Relaxation of mitochondrial hyperfusion in the diabetic retina via N6-furfuryladenosine confers neuroprotection regardless of glycaemic status.

The recovery of mitochondrial quality control (MQC) may bring innovative solutions for neuroprotection, while imposing a significant challenge given the need of holistic approaches to restore mitochondrial dynamics (fusion/fission) and turnover (mitophagy and biogenesis). In diabetic retinopathy, this is compounded by our lack of understanding of human retinal neurodegeneration, but also how MQC processes interact during disease progression. Here, we show that mitochondria hyperfusion is characteristic of retinal neurodegeneration in human and murine diabetes, blunting the homeostatic turnover of mitochondria and causing metabolic and neuro-inflammatory stress. By mimicking this mitochondrial remodelling in vitro, we ascertain that N6-furfuryladenosine enhances mitochondrial turnover and bioenergetics by relaxing hyperfusion in a controlled fashion. Oral administration of N6-furfuryladenosine enhances mitochondrial turnover in the diabetic mouse retina (Ins2Akita males), improving clinical correlates and conferring neuroprotection regardless of glycaemic status. Our findings provide translational insights for neuroprotection in the diabetic retina through the holistic recovery of MQC.

Deep Metagenomic Sequencing for Endophthalmitis Pathogen Detection Using a Nanopore Platform.

PURPOSE: To evaluate the utility of nanopore sequencing for identifying potential causative pathogens in endophthalmitis, comparing culture results against full-length 16S rRNA nanopore sequencing (16S Nanopore), whole genome nanopore sequencing (Nanopore WGS), and Illumina (Illumina WGS). DESIGN: Cross-sectional diagnostic comparison. METHODS: Patients with clinically suspected endophthalmitis underwent intraocular vitreous biopsy as per standard care. Clinical samples were cultured by conventional methods, together with full-length 16S rRNA and WGS using nanopore and Illumina sequencing platforms. RESULTS: Of 23 patients (median age 68.5 years [range 47-88]; 14 males [61%]), 18 cases were culture-positive. Nanopore sequencing identified the same cultured organism in all of the culture-positive cases and identified potential pathogens in two culture-negative cases (40%). Nanopore WGS was able to additionally detect the presence of bacteriophages in three samples. The agreements at genus level between culture and 16S Nanopore, Nanopore WGS, and Illumina WGS were 75%, 100%, and 78%, respectively. CONCLUSIONS: Whole genome sequencing has higher sensitivity and provides a viable alternative to culture and 16S sequencing for detecting potential pathogens in endophthalmitis. Moreover, WGS has the ability to detect other potential pathogens in culture-negative cases. Whilst Nanopore and Illumina WGS provide comparable data, nanopore sequencing provides potential for cost-effective point-of-care diagnostics.

Ex vivo modelling of PD-1/PD-L1 immune checkpoint blockade under acute, chronic, and exhaustion-like conditions of T-cell stimulation.

Blockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1-revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1-to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

Sustained release of decorin to the surface of the eye enables scarless corneal regeneration.

Disorganization of the transparent collagenous matrix in the cornea, as a consequence of a variety of infections and inflammatory conditions, leads to corneal opacity and sight-loss. Such corneal opacities are a leading cause of blindness, according to the WHO. Public health programs target prevention of corneal scarring, but the only curative treatment of established scarring is through transplantation. Although attempts to minimize corneal scarring through aggressive control of infection and inflammation are made, there has been little progress in the development of anti-scarring therapies. This is owing to eye drop formulations using low viscosity or weak gelling materials having short retention times on the ocular surface. In this study, we report an innovative eye drop formulation that has the ability to provide sustained delivery of decorin, an anti-scarring agent. The novelty of this eye drop lies in the method of structuring during manufacture, which creates a material that can transition between solid and liquid states, allowing retention in a dynamic environment being slowly removed through blinking. In a murine model of Pseudomonas keratitis, applying the eye drop resulted in reductions of corneal opacity within 16 days. More remarkably, the addition of hrDecorin resulted in restoration of corneal epithelial integrity with minimal stromal opacity endorsed by reduced α-smooth muscle actin (αSMA), fibronectin, and laminin levels. We believe that this drug delivery system is an ideal non-invasive anti-fibrotic treatment for patients with microbial keratitis, potentially without recourse to surgery, saving the sight of many in the developing world, where corneal transplantation may not be available.

Obese subcutaneous adipose tissue impairs human myogenesis, particularly in old skeletal muscle, via resistin-mediated activation of NFκB.

Adiposity and adipokines are implicated in the loss of skeletal muscle mass with age and in several chronic disease states. The aim of this study was to determine the effects of human obese and lean subcutaneous adipose tissue secretome on myogenesis and metabolism in skeletal muscle cells derived from both young (18-30 yr) and elderly (>65 yr) individuals. Obese subcutaneous adipose tissue secretome impaired the myogenesis of old myoblasts but not young myoblasts. Resistin was prolifically secreted by obese subcutaneous adipose tissue and impaired myotube thickness and nuclear fusion by activation of the classical NFκB pathway. Depletion of resistin from obese adipose tissue secretome restored myogenesis. Inhibition of the classical NFκB pathway protected myoblasts from the detrimental effect of resistin on myogenesis. Resistin also promoted intramyocellular lipid accumulation in myotubes and altered myotube metabolism by enhancing fatty acid oxidation and increasing myotube respiration and ATP production. In conclusion, resistin derived from human obese subcutaneous adipose tissue impairs myogenesis of human skeletal muscle, particularly older muscle, and alters muscle metabolism in developing myotubes. These findings may have important implications for the maintenance of muscle mass in older people with chronic inflammatory conditions, or older people who are obese or overweight.

IL-15 promotes human myogenesis and mitigates the detrimental effects of TNFα on myotube development.

Studies in murine cell lines and in mouse models suggest that IL-15 promotes myogenesis and may protect against the inflammation-mediated skeletal muscle atrophy which occurs in sarcopenia and cachexia. The effects of IL-15 on human skeletal muscle growth and development remain largely uncharacterised. Myogenic cultures were isolated from the skeletal muscle of young and elderly subjects. Myoblasts were differentiated for 8 d, with or without the addition of recombinant cytokines (rIL-15, rTNFα) and an IL-15 receptor neutralising antibody. Although myotubes were 19% thinner in cultures derived from elderly subjects, rIL-15 increased the thickness of myotubes (MTT) from both age groups to a similar extent. Neutralisation of the high-affinity IL-15 receptor binding subunit, IL-15rα in elderly myotubes confirmed that autocrine concentrations of IL-15 also support myogenesis. Co-incubation of differentiating myoblasts with rIL-15 and rTNFα, limited the reduction in MTT and nuclear fusion index (NFI) associated with rTNFα stimulation alone. IL-15rα neutralisation and rTNFα decreased MTT and NFI further. This, coupled with our observation that myotubes secrete IL-15 in response to TNFα stimulation supports the notion that IL-15 serves to mitigate inflammatory skeletal muscle loss. IL-15 may be an effective therapeutic target for the attenuation of inflammation-mediated skeletal muscle atrophy.

The Use of 1α,25-Dihydroxyvitamin D3 as an Anticancer Agent.

The notion that vitamin D can influence the incidence of cancer arose from epidemiological studies. The major source of vitamin D in the organism is skin production upon exposure to ultra violet-B. The very first observation of an inverse correlation between exposure of individuals to the sun and the likelihood of cancer was reported as early as 1941. In 1980, Garland and Garland hypothesised, from findings from epidemiological studies of patients in the US with colon cancer, that vitamin D produced in response to sun exposure is protective against cancer as opposed to sunlight per se. Later studies revealed inverse correlations between sun exposure and the occurrence of prostate and breast cancers. These observations prompted laboratory investigation of whether or not vitamin D had an effect on cancer cells. Vitamin D is not active against cancer cells, but the most active metabolite 1α,25-dihydroxyvitamin D3 (1,25D) has profound biological effects. Here, we review the anticancer action of 1,25D, clinical trials of 1,25D to date and the prospects of the future therapeutic use of new and low calcaemic analogues.

Ciprofloxacin and ceftriaxone alter cytokine responses, but not Toll-like receptors, to Salmonella infection in vitro.

OBJECTIVES: Antibiotics that enhance host natural defences to infection offer an alternative approach to treating infections. However, mechanisms underlying such processes are poorly understood. The aim of this study was to investigate the effects of clinically relevant concentrations of two antibiotics on bacterial interactions with murine macrophages. METHODS: Adhesion of Salmonella Typhimurium SL1344 to and invasion by Salmonella Typhimurium SL1344 of antibiotic-treated or untreated J774 murine macrophages were measured using a tissue culture infection model. Expression of genes central to the Toll-like receptor (TLR) signalling pathway of macrophages infected with Salmonella was analysed using the RT(2) Profiler PCR Array. Cytokine production was measured by ELISA. RESULTS: Adhesion of Salmonella Typhimurium SL1344 to J774 macrophage monolayers was increased when macrophages were exposed to ciprofloxacin and ceftriaxone, while invasion was decreased by ciprofloxacin. Expression of IL-1ß and TNF-α mRNA was greater in SL1344-infected macrophages that had been treated with ciprofloxacin or ceftriaxone than in macrophages exposed to antibiotics alone or SL1344 alone. TLR mRNA was down-regulated by SL1344 infection, a response that was not altered by antibiotic pretreatment. CONCLUSIONS: Clinically relevant concentrations of two antibiotics differentially enhanced the response of immune cells and their interaction with bacteria, increasing bacterial adhesion to macrophages and increasing cytokine production. As increased expression of IL-1ß fosters apoptosis of Salmonella-infected macrophages and clearance by neutrophils, the immunomodulatory potential of these antibiotics may explain, in part, why these two drugs continue to be used to treat salmonellosis successfully.

Systemic lupus erythematosus: An update for ophthalmologists.

Systemic lupus erythematosus (SLE) is a life-threatening multisystem inflammatory condition that may affect almost any part of the eye. We provide an update for the practicing ophthalmologist comprising a systematic review of the recent literature presented in the context of current knowledge of the pathogenesis, diagnosis, and treatment of this condition. We review recent advances in the understanding of the influence of genetic and environmental factors on the development of SLE. Recent changes in the diagnostic criteria for SLE are considered. We assess the potential for novel molecular biomarkers to find a clinical application in disease diagnosis and stratification and in the development of therapeutic agents. We discuss limited forms of SLE and their differentiation from other collagen vascular disorders and review recent evidence underlying the use of established and novel therapeutics in this condition, including specific implications regarding monitoring for ocular toxicity associated with antimalarials.

Genetics of Behçet's disease.

PURPOSE OF REVIEW: This article discusses recent genetic and epigenetic associations involved in the pathogenesis of Behçet's disease. RECENT FINDINGS: Genetic studies have supported the strong association of human leukocyte antigen-B and Behçet's disease, and high production of tumour necrosis factor and low production of interleukin (IL)-10, which have led to therapy based on controlling these effects. Polymorphisms that affect the response to pathogens (TLR and FUT2) are leading to increased interest in responses to microbiomes. Inflammation in Behçet's disease results in vascular damage and several single nucleotide polymorphisms in chemokine and adhesion molecules may be involved in this process. Increased levels of inflammatory cytokines including IL-1ß and IL-17 have been linked to altered expression of microRNAs, miR155, miR21 and miR23b. DNA methylation changes in monocytes and lymphocytes have been described that affect the function of these cells. SUMMARY: Genetic and epigenetic changes affecting cells and molecules involved in Behçet's disease offer new pathways for research, including cytoskeletal protein function, that will provide new targets for therapy, and potentially address the ethnic differences seen in validation of gene studies.

Progenitor cells are mobilized by acute psychological stress but not beta-adrenergic receptor agonist infusion.

OBJECTIVES: Stimuli that activate the sympathetic nervous system, such as acute psychological stress, rapidly invoke a robust mobilization of lymphocytes into the circulation. Experimental animal studies suggest that bone marrow-derived progenitor cells (PCs) also mobilize in response to sympathetic stimulation. Here we tested the effects of acute psychological stress and brief pharmacological ß-adrenergic (ßAR) stimulation on peripheral PC numbers in humans. METHODS: In two studies, we investigated PC mobilization in response to an acute speech task (n=26) and ßAR-agonist (isoproterenol) infusion (n=20). A subset of 8 participants also underwent the infusion protocol with concomitant administration of the ßAR-antagonist propranolol. Flow cytometry was used to enumerate lymphocyte subsets, total progenitor cells, total haematopoietic stem cells (HSC), early HSC (multi-lineage potential), late HSC (lineage committed), and endothelial PCs (EPCs). RESULTS: Both psychological stress and ßAR-agonist infusion caused the expected mobilization of total monocytes and lymphocytes and CD8(+) T lymphocytes. Psychological stress also induced a modest, but significant, increase in total PCs, HSCs, and EPC numbers in peripheral blood. However, infusion of a ßAR-agonist did not result in a significant change in circulating PCs. CONCLUSION: PCs are rapidly mobilized by psychological stress via mechanisms independent of ßAR-stimulation, although the findings do not exclude ßAR-stimulation as a possible cofactor. Considering the clinical and physiological relevance, further research into the mechanisms involved in stress-induced PC mobilization seems warranted.

Cortisol biosynthesis in the human ocular surface innate immune response.

Innate immune responses have a critical role in regulating sight-threatening ocular surface (OcS) inflammation. While glucocorticoids (GCs) are frequently used to limit tissue damage, the role of intracrine GC (cortisol) bioavailability via 11-beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in OcS defense, remains unresolved. We found that primary human corneal epithelial cells (PHCEC), fibroblasts (PHKF) and allogeneic macrophages (M1, GM-CSF; M2, M-CSF) were capable of generating cortisol (M1>PHKF>M2>PHCEC) but in corneal cells, this was independent of Toll-like receptor (TLR) activation. While PolyI∶C induced maximal cytokine and chemokine production from both PHCEC (IFNγ, CCL2, CCL3, and (CCL4), IL6, CXCL10, CCL5, TNFα) and PHKF (CCL2, IL-6, CXCL10, CCL5), only PHKF cytokines were inhibited by GCs. Both Poly I∶C and LPS challenged-corneal cells induced M1 chemotaxis (greatest LPS-PHKF (250%), but down-regulated M1 11ß-HSD1 activity (30 and 40% respectively). These data were supported by clinical studies demonstrating reduced human tear film cortisol∶cortisone ratios (a biomarker of local 11ß-HSD1 activity) in pseudomonas keratitis (1∶2.9) versus healthy controls (1∶1.3; p<0.05). This contrasted with putative TLR3-mediated OcS disease (Stevens-Johnson Syndrome, Mucous membrane pemphigoid) where an increase in cortisol∶cortisone ratio was observed (113.8∶1; p<0.05). In summary, cortisol biosynthesis in human corneal cells is independent of TLR activation and is likely to afford immunoprotection under physiological conditions. Contribution to ocular mucosal innate responses is dependent on the aetiology of immunological challenge.

Characterization of vitamin D production by human ocular barrier cells.

PURPOSE: Vitamin D3 is a secosteroid mainly synthesized from the conversion of the skin precursor 7-dehydrocholesterol (7DHC) to vitamin D3 by ultraviolet (UV) B sunlight. Extrarenal synthesis of vitamin D3 has been reported in many tissues and cells, including barrier sites. This study characterizes the expression of components of vitamin D3 signaling in human ocular barrier cells. METHODS: Primary human scleral fibroblasts (HSF), human corneal endothelial (HCEC-12), nonpigmented ciliary body epithelial (ODM-2), and adult retinal pigment epithelial (ARPE-19) cell lines were analyzed for the expression of vitamin D receptor (VDR), the vitamin D3 activating enzymes 1α-hydroxylase (CYP27B1), 25-hydroxylases (CYP27A1 and CYP2R1), the vitamin D3 inactivating enzyme 24-hydroxylase (CYP24A1), and the endocytic receptors cubilin and megalin using a combination of RT-PCR, immunocytochemistry, and enzyme immunoassay (EIA). RESULTS: The HSF, HCEC-12, ODM-2, and ARPE-19 express mRNA and protein for all vitamin D3 synthesizing and metabolizing components. The cell types tested, except HSF, are able to convert inactive 25-hydroxyvitamin D3 (25[OH]D3) into active 1,25-hydroxyvitamin D3 (1,25[OH]2D3). CONCLUSIONS: This novel study demonstrated that ocular barrier epithelial cells express the machinery for vitamin D3 and can produce 1,25(OH)2D3. We suggest that vitamin D3 might have a role in immune regulation and barrier function in ocular barrier epithelial cells.

Age, gender and disease-related platelet and neutrophil activation ex vivo in whole blood samples from patients with Behçet's disease.

OBJECTIVES: Behçet's disease (BD) is more severe among young males and disease severity decreases with age. Therefore, the effect of disease activity, gender and age on platelet and neutrophil activation in whole blood taken from patients with BD was investigated. METHODS: Using an anti-coagulant Tripotassium ethylenediaminetetra acetic acid (K3EDTA) plus citrate-theophylline-adenosine-dipyridamole (CTAD) (K3EDTA/CTAD) that preserves the degree of platelet activation that exists in vivo, we assessed neutrophil and platelet activation, microparticles, and monocyte and neutrophil-platelet aggregate formation in 43 BD patients using flow cytometry. This is the first description of platelet activation and microparticles in BD patients using this methodology. RESULTS: Inactive [2.78 (0.56)%, P = 0.0009; 3.11 (0.78)%, P < 0.0001] and active [2.28 (0.84)%, P < 0.0001; 3.071 (0.67)%, P = 0.0031] BD patients had significantly higher percentages of CD62P-expressing platelets and CD62P+ platelet microparticles as compared with healthy controls (HCs) [0.84 (0.1)% and 1.23 (0.14)%], respectively. The percentages of CD62P+ platelets and CD62P+ platelet microparticles in female and male BD patients were also significantly higher than those expressed by female and male HCs. The percentages of CD62P+ microparticles were significantly increased in the 20-30-(P = 0.0301) and 31-50-(P < 0.0162) year age ranges, but not in the >50-year age group of BD patients. CONCLUSION: BD is a rare, chronic multi-systemic vasculitis and interaction of activated platelets with leucocytes has been linked to pathological disorders associated with vascular inflammation. Importantly, this study demonstrates that platelet microparticle activation is increased in BD. Also, this is the first report in which changes in platelet activation in BD are concordant with the observations that BD disease activity diminishes with age.

Gene expression and miR profiles of human corneal fibroblasts in response to dexamethasone.

PURPOSE: Dexamethasone (DEX) is commonly used as a therapeutic agent for various ocular inflammatory diseases; however, its effect on resident naive cells is unknown. In this study, genome microarray and microRNA (miR) analyses were used to evaluate the global gene and miR expression of human corneal fibroblasts (HKFs) in response to treatment with DEX. METHODS: Primary HKFs from three donors were treated with DEX for 16 hours. Treated and untreated cells were snap frozen for microarray and miR array analyses. Genes with a more than threefold change were classified into gene families using the DAVID web-based classification database, and six of these genes were validated using quantitative real-time PCR. Five miRs were also validated using miR-detection assays. RESULTS: Of the 41,093 genes examined, 261 were upregulated and 123 were downregulated greater than threefold after DEX treatment. Real-time PCR confirmed upregulation of six genes, including oculocutaneous albinism II (OCA2), angiopoietin-like 7 (ANGPTL7), neuron navigator 2 (NAV2), neurofilament light chain polypeptide (NEFL), solute carrier family 16/member 12 (SLC16A12), and serum amyloid A1 (SAA1). Expression of several miR including miR-16, -21, and -29C were upregulated, whereas miR-100 was downregulated in fibroblasts by DEX. CONCLUSIONS: DEX can greatly change the global gene and miR profile of HKFs. DEX not only downregulates inflammatory genes, but can also induce expression of angiogenic and inflammatory genes. In addition, DEX may exert posttranscriptional gene regulation through miRs. These data support a complex role for DEX-induced changes in resident cells that may have implications in the clinical management of corneal inflammation with topical glucocorticoids.

TIRAP Ser180Leu polymorphism is associated with Behcet's disease.

OBJECTIVES: The initiating cause of Behçet's disease (BD) is unknown, but an aberrant response to infection has been suggested. In this study, single nucleotide polymorphisms in Toll-like receptors (TLRs) and associated molecules that have a sentinel function at mucosal surfaces were analysed in patients with BD. METHODS: TLR expression was determined by immunohistochemistry in buccal mucosal tissue from patients with BD, in tissue from patients with lichen planus (LP) or pyogenic granuloma (PG) as disease controls, or from healthy individuals. Using SSP-PCR we analysed SNP in CD14, TLR2, TLR4 and TIRAP (TIR domain-containing adaptor protein) in patients with BD from different geographical regions. RESULTS: TLR expression was increased in buccal lesions from patients with BD compared with healthy controls; however, a similar increase was seen in lesion tissue from patients with LP or PG, suggesting that this was a generalized inflammatory response as opposed to a BD-specific response. SNP analysis showed no association between CD14, TLR2 or TLR4 polymorphisms. However, TIRAP 180Leu was significantly associated with BD in UK, but not Middle Eastern, patients. CONCLUSION: TLR expression showed no difference in tissue from patients with BD compared with either disease or healthy controls. Likewise, SNPs in TLR genes were no different from healthy controls. The association with the increased function variant of TIRAP suggests that encounter with a pathogen at mucosal sites will lead to increased cytokine production and tissue damage with persistence of mucosal lesions.

Endogenous cortisol and TGF-beta in human aqueous humor contribute to ocular immune privilege by regulating dendritic cell function.

Aqueous humor (AqH) has been shown to have significant immunosuppressive effects on APCs in animal models. We wanted to establish whether, in humans, AqH can regulate dendritic cell (DC) function and to identify the dominant mechanism involved. Human AqH inhibited the capacity of human peripheral blood monocyte-derived DC to induce naive CD4(+) T cell proliferation and cytokine production in vitro, associated with a reduction in DC expression of the costimulatory molecule CD86. This was seen both for DC cultured under noninflammatory conditions (immature DC) and for DC stimulated by proinflammatory cytokines (mature DC). DC expression of MHC classes I/II and CD83 was reduced (mature DC only). Myeloid DC from peripheral blood were similarly sensitive to the effects of human AqH, but only under inflammatory conditions. The addition of α-melanocyte stimulating hormone and vasoactive intestinal peptide did not cause significant inhibition at physiological levels. However, the addition of exogenous cortisol at physiological levels recapitulated the AqH-induced reduction in CD86 and inhibition of DC-induced T cell proliferation, and blockade of cortisol in AqH partially reversed its suppressive effects. TGF-ß2 had an additional effect with cortisol, and although simultaneous blockade of cortisol and TGF-ß2 in AqH reduced its effectiveness, there was still a cortisol- and TGF-ß-independent component. In humans, AqH regulates DC maturation and function by the combined actions of cortisol and TGF-ß2, a pathway that is likely to contribute to the maintenance of immune privilege in the eye.

Soluble gp130, an antagonist of IL-6 transsignaling, is elevated in uveitis aqueous humor.

PURPOSE: To determine the concentrations of soluble gp130, a natural antagonist of IL-6 transsignaling, in the serum and aqueous humor (AqH) of patients with uveitis. METHODS: Serum was obtained from the peripheral blood of patients with active uveitis and healthy control subjects. AqH samples were collected from patients with active uveitis and those without uveitis who were undergoing routine cataract surgery. Samples were centrifuged and the cell-free supernatant frozen at -80 degrees C. Concentrations of sgp130, sIL-6R, and IL-6 were determined by a sandwich ELISA or multiplex bead immunoassay, using standard curves of known concentrations of recombinant cytokines. RESULTS: Serum concentrations of sgp130 were not significantly different between control individuals and patients with active anterior uveitis, regardless of the degree of intraocular inflammation cells. By contrast, the concentration of sgp130 in AqH was very low in patients with no or little inflammation, but increased significantly with disease severity. The greatest elevations of AqH sgp130 were found in patients with the highest cellular activity. Simultaneous measurement of IL-6, sIL-6R, and sgp130 revealed a high degree of correlation between the levels of these molecules, especially for sIL-6R and sgp130. CONCLUSIONS: Soluble gp130 is increased in the AqH of patients with active uveitis. It is likely that sgp130 partially inhibits the process of IL-6 transsignaling during inflammation. However, the concentration found is still far below that in serum, suggesting that increasing the level of sgp130 further may assist in reducing the inflammatory changes induced by IL-6 transsignaling.

Behçet's disease: ocular effects and treatment.

Behçet's disease (BD) is a systemic immune-mediated vasculitis of unclear origin. Major symptoms include oral aphthous ulcers, genital ulcerations, skin lesions, and ocular lesions. Eye involvement, which affects 60-80% of BD patients, is characterized by posterior or panuveitis with occlusive retinal vasculitis. The pathogenesis of BD remains unclear, but research of the last decades has shown a complex role of genetic factors (HLA-B51) predisposing to inflammation with involvement of the innate-immune system (neutrophils, NK cells), perpetuated by the adaptive immune response, most importantly T cells, against infectious- and/or auto-antigens. Despite aggressive immunosuppressive treatment, the visual prognosis of ocular BD was generally poor to date. Recently, novel biologic drugs, including interferon-alpha and tumour necrosis factor (TNF)-alpha-antagonists have been introduced in the treatment of ocular BD with very promising results and seem for the first time to improve the prognosis of the disease. This article will provide a current review of BD including recent developments in epidemiology, immunology, genetics, and treatment.

Multiplex bead analysis of vitreous humor of patients with vitreoretinal disorders.

PURPOSE: Vitreoretinal disorders are frequently characterized by increased vitreous levels of cellular mediators, including cytokines, chemokines, and growth factors. The study was conducted to investigate whether multiplex bead analysis could identify disease-specific profiles of these mediators in a variety of vitreoretinal diseases. METHODS: Levels of 19 mediators were measured: the cytokines IL-6, IL-10, IL-12, IL-13, IL-15, IL-17, TNF, IFN-gamma, granulocyte-macrophage-colony-stimulating factor (GM-CSF), and granulocyte-stimulating factor (G-CSF); the chemokines CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL8; and the growth factors epidermal growth factor (EGF), FGF, and VEGF, by using multiplex bead analysis of vitreous humor of 58 eyes undergoing vitrectomy for a variety of vitreoretinal disorders. RESULTS: The predominant mediators detected were IL-6, CXCL8, and CCL2. The most complex pattern of mediators was seen in patients with proliferative vitreoretinopathy (PVR) and included a mixture of cytokines, chemokines, and growth factors. Patients with chronic uveitis showed a limited mediator pattern that did not suggest either a Th1 or Th2 response. By comparison, patients with lens-induced uveitis (LIU) showed significantly greater levels of cytokines than did patients with chronic uveitis, including IFN-gamma and IL-12, with a trend toward an acute Th1 inflammatory response. Moreover, in samples from patients with LIU, CXCL8 inversely correlated with time after initial surgery and duration of treatment. CONCLUSIONS: Multiplex bead analysis allows the measurement of multiple mediators from a single vitreous sample. The data confirm patterns of mediators previously described in different vitreoretinal conditions. In addition, LIU mediator levels correlate with duration of treatment and time after cataract surgery.