Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

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

Adhesion Molecule Targeted Therapy for Non-Infectious Uveitis

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

Immune-Mediated Retinal Vasculitis in Posterior Uveitis and Experimental Models: The Leukotriene (LT)B4-VEGF Axis

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

Leukotriene B4 (LTB4) and its receptor in Experimental Autoimmune Uveitis (EAU) and in human retinal tissues: clinical severity and LTB4-dependence of retinal Th17 cells

Nomacopan, a drug originally derived from tick saliva, has dual functions of sequestering leukotriene B4 (LTB4) and inhibiting complement component 5 (C5) activation. It was demonstrated that nomacopan provides therapeutic benefit in experimental autoimmune uveitis (EAU). The 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 significant reduction of effector Th17 cells and inflammatory macrophages. Increased levels of LTB4 and C5a (produced upon C5 activation) were detected during disease progression. Retinal activated lymphocytes were shown to express LTB4 receptors (R) in vitro and in inflamed draining lymph nodes (dLN). Levels of LTB4R-expressing retinal active/inflammatory macrophages were also increased. Within the dLN CD4+T cell population, 30% expressed LTB4R+ following activation in vitro, while 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-BLT1 pathway as a novel therapeutic approach. In conclusion, 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

An Interleaved Configuration of Modified KY Converter with High Conversion Ratio for Renewable Energy Applications; Design, Analysis and Implementation

In this paper, a new high efficiency, high step-up, non-isolated, interleaved DC-DC converter for renewable energy applications is presented. In the suggested topology, two modified step-up KY converters are interleaved to obtain a high conversion ratio without the use of coupled inductors. In comparison with the conventional interleaved DC-DC converters such as boost, buck-boost, SEPIC, ZETA and CUK, the presented converter has higher voltage gain that is obtained with a suitable duty cycle. Despite the high voltage gain of the proposed converter, the voltage stress of the power switches and diodes is low. Therefore, switches with low conduction losses can be applied to improve the converter efficiency. Moreover, due to utilization of interleaving techniques, the input current ripple is low which makes the suggested converter a good candidate for renewable energy applications such as PV power system. Operation principle and steady-state analysis of the proposed converter in continuous conduction mode (CCM) and discontinuous conduction mode (DCM) are discussed in detail. Also, theoretical efficiency of the proposed converter is calculated. Finally, in order to evaluate the proposed converter operation by a renewable energy source such as a PV, the simulation results are presented. Moreover, a 220W prototype of the presented DC-DC converter is designed and implemented in the laboratory to verify its performance

Small-molecule antagonist of VLA-4 (GW559090) attenuated neuro-inflammation by targeting Th17 cell trafficking across the blood-retinal barrier in experimental autoimmune uveitis

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

Formulation and solution of a two-stage capacitated facility location problem with multilevel capacities

In this paper, the multi-product facility location problem in a two-stage supply chain is investigated. In this problem, the locations of depots (distribution centres) need to be determined along with their corresponding capacities. Moreover, the product flows from the plants to depots and onto customers must also be optimised. Here, plants have a production limit whereas potential depots have several possible capacity levels to choose from, which are defined as multilevel capacities. Plants must serve customer demands via depots. Two integer linear programming (ILP) models are introduced to solve the problem in order to minimise the fixed costs of opening depots and transportation costs. In the first model, the depot capacity is based on the maximum number of each product that can be stored whereas in the second one, the capacity is determined by the size (volume) of the depot. For large problems, the models are very difficult to solve using an exact method. Therefore, a matheuristic approach based on an aggregation approach and an exact method (ILP) is proposed in order to solve such problems. The methods are assessed using randomly generated data sets and existing data sets taken from the literature. The solutions obtained from the computational study confirm the effectiveness of the proposed matheuristic approach which outperforms the exact method. In addition, a case study arising from the wind energy sector in the UK is presented

An evaluation of three DoE-guided meta-heuristic-based solution methods for a three-echelon sustainable distribution network

This article evaluates the efficiency of three meta-heuristic optimiser (viz. MOGA-II, MOPSO and NSGA-II)-based solution methods for designing a sustainable three-echelon distribution network. The distribution network employs a bi-objective location-routing model. Due to the mathematically NP-hard nature of the model a multi-disciplinary optimisation commercial platform, modeFRONTIER®, is adopted to utilise the solution methods. The proposed Design of Experiment (DoE)-guided solution methods are of two phased that solve the NP-hard model to attain minimal total costs and total CO2 emission from transportation. Convergence of the optimisers are tested and compared. Ranking of the realistic results are examined using Pareto frontiers and the Technique for Order Preference by Similarity to Ideal Solution approach, followed by determination of the optimal transportation routes. A case of an Irish dairy processing industry’s three-echelon logistics network is considered to validate the solution methods. The results obtained through the proposed methods provide information on open/closed distribution centres (DCs), vehicle routing patterns connecting plants to DCs, open DCs to retailers and retailers to retailers, and number of trucks required in each route to transport the products. It is found that the DoE-guided NSGA-II optimiser based solution is more efficient when compared with the DoE-guided MOGA-II and MOPSO optimiser based solution methods in solving the bi-objective NP-hard three-echelon sustainable model. This efficient solution method enable managers to structure the physical distribution network on the demand side of a logistics network, minimising total cost and total CO2 emission from transportation while satisfying all operational constraints

Post-translational regulation contributes to the loss of LKB1 expression through SIRT1 deacetylase in osteosarcomas

background: The most prevalent form of bone cancer is osteosarcoma (OS), which is associated with poor prognosis in case of metastases formation. Mice harbouring liver kinase B1 (LKB1+/−) develop osteoblastoma-like tumours. Therefore, we asked whether loss of LKB1 gene has a role in the pathogenesis of human OS. methods: Osteosarcomas (n=259) were screened for LKB1 and sirtuin 1 (SIRT1) protein expression using immunohistochemistry and western blot. Those cases were also screened for LKB1 genetic alterations by next-generation sequencing, Sanger sequencing, restriction fragment length polymorphism and fluorescence in situ hybridisation approaches. We studied LKB1 protein degradation through SIRT1 expression. MicroRNA expression investigations were also conducted to identify the microRNAs involved in the SIRT1/LKB1 pathway. results: Forty-one per cent (106 out of 259) OS had lost LKB1 protein expression with no evident genetic anomalies. We obtained evidence that SIRT1 impairs LKB1 protein stability, and that SIRT1 depletion leads to accumulation of LKB1 in OS cell lines resulting in growth arrest. Further investigations revealed the role of miR-204 in the regulation of SIRT1 expression, which impairs LKB1 stability. conclusions: We demonstrated the involvement of sequential regulation of miR-204/SIRT1/LKB1 in OS cases and showed a mechanism for the loss of expression of LKB1 tumour suppressor in this malignancy