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Summary: Regulatory B (Breg) cells are potentially implicated in the pathogenesis of immune thrombocytopenia (ITP). We analysed a prospective cohort of newly diagnosed steroid naïve ITP patients enrolled in the multicentre FLIGHT trial and found that the numbers of Bregs in their peripheral blood were similar to healthy controls. In contrast, Breg numbers were significantly reduced in ITP patients treated with systemic immunosuppression (glucocorticoids or mycophenolate mofetil). We also demonstrate that glucocorticoid treatment impairs Breg interleukin‐10 production via an indirect T‐cell‐mediated mechanism. [ABSTRACT FROM AUTHOR]

Graves orbitopathy is both disabling and disfiguring. Medical therapies to reduce inflammation are widely used, but there is limited trial data beyond 18 months of follow-up. Methods: Three-year follow-up of a subset of the CIRTED trial (N = 68), which randomized patients to receive high-dose oral steroid with azathioprine/placebo and radiotherapy/sham radiotherapy. Results: Data were available at 3 years from 68 of 126 randomized subjects (54%). No additional benefit was seen at 3 years for patients randomized to azathioprine or radiotherapy with regard to a binary clinical composite outcome measure (BCCOM), modified European Group on Graves’ Orbitopathy score, or Ophthalmopathy Index. Clinical Activity Score (CAS), Ophthalmopathy Index, and Total Eye Score improved over 3 years (P < .001). However, quality of life at 3 years remained poor. Of 64 individuals with available surgical outcome data, 24 of 64 (37.5%) required surgical intervention. Disease duration of greater than 6 months before treatment was associated with increased need for surgery [odds ratio (OR) 16.8; 95% CI 2.95, 95.0; P = .001]. Higher baseline levels of CAS, Ophthalmopathy Index, and Total Eye Score but not early improvement in CAS were associated with increased requirement for surgery. Conclusion: In this long-term follow-up from a clinical trial, 3-year outcomes remained suboptimal with ongoing poor quality of life and high numbers requiring surgery. Importantly, reduction in CAS in the first year, a commonly used surrogate outcome measure, was not associated with improved long-term outcomes. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

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 IL- 10: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 stratification. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The specific pathogenesis of idiopathic nephrotic syndrome (NS) is poorly understood, and the role of immune mediators remains contentious. However, there is good evidence for the role of a circulating factor, and we recently postulated circulating proteases as candidate factors. Immunosuppressive therapy with glucocorticoids (GCs) and T cell inhibitors are widely used in the clinical treatment of NS. Given that T helper (CD4+) cells expressing IL-17A (so-called Th17 cells) have recently been reported to be resistant to GC treatment, and GC resistance remains a major challenge in the management of NS, we hypothesized that Th17 cells produce a circulating factor that is capable of signaling to the podocyte and inducing deleterious phenotypic changes. To test this, we generated human Th17 cells from healthy volunteers and added the supernatants from these T cell cultures to conditionally immortalized human podocytes in vitro. This demonstrated that podocytes treated with Th17 cell culture supernatant, as well as with patient disease plasma, showed significant stimulation of JNK and p38 MAPK pathways and an increase in motility, which was blocked using a JNK inhibitor. We have previously shown that nephrotic plasma elicits a podocyte response via protease-activated receptor-1 (PAR-1). Stimulation of PAR-1 inpodocytes elicited the same signaling response as Th17 cell culture supernatant treatment. Equally, protease inhibitors with Th17 cell culture treatment blocked the signaling response. This was not replicated by the reagents added to Th17 cell cultures or by IL-17A. Hence, we conclude that an undefined soluble mediator produced by Th17 cells mimics the deleterious effect of PAR-1 activation in vitro. Given the association between pathogenic subsets of Th17 cells and GC resistance, these observations have potential therapeutic relevance for patients with NS. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Purpose: To report a case of juxtafoveal choroidal neovascularization in a patient with candida chorioretinitis successfully treated with intravitreal bevacizumab.Methods: Case report.Results: A 45-year-old woman previously treated for candida chorioretinitis was presented with reduced vision in the left eye. The patient was investigated with ophthalmoscopy, fluorescein angiography, and optical coherence tomography (OCT). Following initial treatment, fundus examination, fluorescein angiography, and OCT of the right eye revealed a secondary juxtafoveal classic choroidal neovascularization. Following a single intravitreal injection of bevacizumab, the patient had excellent visual recovery, with absence of subretinal or intraretinal fluid in the OCT.Conclusions: Bevacizumab was effective in treatment of choroidal neovascularization associated with candida chorioretinitis. [ABSTRACT FROM AUTHOR]

Aim To assess the efficacy of intravitreal ranibizumab for the treatment of new onset in ammatory choroidal neovascularisation (iCNV), including both uveitic and idiopathic CNVs. Methods Single-centre, open-label, non-randomised Phase IIb clinical trial. Patients fulfilling strict entry criteria of new onset iCNV were given monthly intravitreal ranibizumab injections for 3 months. Thereafter, re-treatment was based on evidence of persisting activity. All patients completed trial follow-up. Optical coherence tomography (OCT) and best-corrected visual acuity (BCVA) were performed at every visit. Fluorescein angiography was performed at baseline, months 4 and 12. Descriptive analysis and Wilcoxon non-parametric test were performed for analysis. Results 15 patients, 10 women with a mean age of 48.8 years (range 24-85 years) were included in the study. The mean number of injections was 4.33 (range 3-7). There was a statistically significant difference in the BCVA at month 4 (p=0.001) and at month 12 (p=0.001) compared with baseline. The mean gain in BCVA at month 4 compared with baseline was 20 ±15.36 letters (mean±SD), and at month 12 was 21 ±16.97 letters. There was a statistically significant difference in the mean central sub field thickness (CST) at baseline versus month 4 (p=0.003) and month 12 (p=0.001). Conclusion Patients gained vision (mean of 21 letters at 12 months) and showed reduced CST. These results support the continued use of ranibizumab in the treatment of iCNV. Trial registration number 2008-007476-19, results. [ABSTRACT FROM AUTHOR]

Purpose To determine whether patients with juvenile systemic granulomatous disease ( JSGD) (Blau syndrome) and uveitis have a characteristic ocular phenotype. Methods Clinical and imaging data were collected retrospectively from patients attending the Regional Combined Paediatric Rheumatology and Ocular Inflammatory Service, Bristol Eye Hospital. General demographic information, laterality of the uveitis, age at onset, anatomical classification and course of the uveitis, clinical phenotype and specific NOD2 mutation were recorded for each patient. Results Seventeen eyes from nine patients (five males; four females) were included in the study. Mean age at the disease onset was 15 months, range 1-84 months. Eight patients had bilateral uveitis. Anterior uveitis was present in five eyes, intermediate uveitis in two eyes, and there were 10 eyes with panuveitis, manifesting as multifocal choroiditis. Appearance of optic disc included indistinct disc margins in six eyes, optic nerve head pallor in six eyes, optic disc vessel sheathing in four eyes, and there was peripapillary hypo/hyperpigmentation in 13 eyes accompanied with characteristic peripapillary nodular excrescences. Among NOD2 mutations, the p.R334W was the most commonly detected (n: four cases), and three patients carried novel variants, the p.E338D and p.D390V variants in one patient, and the p.H520Y and p.Q809K variants in two different patients. Conclusions Chronic bilateral panuveitis and a nodular peripapillary appearance in childhood onset uveitis are characteristic features of JSGD, which support the need for an appropriate genetic NOD2 analysis. [ABSTRACT FROM AUTHOR]

Understanding immune mechanisms of noninfectious intraocular inflammation via animal models, and the relatively more restricted insights that can be achieved through studies in humans, continues to generate successful immunotherapies. This translational conduit elaborates immunopathogenic mechanisms, and illuminates further prospects of tailored therapies and biomarkers of disease activity and prognosis. More recently, our increased understanding has moved on from the success of previous biologic therapies, such as anti-TNF and IFN-α treatments, revealing other possible avenues to target; for example, Th17 cells, immune cell migration and the use of T-regulatory and dendritic cells to induce immunological tolerance. We now recognize that the ocular environment is endowed with many regulatory mechanisms but is hardly privileged in as much as ocular inflammation remains prevalent. Nevertheless, future therapeutic and diagnostic developments will harness our understanding of the local immunoregulatory networks to not only restrain immune-mediated damage, but also to restore homeostasis and neuronal function. This article attempts to crystallize our understanding of local immune regulation and immune mechanisms leading to intraocular inflammation. [ABSTRACT FROM AUTHOR]

Despite a lack of robust evidence, anti-TNF therapies are in wide use for the treatment of noninfectious ocular inflammatory diseases. There is a clear rationale, based on mechanistic and preclinical efficacy data, for their use in posterior segment intraocular inflammation. However, their increasing use for other indications has been largely extrapolated from the benefit observed in autoinflammatory and autoimmune systemic diseases. Given their cost and the potential for significant adverse events, this review highlights the evidence for their continued use, possibilities for switching anti-TNF agents, and ways of reducing the risk of therapy. [ABSTRACT FROM AUTHOR]

Glucocorticoids remain the first-line treatment for a range of autoimmune and allergic diseases. However, 30% of patients fail to achieve disease control at tolerable systemic doses and continue to have an increased immune response with poor clinical outcome. This steroid refractory (SR) phenotype has previously been attributed to enhanced expression of inactive glucocorticoid receptor isoforms and cytokine-mediated suppression of glucocorticoid (GC) signaling, in particular by interleukin-2. These mechanisms are discussed, with emphasis on recent evidence for the role of the CD4+CD25intand GC-induced T regulatory cell subsets in perpetrating SR disease. [ABSTRACT FROM AUTHOR]

Background: Medical management of thyroid eye disease remains controversial due to a paucity of high quality evidence on long-term treatment outcomes. Glucocorticoids are known to be effective initially but have significant side-effects with long-term use and recrudescence can occur on cessation. Current evidence is conflicting on the efficacy of radiotherapy and non-steroid systemic immunosuppression, and the majority of previous studies have been retrospective, uncontrolled, small or poorly designed. The Combined Immunosuppression and Radiotherapy in Thyroid Eye Disease (CIRTED) trial was designed to investigate the efficacy of radiotherapy and azathioprine in combination with a standard course of oral prednisolone in patients with active thyroid eye disease. Methods/design: Patients with active thyroid eye disease will be randomised to receive (i) azathioprine or oral placebo and (ii) radiotherapy or sham-radiotherapy in this multi-centre, factorial randomised control trial. The primary outcome is improvement in disease severity (assessed using a composite binary measure) at 12 months and secondary end-points include quality of life scores and health economic measures. Discussion: The CIRTED trial is the first study to evaluate the role of radiotherapy and azathioprine as part of a long-term, combination immunosuppressive treatment regime for Thyroid Eye Disease. It will provide evidence for the role of radiotherapy and prolonged immunosuppression in the management of this condition, as well as pilot data on their use in combination. We have paid particular attention in the trial design to establishing (a) robust placebo controls and masking protocols which are effective and safe for both radiotherapy and the systemic administration of an antiproliferative drug; (b) constructing effective inclusion and exclusion criteria to select for active disease; and (c) selecting pragmatic outcome measures. Trial registration: Current controlled trials ISRCTN22471573 [ABSTRACT FROM AUTHOR]

Orbital involvement in chronic lymphocytic leukemia (CLL) is highly unusual and most commonly involves hemorrhage or soft tissue infiltration in advanced disease. We report a case of rapid onset bilateral orbital muscle infiltration as the presenting feature of early stage CLL. In addition, we demonstrate clinico-pathological correlation with an identical chronic B-cell lymphocytic infiltrate in both orbit and bone marrow, with good response of the orbital disease to local radiotherapy. [ABSTRACT FROM AUTHOR]

The author reflects on the need to embrace medical developments in favor of the anti-tumour necrosis factor therapy for the treatment of Behcet's uveitis.