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Nomacopan, a drug originally derived from tick saliva, has dual functions of sequestering leukotriene B4 (LTB4) and inhibiting complement component 5 (C5) activation. Nomacopan has been shown to provide therapeutic benefit in experimental autoimmune uveitis (EAU). Longer acting forms of nomacopan were more efficacious in mouse EAU models, and the long-acting variant that inhibited only LTB4 was at least as effective as the long-acting variant that inhibited both C5 and LTB4, preventing structural damage to the retina and a significantly reducing effector T helper 17 cells and inflammatory macrophages. Increased levels of LTB4 and C5a (produced upon C5 activation) were detected during disease progression. Activated retinal lymphocytes were shown to express LTB4 receptors (R) in vitro and in inflamed draining lymph nodes. Levels of LTB4R-expressing active/inflammatory retinal macrophages were also increased. Within the draining lymph node CD4+ T-cell population, 30% expressed LTB4R+ following activation in vitro, whereas retinal infiltrating cells expressed LTB4R and C5aR. Validation of expression of those receptors in human uveitis and healthy tissues suggests that infiltrating cells could be targeted by inhibitors of the LTB4-LTB4 receptor 1 (BLT1) pathway as a novel therapeutic approach. This study provides novel data on intraocular LTB4 and C5a in EAU, their associated receptor expression by retinal infiltrating cells in mouse and human tissues, and in attenuating EAU via the dual inhibitor nomacopan.

Immunopathogenic roles for both Th1 (CD4+ IFN-γ+ ) and Th17 (CD4+ IL-17A+ ) cells have been demonstrated in experimental autoimmune uveitis (EAU). However, the role for Th17/Th1 (CD4+ T cells co-expressing IFN-γ and IL-17A) cells in EAU is not yet understood. Using interphotoreceptor retinoid-binding protein peptide-induced EAU in mice, we found increased levels of Th17/Th1 cells in EAU retinae (mean 9.6 ± 4.2%) and draining LNs (mean 8.4 ± 3.9%; p = 0.01) relative to controls. Topical dexamethasone treatment effectively reduced EAU severity and decreased retinal Th1 cells (p = 0.01), but had no impact on retinal Th17/Th1 or Th17 cells compared to saline controls. Using in vitro migration assays with mouse CNS endothelium, we demonstrated that Th17/Th1 cells were significantly increased within the migrated population relative to controls (mean 15.6 ± 9.5% vs. 1.9 ± 1.5%; p = 0.01). Chemokine receptor profiles of Th17/Th1 cells (CXCR3 and CCR6) did not change throughout the transendothelial migration process and were unaffected by dexamethasone treatment. These findings support a role for Th17/Th1 cells in EAU and their resistance to steroid inhibition suggests the importance of targeting both Th17 and Th17/Th1 cells for improving therapy.

Context Insulin autoimmune syndrome (IAS) is characterized by hyperinsulinemic hypoglycemia with elevated anti-insulin antibodies. Most commonly observed in the Japanese population, elsewhere it is rare and associated with autoimmune diseases, plasma cell dyscrasias, or sulfhydryl group medications. The active metabolite of clopidogrel has a sulfhydryl group and here we report a case of clopidogrel-induced IAS. Case Description A 67-year-old man was admitted with severe hyperinsulinemic hypoglycemia requiring continuous intravenous infusion of 10% dextrose to sustain euglycemia. His symptoms of hypoglycemia had started after commencing dual antiplatelet therapy (including clopidogrel) for ischemic heart disease 9 months earlier. The hypoglycemia was associated with elevated insulin, proinsulin, c-peptide, and anti-insulin antibody titers as well as the HLA-DRB1*04 haplotype. Multiple localizing studies were negative for an insulinoma. A diagnosis of IAS was thus made. Clopidogrel cessation, oral dexamethasone, and diazoxide therapy were not sufficient to safely wean the dextrose infusion. Plasma exchange was ultimately effective. Conclusions This case highlights a case of severe IAS. Given the ubiquity of clopidogrel, IAS should be remembered as a rare adverse effect.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund's Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

Non-infectious uveitis (NIU) is an inflammatory eye disease initiated via CD4+ T-cell activation and transmigration, resulting in focal retinal tissue damage and visual acuity disturbance. Cell adhesion molecules (CAMs) are activated during the inflammatory process to facilitate the leukocyte recruitment cascade. Our review focused on CAM-targeted therapies in experimental autoimmune uveitis (EAU) and NIU. We concluded that CAM-based therapies have demonstrated benefits for controlling EAU severity with decreases in immune cell migration, especially via ICAM-1/LFA-1 and VCAM-1/VLA-4 (integrin) pathways. P-selectin and E-selectin are more involved specifically in uveitis related to vasculitis. These therapies have potential clinical applications for the development of a more personalized and specific treatment. Localized therapies are the future direction to avoid serious systemic side effects.

Non-infectious uveitis (NIU) is a potentially sight-threatening disease. Effector CD4+ T cells, especially interferon-γ-(IFNγ) producing Th1 cells and interleukin-17-(IL-17) producing Th17 cells, are the major immunopathogenic cells, as demonstrated by adoptive transfer of disease in a model of experimental autoimmune uveitis (EAU). CD4+FoxP3+CD25+ regulatory T cells (Tregs) were known to suppress function of effector CD4+ T cells and contribute to resolution of disease. It has been recently reported that some CD4+ T-cell subsets demonstrate shared phenotypes with another CD4+ T-cell subset, offering the potential for dual function. For example, Th17/Th1 (co-expressing IFNγ and IL-17) cells and Th17/Treg (co-expressing IL-17 and FoxP3) cells have been identified in NIU and EAU. In this review, we have investigated the evidence as to whether these ‘plastic CD4+ T cells’ are functionally active in uveitis. We conclude that Th17/Th1 cells are generated locally, are resistant to the immunosuppressive effects of steroids, and contribute to early development of EAU. Th17/Treg cells produce IL-17, not IL-10, and act similar to Th17 cells. These cells were considered pathogenic in uveitis. Future studies are needed to better clarify their function, and in the future, these cell subsets may in need to be taken into consideration for designing treatment strategies for disease.

PubMed: 30887530, The treatment and management of ocular allergy (OA) remain a major concern for different specialties, including allergists, ophthalmologists, primary care physicians, rhinologists, pediatricians, dermatologists, clinical immunologists, and pharmacists. We performed a systematic review of all relevant publications in MEDLINE, Scopus, and Web Science including systematic reviews and meta-analysis. Publications were considered relevant if they addressed treatments, or management strategies of OA. A further wider systematic literature search was performed if no evidence or good quality evidence was found. There are effective drugs for the treatment of OA; however, there is a lack an optimal treatment for the perennial and severe forms. Topical antihistamines, mast cell stabilizers, or double-action drugs are the first choice of treatment. All of them are effective in reducing signs and symptoms of OA. The safety and optimal dosing regimen of the most effective topical anti-inflammatory drugs, corticosteroids, are still a major concern. Topical calcineurin inhibitors may be used in steroid-dependent/resistant cases of severe allergic keratoconjunctivitis. Allergen-specific immunotherapy may be considered in cases of failure of first-line treatments or to modify the natural course of OA disease. Based on the current wealth of publications and on the collective experience, recommendations on management of OA have been proposed. © 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd., Funding information This work was done under the approval of EAACI with a TF budget 2015-18.

CONTEXT: Diabetic ketoacidosis (DKA) has been associated with the use of sodium glucose cotransporter 2 inhibitors (SGLT2is). OBJECTIVE: To determine the incidence, characteristics, and outcomes of DKA in SGLT2i users vs nonusers with type 2 diabetes. DESIGN: Retrospective, multicenter, controlled cohort study. SETTING: All public hospitals in Melbourne and Geelong (combined population of 5 million), Australia, from 1 September 2015 to 31 October 2017. PATIENTS: Consecutive cases of DKA that developed in the community, or during the course of hospital admission, in patients with type 2 diabetes. MAIN OUTCOME MEASURES: In SGLT2i users vs nonusers: (i) OR of DKA developing during hospital admission, and (ii) incidence of DKA. RESULTS: There were 162 cases of DKA (37 SGLT2i users and 125 non-SGLT2i users) with a physician-adjudicated diagnosis of type 2 diabetes. Of these, DKA developed during the course of inpatient admission in 14 (38%) SGLT2i users vs 2 (2%) non-SGLT2i users (OR, 37.4; 95% CI, 8.0 to 175.9; P < 0.0001). The incidence of DKA was 1.02 per 1000 (95% CI, 0.74 to 1.41 per 1000) in SGLT2i users vs 0.69 per 1000 (95% CI, 0.58 to 0.82 per 1000) in non-SGLT2i users (OR, 1.48; 95% CI, 1.02 to 2.15; P = 0.037). Fifteen SGLT2i users (41%) had peak blood glucose

The mechanistic basis of gliogenesis, which occurs late in human development, is poorly understood. Here we identify nuclear factor IA (NFIA) as a molecular switch inducing human glial competency. Transient expression of NFIA is sufficient to trigger glial competency of human pluripotent stem cell-derived neural stem cells within 5 days and to convert these cells into astrocytes in the presence of glial-promoting factors, as compared to 3–6 months using current protocols. NFIA-induced astrocytes promote synaptogenesis, exhibit neuroprotective properties, display calcium transients in response to appropriate stimuli and engraft in the adult mouse brain. Differentiation involves rapid but reversible chromatin remodeling, glial fibrillary acidic protein (GFAP) promoter demethylation and a striking lengthening of the G1 cell cycle phase. Genetic or pharmacological manipulation of G1 length partially mimics NFIA function. We used the approach to generate astrocytes with region-specific or reactive features. Our study defines key mechanisms of the gliogenic switch and enables the rapid production of human astrocytes for disease modeling and regenerative medicine. A new method generates astrocytes from human pluripotent stem cells in 5 days.

Non-infectious uveitis (NIU) is a potentially sight-threatening disease. Effector CD4+ T cells, especially interferon-γ-(IFNγ) producing Th1 cells and interleukin-17-(IL-17) producing Th17 cells, are the major immunopathogenic cells, as demonstrated by adoptive transfer of disease in a model of experimental autoimmune uveitis (EAU). CD4+FoxP3+CD25+ regulatory T cells (Tregs) were known to suppress function of effector CD4+ T cells and contribute to resolution of disease. It has been recently reported that some CD4+ T-cell subsets demonstrate shared phenotypes with another CD4+ T-cell subset, offering the potential for dual function. For example, Th17/Th1 (co-expressing IFNγ and IL-17) cells and Th17/Treg (co-expressing IL-17 and FoxP3) cells have been identified in NIU and EAU. In this review, we have investigated the evidence as to whether these ‘plastic CD4+ T cells’ are functionally active in uveitis. We conclude that Th17/Th1 cells are generated locally, are resistant to the immunosuppressive effects of steroids, and contribute to early development of EAU. Th17/Treg cells produce IL-17, not IL-10, and act similar to Th17 cells. These cells were considered pathogenic in uveitis. Future studies are needed to better clarify their function, and in the future, these cell subsets may in need to be taken into consideration for designing treatment strategies for disease.

Retinal vascular diseases have distinct, complex and multifactorial pathogeneses yet share several key pathophysiological aspects including inflammation, vascular permeability and neovascularisation. In non-infectious posterior uveitis (NIU), retinal vasculitis involves vessel leakage leading to retinal enlargement, exudation, and macular oedema. Neovascularisation is not a common feature in NIU, however, detection of the major angiogenic factor—vascular endothelial growth factor A (VEGF-A)—in intraocular fluids in animal models of uveitis may be an indication for a role for this cytokine in a highly inflammatory condition. Suppression of VEGF-A by directly targeting the leukotriene B4 (LTB4) receptor (BLT1) pathway indicates a connection between leukotrienes (LTs), which have prominent roles in initiating and propagating inflammatory responses, and VEGF-A in retinal inflammatory diseases. Further research is needed to understand how LTs interact with intraocular cytokines in retinal inflammatory diseases to guide the development of novel therapeutic approaches targeting both inflammatory mediator pathways.

Treatment of posterior eye diseases with intravitreal injections of drugs, while effective, is invasive and associated with side effects such as retinal detachment and endophthalmitis. In this work, we have formulated a model compound, rapamycin (RAP), in nanoparticle-based eye drops and evaluated the delivery of RAP to the posterior eye tissues in a healthy rabbit. We have also studied the formulation in experimental autoimmune uveitis (EAU) mouse model with retinal inflammation. Aqueous RAP eye drops were prepared using N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (Molecular Envelope Technology - MET) containing 0.23 ± 0.001% w/v RAP with viscosity, osmolarity, and pH within the ocular comfort range, and the formulation (MET-RAP) was stable in terms of drug content at both refrigeration and room temperature for one month. The MET-RAP eye drops delivered RAP to the choroid-retina with a Cmax of 145 ± 49 ng/g (tmax = 1 h). The topical application of the MET-RAP eye drops to the EAU mouse model resulted in significant disease suppression compared to controls, with activity similar to dexamethasone eye drops. The MET-RAP eye drops also resulted in a reduction of RORγt and an increase in both Foxp3 expression and IL-10 secretion, indicating a mechanism involving the inhibition of Th17 cells and the up-regulation of T-reg cells. The MET-RAP formulation delivers RAP to the posterior eye segments, and the formulation is active in EAU.

Amyotrophic lateral sclerosis is a disease characterized by progressive paralysis and death. Most ALS-cases are sporadic (sALS) and patient heterogeneity poses challenges for effective therapies. Applying metabolite profiling on 77-sALS patient-derived-fibroblasts and 43-controls, we found ~25% of sALS cases (termed sALS-1) are characterized by transsulfuration pathway upregulation, where methionine-derived-homocysteine is channeled into cysteine for glutathione synthesis. sALS-1 fibroblasts selectively exhibited a growth defect under oxidative conditions, fully-rescued by N-acetylcysteine (NAC). [U–13C]-glucose tracing showed transsulfuration pathway activation with accelerated glucose flux into the Krebs cycle. We established a four-metabolite support vector machine model predicting sALS-1 metabotype with 97.5% accuracy. Both sALS-1 metabotype and growth phenotype were validated in an independent cohort of sALS cases. Importantly, plasma metabolite profiling identified a system-wide cysteine metabolism perturbation as a hallmark of sALS-1. Findings reveal that sALS patients can be stratified into distinct metabotypes with differential sensitivity to metabolic stress, providing novel insights for personalized therapy.

Background The integrin VLA-4 (α4β1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4β1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets. Methods Mice (female; B10.RIII or C57Bl/6; aged 6–8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry. Results There was a significant reduction in clinical and histological scores in GW10- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers. Conclusions This α4β1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4β1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.

Nomacopan, a drug originally derived from tick saliva, has dual functions of sequestering leukotriene B

Aim: To investigate the role of interleukin-17 in ocular surface and corneal disease. Ocular surface and corneal disease is a leading cause of blindness and is an ongoing challenge for the public health sector to implement effective therapies. The majority of cells in corneal lesions are derived primarily from neutrophils that induce inflammatory events that lead to tissue damage. One of the key pro-inflammatory cytokines is IL-17, and it has been investigated in order to facilitate the understanding of the pathogenesis of ocular surface lesion development. Method: A review of the literature was performed through a systematic approach. Results: IL-17 has been shown to exacerbate dry eye disease, viral and bacterial keratitis lesion severity, although it was found to be protective for Acanthamoeba. Antibodies developed to neutralize IL-17 have shown some promise in reducing the severity of some diseases. Conclusion: IL-17 plays a role in the pathogenesis of ocular surface and corneal disease and targeting this cytokine may provide a useful treatment option in the future.

BACKGROUND: Diabetic ketoacidosis (DKA) is an acute life-threatening metabolic complication of diabetes that imposes substantial burden on our healthcare system. There is a paucity of published data in Australia assessing factors influencing time to resolution of DKA and length of stay (LOS). AIMS: To identify factors that predict a slower time to resolution of DKA in adults with diabetes. METHODS: Retrospective audit of patients admitted to St Vincent's Hospital Melbourne between 2010 to 2014 coded with a diagnosis of 'Diabetic Ketoacidosis'. The primary outcome was time to resolution of DKA based on normalisation of biochemical markers. Episodes of DKA within the wider Victorian hospital network were also explored. RESULTS: Seventy-one patients met biochemical criteria for DKA; median age 31 years (26-45 years), 59% were male and 23% had newly diagnosed diabetes. Insulin omission was the most common precipitant (42%). Median time to resolution of DKA was 11 h (6.5-16.5 h). Individual factors associated with slower resolution of DKA were lower admission pH (P < 0.001) and higher admission serum potassium level (P = 0.03). Median LOS was 3 days (2-5 days), compared to a Victorian state-wide LOS of 2 days. Higher comorbidity scores were associated with longer LOS (P < 0.001). CONCLUSIONS: Lower admission pH levels and higher admission serum potassium levels are independent predictors of slower time to resolution of DKA. This may assist to stratify patients with DKA using markers of severity to determine who may benefit from closer monitoring and to predict LOS.

Experimental autoimmune uveitis (EAU), in which CD4+ Th1 and/or Th17 cells are immunopathogenic, mimics various clinical features of noninfectious uveitis in humans. The impact of bromodomain extraterminal (BET) inhibitors on Th17 cell function was studied in a mouse model of EAU in vivo and in mouse and human Th17 cells in vitro. Two BET inhibitors (GSK151 and JQ1) were able to ameliorate the progression of inflammation in EAU and in mouse CD4+ T cells in vitro, downregulating levels of Th17 cells. Additionally, the uveitogenic capacity of Th17 cells to transfer EAU was abrogated by BET inhibitors in an adoptive transfer model. In human CD4+ T cells, a 5-d exposure to BET inhibitors was accompanied by a significant downregulation of Th17-associated genes IL-17A, IL-22, and retinoic acid–related orphan receptor γt. However, in vitro, the inhibitors had no effect on already polarized Th17 cells. The key finding is that, in response to BET inhibitors, Th17-enriched cultures developed a regulatory phenotype, upregulated FOXP3 expression and IL-10 secretion, and lost pathogenicity in vivo. We conclude that BET targeting of Th17 cells is a potential therapeutic opportunity for a wide range of inflammatory and autoimmune diseases, including uveitis.

Objective: Adherence to combination antiretroviral therapy (ART) among pregnant women is essential to attaining the goal of eliminating mother-to-child HIV transmission. The objective of this study was to determine which factors affect adherence to ART among HIV-positive women enrolled in a large prevention of mother-to-child HIV transmission (PMTCT) trial in rural north-central Nigeria. Methods: The parent study included 372 HIV-positive pregnant women enrolled in a cluster-randomized control trial conducted at 12 health facilities in Nigeria between 2013 and 2015. This secondary analysis included HIV-positive women (and their infants) from the original trial with documented adherence data (n=210, 56.5%). The primary outcome was maternal adherence to ART, determined by self-report and based on the visual analogue scale (VAS) of a validated medication adherence tool. Participants with a VAS score of ? 95% were classified as adherent. We employed multivariate logistic regression to evaluate the predictors of maternal ART adherence in the study sample. Results: Approximately 61.0% of study participants (128/210) were adherent to ART. The majority of adherent participants (62.5%, 80/128) were enrolled in the trial intervention arm. The most common cited response for non-adherence was fear of status disclosure. Adherence to ART was associated with study arm (intervention arm vs. control arm, adjusted Odds Ratio (aOR) [95% CI]: 16.95 [5.30-54.23]), maternal ethnicity (Gwari vs. Other, aOR = 0.13 [0.05-0.38]), and partner HIV status (HIV-positive vs. unknown, aOR = 3.14 [1.22-8.07]). Conclusion and Global Health Implications: Adherence to ART among a cohort of pregnant women enrolled in a PMTCT trial in rural North-Central Nigeria was associated with trial arm, maternal self- reported ethnicity, and partner’s HIV status. Increased understanding of the interplay between these factors will enable the development of more targeted and effective interventions. Key words: • HIV/AIDS • PMTCT • Combination antiretroviral therapy • Adherence • PMCTC • Nigeria Copyright © 2020 Calder et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Amyotrophic lateral sclerosis (ALS) is a disease characterized by progressive paralysis and death. Most ALS cases are sporadic (sALS) and patient heterogeneity poses a formidable challenge for the development of viable biomarkers and effective therapies. Applying untargeted metabolite profiling on 77 sALS patient-derived primary dermal fibroblast lines and 45 sex/age matched controls, we found that ∼25% of cell lines (termed sALS-1) are characterized by upregulated trans-sulfuration, where methionine-derived homocysteine is channeled into cysteine and glutathione synthesis. sALS-1 fibroblasts exhibit a growth defect when grown under oxidative conditions, that can be fully-rescued by N-acetylcysteine. [U-13C]-glucose tracing shows that activation of the trans-sulfuration pathway is associated with accelerated glucose flux into the TCA cycle. Based on four metabolites, we developed a support vector machine model capable of distinguishing sALS-1 with 97.5% accuracy. Importantly, plasma metabolite profiling identifies a systemic perturbation of cysteine metabolism as a hallmark of sALS-1. These results indicate that sALS patients can be stratified into distinct metabotypes, differently sensitive to metabolic stress, and provides new insights into metabolic biomarkers for personalized sALS therapy.

Exploratory analysis to assess the association of single nucleotide polymorphisms (SNPs) in the interleukin (IL) 10 and IL-17 genes with severity of contact lens keratitis.This was a retrospective case control study of 88 contact lens keratitis cases (25 severe) and 185 healthy contact lens wearers recruited from studies conducted at Moorfields Eye Hospital and in Australia-wide during 2003-2005. Buccal swab samples were collected on Whatman FTA cards and mailed by post for DNA extraction and SNP genotyping. IL-10 (rs1800871; rs1800896; rs1800872) and IL-17 (rs1800871; rs1800896; rs1800872) SNPs were screened by pyrosequencing. Genetic association analyses were performed via Cochran-Armitage trend tests and logistic regression models using PLINK software.None of the SNPs tested showed evidence of association with severity of contact lens keratitis at P 0.05. Nevertheless, minor allele G in SNP rs2397084 of the IL-17F gene was associated with increased risk of severe MK, with OR=2.1 (95% CI=0.9-4.8, P = 0.066).Our study cannot exclude with confidence that genetic variation in the IL-17 F proinflammatory cytokine is associated with more severe outcomes of MK. However, there is general body of information that the IL-17 pathway is important in the mechanisms of MK. Studies with larger power and the expanded array of laboratory tools will elucidate the exact role of IL-17 in MK.

Purpose: Over a third of patients with Acanthamoeba keratitis (AK) experience severe inflammatory complications (SICs). This study aimed to determine if some contact lens (CL) wearers with AK were predisposed to SICs due to variations in key immune genes. Methods: CL wearers with AK who attended Moorfields Eye Hospital were recruited prospectively between April 2013 and October 2014. SICs were defined as scleritis and/or stromal ring infiltrate. Genomic DNA was processed with an Illumina Low Input Custom Amplicon assay of 58 single nucleotide polymorphism (SNP) targets across 18 genes and tested for association in PLINK. Results: Genomic DNA was obtained and analyzed for 105 cases of AK, 40 (38%) of whom experienced SICs. SNPs in the CXCL8 gene encoding IL-8 was significantly associated with protection from SICs (chr4: rs1126647, odds ratio [OR] = 0.3, P = 0.005, rs2227543, OR = 0.4, P = 0.007, and rs2227307, OR = 0.4, P = 0.02) after adjusting for age, sex, steroids prediagnosis, and herpes simplex keratitis (HSK) misdiagnosis. Two TLR-4 SNPs were associated with increased risk of SICs (chr9: rs4986791 and rs4986790, both OR = 6.9, P = 0.01). Th-17 associated SNPs (chr1: IL-23R rs11209026, chr2: IL-1β rs16944, and chr12: IL-22 rs1179251) were also associated with SICs. Conclusions: The current study identifies biologically relevant genetic variants in patients with AK with SICs; IL-8 is associated with a strong neutrophil response in the cornea in AK, TLR-4 is important in early AK disease, and Th-17 genes are associated with adaptive immune responses to AK in animal models. Genetic screening of patients with AK to predict severity is viable and this would be expected to assist disease management.

Aceruloplasminaemia is an autosomal recessive disorder of iron metabolism which is characterised by diabetes, neurodegeneration and anaemia. It should be considered in the differential diagnosis of adult onset, antibody-negative diabetes associated with persistent mild anaemia and hyperferritinaemia and/or progressive neuropsychiatric impairments.

Survival of L. monocytogenes (LM) and shigatoxigenic Escherichia coli (STEC) during manufacture and aging of unpasteurized, farmstead-style cheese was investigated. Cheese produced with fresh, unpasteurized milk inoculated with a two-strain cocktail of LM and STEC (both ∼4.5 log cfu mL−1) was aged for 84 days at 4, 10, or 22 °C. Bacterial populations were assessed weekly. Pathogen populations were unchanged throughout cheese manufacture, but were significantly lower in whey than in curd. Aging at 4 °C caused a significant reduction in STEC and LM populations to below the enumeration limit on day 84. At 10 °C, a significant decrease in STEC was observed, but LM levels increased significantly; at 22 °C, populations of both pathogens increased. Regardless of aging temperature or time, both pathogens were detected in every cheese sample analyzed. Results suggest that aging cheese at the lowest temperature possible will provide the best control of STEC and LM.

Purpose To evaluate non-contact infrared meibography and anterior segment optical coherence tomography (AS-OCT) to detect meibomian gland (MG) and corneal changes in children with blepharokeratoconjunctivitis (BKC). Methods We acquired infrared meibography images of upper and lower lids and AS-OCT corneal scans. One masked observer graded meiboscore, full/partial MG dropout, and measured total corneal volume and differential corneal volume per quadrant and central corneal thickness (CCT). Results We enrolled 63 children, 31 with BKC and 32 without ocular surface inflammation; median (interquartile range) age BKC 10.6 (7.2-13.9) years, healthy volunteers (HV) 11.4 (9.5-13.8) years. Likert scale scores for meibography and OCT indicate no to low discomfort. Meiboscores for upper and lower lids as well as the total meiboscore were significantly higher in children with BKC than in HV. Subscores for full and partial MG dropout were also significantly higher in children with BKC than in healthy volunteers. There was no statistically significant difference between upper and lower lid for meiboscore nor full/partial MG dropout scores. The corneal volume in the superior quadrant was significantly higher in children with BKC than in HV, whereas the corneal volume in the nasal and inferior quadrants was significantly lower. Conclusions Non-contact imaging technologies objectively demonstrate damage to meibomian glands and changes in corneal volume secondary to BKC. The tests are well tolerated by children with mild/moderate ocular surface inflammation and can detect changes without the requirement for routine eversion of the upper lid. These parameters may be useful both for clinical follow-up and clinical trials.

Sporadic amyotrophic lateral sclerosis (sALS) is a progressive motor neuron disease resulting in paralysis and death. Genes responsible for familial ALS have been identified, however the molecular basis for sALS is unknown. To discover metabotypic biomarkers that inform on disease etiology, untargeted metabolite profiling was performed on 77 patient-derived dermal fibroblast lines and 45 age/sex-matched controls. Surprisingly, 25% of sALS lines showed upregulated methionine-derived homocysteine, channeled to cysteine and glutathione (GSH). Stable isotope tracing of [U-13C]-glucose showed activation of the trans-sulfuration pathway, associated with accelerated glucose flux into the TCA cycle, glutamate, GSH, alanine, aspartate, acylcarnitines and nucleotide phosphates. A four-molecule support vector machine model distinguished the sALS subtype from controls with 97.5% accuracy. Plasma metabolite profiling identified increased taurine as a hallmark metabolite for this sALS subset, suggesting systemic perturbation of cysteine metabolism. Furthermore, integrated multiomics (mRNAs/microRNAs/metabolites) identified the super-trans-sulfuration pathway as a top hit for the sALS subtype. We conclude that sALS can be stratified into distinct metabotypes, providing for future development of personalized therapies that offer new hope to sufferers.

Capturing the full potential of human pluripotent stem cell (PSC)-derived neurons in disease modeling and regenerative medicine requires analysis in complex functional systems. Here we establish optogenetic control in human PSC-derived spinal motorneurons and show that co-culture of these cells with human myoblast-derived skeletal muscle builds a functional all-human neuromuscular junction that can be triggered to twitch upon light stimulation. To model neuromuscular disease we incubated these co-cultures with IgG from myasthenia gravis patients and active complement. Myasthenia gravis is an autoimmune disorder that selectively targets neuromuscular junctions. We saw a reversible reduction in the amplitude of muscle contractions, representing a surrogate marker for the characteristic loss of muscle strength seen in this disease. The ability to recapitulate key aspects of disease pathology and its symptomatic treatment suggests that this neuromuscular junction assay has significant potential for modeling of neuromuscular disease and regeneration.

Foodborne illness is a global public health issue. Young adults may work in foodservice while they are university students, and their habits may later shape the practices and well-being of their children. The objective of this study was to establish baseline data and assess the food safety knowledge of 18- to 26-year-old Univ. of Maine students. Demographic questions and the previously validated Food Safety Knowledge Questionnaire (FSKQ) were placed online. Of 123 people who responded to the email recruitment notice, 104 Univ. of Maine undergraduates aged 18 to 26 years completed the survey. The average score among all participants was 60% correct (53 points out of a possible 89 points). Survey questions that required participants to identify common sources of foodborne pathogens had the lowest average percent correct (31%). Less than 50% of participants were able to correctly identify several high-risk foods, including sliced melon, raw sprouts, and unpasteurized fruit juice. Our findings indicate a need for educational programs for 18- to 26-year-old Univ. of Maine students in regards to common sources of foodborne pathogens and proper handling of fresh produce and that food safety knowledge among university students has not improved since publication of a national survey using the FSKQ in 2006. Effective educational programs are needed to ensure that young adults understand food risks and appropriate food handling practices.

The derivation of somatic motoneurons (MNs) from ES cells (ESCs) after exposure to sonic hedgehog (SHH) and retinoic acid (RA) is one of the best defined, directed differentiation strategies to specify fate in pluripotent lineages. In mouse ESCs, MN yield is particularly high after RA + SHH treatment, whereas human ESC (hESC) protocols have been generally less efficient. In an effort to optimize yield, we observe that functional MNs can be derived from hESCs at high efficiencies if treated with patterning molecules at very early differentiation steps before neural induction. Remarkably, under these conditions, equal numbers of human MNs were obtained in the presence or absence of SHH exposure. Using pharmacological and genetic strategies, we demonstrate that early RA treatment directs MN differentiation independently of extrinsic SHH activation by suppressing the induction of GLI3. We further demonstrate that neural induction triggers a switch from a poised to an active chromatin state at GLI3. Early RA treatment prevents this switch by direct binding of the RA receptor at the GLI3 promoter. Furthermore, GLI3 knock-out hESCs can bypass the requirement for early RA patterning to yield MNs efficiently. Our data demonstrate that RA-mediated suppression of GLI3 is sufficient to generate MNs in an SHH-independent manner and that temporal changes in exposure to patterning factors such as RA affect chromatin state and competency of hESC-derived lineages to adopt specific neuronal fates. Finally, our work presents a streamlined platform for the highly efficient derivation of human MNs from ESCs and induced pluripotent stem cells. SIGNIFICANCE STATEMENT Our study presents a rapid and efficient protocol to generate human motoneurons from embryonic and induced pluripotent stem cells. Surprisingly, and in contrast to previous work, motoneurons are generated in the presence of retinoic acid but in the absence of factors that activate sonic hedgehog signaling. We show that early exposure to retinoic acid modulates the chromatin state of cells to be permissive for motoneuron generation and directly suppresses the induction of GLI3, a negative regulator of SHH signaling. Therefore, our data point to a novel mechanism by which retinoic acid exposure can bypass the requirement for extrinsic SHH treatment during motoneuron induction.

Elevated tumor necrosis factor (TNF) α levels are associated with chronic autoimmune diseases in which effects of TNFα on immune cells are multiple and complex. Analysis of uveitis in mice exhibiting severe autoimmune inflammation, resulting in a destructive subtotal loss of photoreceptors, revealed the presence of high plasma levels of TNFα and a significant population of CD4 + TNFα + cells in the periphery and the eye at peak disease (TNFα hi ). We have shown previously by pharmacological activation that the deacetylase Sirtuin 1 (SIRT1) has an anti-inflammatory role in a less severe, TNFα lo model of uveitis. We now show that SIRT1 activation fails to clinically suppress severe TNFα hi disease, whereas glucocorticoid treatment is successful. TNFα has been reported to mediate cleavage and inactivation of SIRT1 during inflammation, and at peak disease we observed both full-length and cleaved SIRT1 in draining lymph node cells. In vivo systemic TNFα blockade suppressed severe ocular disease and restricted SIRT1 cleavage in the periphery, maintaining full-length active SIRT1 protein. When combining a suboptimal TNFα blockade with SIRT1 activation, a synergistic suppression of severe disease compared with TNFα blockade alone occurred. Our data suggest a new role for TNFα in exacerbating the severity of autoimmune disease by regulating SIRT1 cleavage in draining lymph node effector cells. SIRT1 activation may be effective as an adjunctive treatment for inflammatory conditions not fully controlled by TNFα inhibitors.

Background Non-infectious uveitis can cause chronic relapsing and remitting ocular inflammation, which may require high dose systemic immunosuppression to prevent severe sight loss. It has been classically described as an autoimmune disease, mediated by pro-inflammatory Th1 and Th17 T-cell subsets. Studies suggest that natural immunosuppressive CD4+CD25+FoxP3+ T-regulatory cells (Tregs) are involved in resolution of inflammation and may be involved in the maintenance of clinical remission. Objective To investigate whether there is a peripheral blood immunoregulatory phenotype associated with clinical remission of sight-threatening non-infectious uveitis by comparing peripheral blood levels of Treg, Th1, and Th17, and associated DNA methylation and cytokine levels in patients with active uveitic disease, control subjects and patients (with previously active disease) in clinical remission induced by immunosuppressive drugs. Methods Isolated peripheral blood mononuclear cells (PBMC) from peripheral blood samples from prospectively recruited subjects were analyzed by flow cytometry for CD3, CD4, FoxP3, TIGIT, T-bet, and related orphan receptor γt. Epigenetic DNA methylation levels of FOXP3 Treg-specific demethylated region (TSDR), FOXP3 promoter, TBX21, RORC2, and TIGIT loci were determined in cryopreserved PBMC using a next-generation sequencing approach. Related cytokines were measured in blood sera. Functional suppressive capacity of Treg was assessed using T-cell proliferation assays. Results Fifty patients with uveitis (intermediate, posterior, and panuveitis) and 10 control subjects were recruited. The frequency of CD4+CD25+FoxP3+ Treg, TIGIT+ Treg, and T-bet+ Treg and the ratio of Treg to Th1 were significantly higher in remission patients compared with patients with active uveitic disease; and TIGIT+ Tregs were a significant predictor of clinical remission. Treg from patients in clinical remission demonstrated a high level of in vitro suppressive function compared with Treg from control subjects and from patients with untreated active disease. PBMC from patients in clinical remission had significantly lower methylation levels at the FOXP3 TSDR, FOXP3 promoter, and TIGIT loci and higher levels at RORC loci than those with active disease. Clinical remission was also associated with significantly higher serum levels of transforming growth factor β and IL-10, which positively correlated with Treg levels, and lower serum levels of IFNγ, IL-17A, and IL-22 compared with patients with active disease. Conclusion Clinical remission of sight-threatening non-infectious uveitis has an immunoregulatory phenotype characterized by upregulation of peripheral Treg, polarized toward T-bet and TIGIT. These findings may assist with individualized therapy of uveitis, by informing whether drug therapy has induced phenotypically stable Treg associated with long-term clinical remission.

PURPOSE: To determine differences in key tear film cytokines between mild and severe cases of acanthamoeba keratitis (AK) and control contact lens (CL) wearers. METHODS: This was a prospective study of CL wearers with AK attending Moorfields Eye Hospital and control CL wearers from the Institute of Optometry, London. Basal tear specimens were collected by 10-μL capillary tubes (BLAUBRAND intraMark, Wertheim, Germany), and tear protein levels were measured with a multiplex magnetic bead array (Luminex 100; Luminex Corporation, Austin, TX) for cytokines interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-17A, IL-17E, IL-17F, IL-22, and interferon gamma and with enzyme-linked immunosorbent assay (Abcam, Cambridge, United Kingdom) for CXCL2. Severe cases of AK were defined as having active infection for over 12 months and at least 1 severe inflammatory event. RESULTS: One hundred and thirty-two tear samples were collected from a total of 61 cases (15 severe and 46 mild–moderate) and 22 controls. IL-8, part of the Toll-like receptor 4 cytokine cascade, was found to be expressed at a detectable level more often in cases of AK than in control CL wearers (P = 0.003) and in higher concentrations in severe cases than in milder forms of the disease (z = −2.35). IL-22, part of the IL-10 family, and a proinflammatory Th17 cytokine, was detected more often in severe cases than in milder forms of AK (P < 0.02). CONCLUSIONS: Profiling patients with AK during disease shows differences in cytokine levels between severe and milder disease that may inform clinical management. The Toll-like receptor 4 and IL-10/Th17 inflammatory pathways should be included in further investigations of this disease.

Display Omitted A large situation display can support teamwork.The display should integrate all high-priority information critical to all team members.The context of the situation can influence the extent that such a display is effective.A pilot study demonstrated that a team display supported ED resuscitation teamwork and situational awareness. Effective teamwork in ED resuscitations, including information sharing and situational awareness, could be degraded. Technological cognitive aids can facilitate effective teamwork. Objective: This study focused on the design of an ED situation display and pilot test its influence on teamwork and situational awareness during simulated resuscitation scenarios. Material and methods: The display design consisted of a central area showing the critical dynamic parameters of the interventions with an events time-line below it. Static information was placed at the sides of the display. We pilot tested whether the situation display could lead to higher scores on the Clinical Teamwork Scale (CTS), improved scores on a context-specific Situational Awareness Global Assessment Technique (SAGAT) tool, and team communication patterns that reflect teamwork and situational awareness. Results: Resuscitation teamwork, as measured by the CTS, was overall better with the presence of the situation display as compared with no situation display. Team members discussed interventions more with the situation display compared with not having the situation display. Situational awareness was better with the situation display only in the trauma scenario. Discussion: The situation display could be more effective for certain ED team members and in certain cases. Conclusions: Overall, this pilot study implies that a situation display could facilitate better teamwork and team communication in the resuscitation event.

Summary Background Secondary progressive multiple sclerosis, for which no satisfactory treatment presently exists, accounts for most of the disability in patients with multiple sclerosis. Simvastatin, which is widely used for treatment of vascular disease, with its excellent safety profile, has immunomodulatory and neuroprotective properties that could make it an appealing candidate drug for patients with secondary progressive multiple sclerosis. Methods We undertook a double-blind, controlled trial between Jan 28, 2008, and Nov 4, 2011, at three neuroscience centres in the UK. Patients aged 18–65 years with secondary progressive multiple sclerosis were randomly assigned (1:1), by a centralised web-based service with a block size of eight, to receive either 80 mg of simvastatin or placebo. Patients, treating physicians, and outcome assessors were masked to treatment allocation. The primary outcome was the annualised rate of whole-brain atrophy measured from serial volumetric MRI. Analyses were by intention to treat and per protocol. This trial is registered with ClinicalTrials.gov, number NCT00647348. Findings 140 participants were randomly assigned to receive either simvastatin (n=70) or placebo (n=70). The mean annualised atrophy rate was significantly lower in patients in the simvastatin group (0·288% per year [SD 0·521]) than in those in the placebo group (0·584% per year [0·498]). The adjusted difference in atrophy rate between groups was −0·254% per year (95% CI −0·422 to −0·087; p=0·003); a 43% reduction in annualised rate. Simvastatin was well tolerated, with no differences between the placebo and simvastatin groups in proportions of participants who had serious adverse events (14 [20%] vs nine [13%]). Interpretation High-dose simvastatin reduced the annualised rate of whole-brain atrophy compared with placebo, and was well tolerated and safe. These results support the advancement of this treatment to phase 3 testing. Funding The Moulton Foundation [charity number 1109891], Berkeley Foundation [268369], the Multiple Sclerosis Trials Collaboration [1113598], the Rosetrees Trust [298582] and a personal contribution from A Pidgley, UK National Institute of Health Research (NIHR) University College London Hospitals/UCL Biomedical Research Centres funding scheme.

WOS: 000412545200005, PubMed: 28387947, Ocular allergy (OA) includes a group of common and less frequent hypersensitivity disorders frequently misdiagnosed and not properly managed. The diagnosis of OA is usually based on clinical history and signs and symptoms, with the support of in vivo and in vitro tests when identification of the specific allergen is required. To date, no specific test is available for the diagnosis of the whole spectrum of the different forms of OA. The lack of recommendations on diagnosis of OA is considered a medical need not only for allergists but also for ophthalmologists. This position paper aims to provide a comprehensive overview of the currently available tools for diagnosing OA to promote a common nomenclature and procedures to be used by different specialists. Questionnaires, sign and symptom grading scales, tests, and potential biomarkers for OA are reviewed. We also identified several unmet needs in the diagnostic tools to generate interest, increase understanding, and inspire further investigations. Tools, recommendations, and algorithms for the diagnosis of OA are proposed for use by both allergists and ophthalmologists. Several unmet needs in the diagnostic tools should be further improved by specific clinical research in OA., EAACI BoO; ExCom TF budget, Supported by EAACI BoO and ExCom TF budget 2014-16. Final Approval by EAACI ExCom on December 5, 2016. The number of authors exceeds the suggested maximum 8 because of the need for a wide consensus from different European countries and specialties.

The prevalence of allergy is rising globally at a very significant rate, which is currently at 20-40% of individuals in westernized nations. In the eye, allergic conditions can take on the acute form such as in seasonal and perennial allergic conjunctivitis, or a more severe and debilitating chronic form such as in vernal and atopic keratoconjunctivitis. Indeed, some key aspects of allergic eye disease pathophysiology are understood, such as the role of mast cells in the acute allergic reaction, and the contribution of eosinophils in late-onset and chronic allergy. However, recent developments in animal models and clinical studies have uncovered new and important roles for previously underappreciated players, including chemokine receptors on ocular surface dendritic cells such as CCR7, the contribution of conjunctival epithelium to immunity, histamine and leukotriene receptors on conjunctival goblet cells and a role for mast cells in late-onset manifestations. Furthermore, recent work in animal models has delineated the contribution of IL-4 in the increased incidence of corneal graft rejection in hosts with allergic conjunctivitis. Recent studies such as these mean that conventional paradigms and concepts should be revisited. The aim of this review is to highlight some of the most recent advances and insights on newly appreciated players in the pathogenesis of allergic eye disease.

Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy.