onset APMPPE referred to a regional specialist centre from October 2015 to October 2016 were included. Multimodal imaging employed on all patients from diagnosis included the following: fundus fluorescein angiography, indocyanine green angiography, fundus autofluorescence, spectral domain-OCT (SD-OCT), and OCT-A. All non-invasive imaging processes were repeated during follow-up.ResultsTen eyes of five patients were included in the study, three males and two females, with a mean age of 26.2 years (range: 21-32) and a mean follow-up of 6.4 months (range: 2.6-13.3). All patients presented with bilateral disease and macular involving lesions. OCT-A imaging of the choriocapillaris was supportive of hypoperfusion at the site of APMPPE lesions during the acute phase of this condition with normalisation of choroidal vasculature during follow-up. Multimodal imaging consistently highlighted four sequential phases from presentation to resolution of active disease.Conclusions: Multimodal imaging in patients with APMPPE in acute and long-term follow-up demonstrates a reversible choroidal hypoperfusion supporting the primary inciting pathology as a choriocapillaritis. The evolution shows resolution of the ischaemia through a defined sequence that results in persistent changes at the level of the retinal pigment epithelium and outer retina. OCT-A was able to detect preclinical changes and chart resolution at the level of the choriocapillaris

Identification of an intraocular microbiota

The current dogma in ophthalmology and vision research presumes the intraocular environment to be sterile. However, recent evidence of intestinal bacterial translocation into the bloodstream and many other internal organs including the eyes, found in healthy and diseased animal models, suggests that the intraocular cavity may also be inhabited by a microbial community. Here, we tested intraocular samples from over 1000 human eyes. Using quantitative PCR, negative staining transmission electron microscopy, direct culture, and high-throughput sequencing technologies, we demonstrated the presence of intraocular bacteria. The possibility that the microbiome from these low-biomass communities could be a contamination from other tissues and reagents was carefully evaluated and excluded. We also provide preliminary evidence that a disease-specific microbial signature characterized the intraocular environment of patients with age-related macular degeneration and glaucoma, suggesting that either spontaneous or pathogenic bacterial translocation may be associated with these common sight-threatening conditions. Furthermore, we revealed the presence of an intraocular microbiome in normal eyes from non-human mammals and demonstrated that this varied across species (rat, rabbit, pig, and macaque) and was established after birth. These findings represent the first-ever evidence of intraocular microbiota in humans

Composite polyester membranes with embedded dendrimer hosts and bimetallic Fe/Ni nanoparticles: synthesis, characterisation and application to water treatment

This study describes the preparation, characterization and evaluation of new composite membranes with embedded dendrimer hosts and Fe/Ni nanoparticles. These new reactive membranes consist of films of cyclodextrin–poly(propyleneimine) dendrimers (β-CD–PPI) that are deposited onto commercial polysulfone microporous supports and crosslinked with trimesoyl chloride (TMC). The membranes were subsequently loaded with Fe/Ni nanoparticles and evaluated as separation/reactive media in aqueous solutions using 2,4,6-trichlorophenol as model pollutant. The morphology and physicochemical properties of the composite membranes were characterised using high-resolution transmission electron microscopy (HR-TEM), atomic force microscopy and measurements of contact angle, water intake, porosity and water permeability. The sorption capacity and catalytic activity of the membranes were evaluated using ion chromatography, atmospheric pressure chemical ionisation-mass spectrometry and UV–Vis spectroscopy (UV–Vis). The sizes of the embedded Fe/Ni nanoparticles in the membranes ranged from 40 to 66 nm as confirmed by HR-TEM. The reaction rates for the dechlorination of 2,4,6-trichlorophenol ranged from 0.00148 to 0.00250 min−1. In all cases, we found that the reaction by-products consisted of chloride ions and mixtures of compounds including phenol (m/z = 93), 2,4-dichlorophenol (m/z = 163) and 4-chlorophenol (m/z = 128). The overall results of this study suggest that β-CD–PPI dendrimers are promising building blocks for the synthesis of composite and reactive membranes for the efficient removal of chlorinated organic pollutants from water