Background and ObjectivesBlood-brain barrier (BBB) dysfunction is an integral feature of neurological disorders and involves the action of multiple proinflammatory cytokines on the microvascular endothelial cells lining cerebral capillaries. There is still however, considerable ambiguity throughout the scientific literature regarding the mechanistic role(s) of cytokines in this context, thereby warranting a comprehensive in vitro investigation into how different cytokines may cause dysregulation of adherens and tight junctions leading to BBB permeabilization.MethodsThe present study employs human brain microvascular endothelial cells (HBMvECs) to compare/contrast the effects of TNF-α and IL-6 on BBB characteristics ranging from the expression of interendothelial junction proteins (VE-cadherin, occludin and claudin-5) to endothelial monolayer permeability. The contribution of cytokine-induced NADPH oxidase activation to altered barrier phenotype was also investigated.ResultsIn response to treatment with either TNF-α or IL-6 (0–100 ng/ml, 0–24 hrs), our studies consistently demonstrated significant dose- and time-dependent decreases in the expression of all interendothelial junction proteins examined, in parallel with dose- and time-dependent increases in ROS generation and HBMvEC permeability. Increased expression and co-association of gp91 and p47, pivotal NADPH oxidase subunits, was also observed in response to either cytokine. Finally, cytokine-dependent effects on junctional protein expression, ROS generation and endothelial permeability could all be attenuated to a comparable extent using a range of antioxidant strategies, which included ROS depleting agents (superoxide dismutase, catalase, N-acetylcysteine, apocynin) and targeted NADPH oxidase blockade (gp91 and p47 siRNA, NSC23766).ConclusionA timely and wide-ranging investigation comparing the permeabilizing actions of TNF-α and IL-6 in HBMvECs is presented, in which we demonstrate how either cytokine can similarly downregulate the expression of interendothelial adherens and tight junction proteins leading to elevation of paracellular permeability. The cytokine-dependent activation of NADPH oxidase leading to ROS generation was also confirmed to be responsible in-part for these events.

Heparan sulfate is found on the surface of most cell types, as well as in basement membranes and extracellular matrices. Its strong anionic properties and highly variable structure enable this glycosaminoglycan to provide binding sites for numerous protein ligands, including many soluble mediators of the immune system, and may promote or inhibit their activity. The formation of ligand binding sites on heparan sulfate (HS) occurs in a tissue-and context-specific fashion through the action of several families of enzymes, most of which have multiple isoforms with subtly different specificities. Changes in the expression levels of these biosynthetic enzymes occur in response to inflammatory stimuli, resulting in structurally different HS and acquisition or loss of binding sites for immune mediators. In this review, we discuss the multiple roles for HS in regulating immune responses, and the evidence for inflammation-associated changes to HS structure. K E Y W O R D Schemokines, cytokines, heparan sulfate, inflammation, leukocyte chains undergo extensive enzymatic modification beginning with Nde-acetylation/N-sulfation, and epimerization of some glucuronic acid residues to iduronic acid by the D-glycuronyl C5-epimerase (GLCE). Sequence complexity is further increased by sulfation at various

The co-involvement of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during blood-brain barrier (BBB) injury has been reported in various models of neuroinflammation, although the precise functional interplay between these archetypal proinflammatory cytokines remains largely undefined within this context. In the current paper, we tested the hypothesis that TNF-α-mediated BBB disruption is measurably attributable in-part to induction of microvascular endothelial IL-6 production. In initial experiments, we observed that treatment of human brain microvascular endothelial cells (HBMvECs) with TNF-α (0-100 ng/mL, 0-24 h) robustly elicited both time- and dose-dependent induction of IL-6 expression and release, as well as expression of the IL-6 family receptor, GP130. Further experiments demonstrated that the TNF-α-dependent generation of reactive oxygen species, down-regulation of adherens/tight junction proteins, and concomitant elevation of HBMvEC permeability, were all significantly attenuated by blockade of IL-6 signalling using either an anti-IL-6 neutralizing antibody or an IL-6 siRNA. Based on these observations, we conclude that TNF-α treatment of HBMvECs in vitro activates IL-6 production and signalling, events that were shown to synergize with TNF-α actions to elicit HBMvEC permeabilization. These novel findings offer a constructive insight into the specific contribution of downstream cytokine induction to the injurious actions of TNF-α at the BBB microvascular endothelium interface. The co-involvement of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during blood-brain barrier (BBB) injury has been widely reported. Using human brain microvascular endothelial cells (HBMvEC), we show that TNF-α-mediated BBB disruption is measurably attributable in-part to induction of endothelial IL-6 production and signalling. We demonstrate that the TNF-α-dependent generation of reactive oxygen species (ROS), down-regulation of interendothelial junctions, and concomitant elevation of HBMvEC permeability, could be significantly attenuated by using either an IL-6 neutralizing antibody or an IL-6-specific siRNA. These findings provide insight into the complex nature of proinflammatory cytokine injury at the BBB microvascular endothelium interface.

In patients with previously treated metastatic uveal melanoma, the historical 1 year overall survival rate is 37% with a median overall survival of 7.8 months. We conducted a multicenter, single-arm, open-label phase 2 study of tebentafusp, a soluble T cell receptor bispecific (gp100×CD3), in 127 patients with treatment-refractory metastatic uveal melanoma (NCT02570308). The primary endpoint was the estimation of objective response rate based on RECIST (Response Evaluation Criteria in Solid Tumours) v1.1. Secondary objectives included safety, overall survival, progression-free survival and disease control rate. All patients had at least one treatment-related adverse event, with rash (87%), pyrexia (80%) and pruritus (67%) being the most common. Toxicity was mostly mild to moderate in severity but was greatly reduced in incidence and intensity after the initial three doses. Despite a low overall response rate of 5% (95% CI: 2–10%), the 1 year overall survival rate was 62% (95% CI: 53–70%) with a median overall survival of 16.8 months (95% CI: 12.9–21.3), suggesting benefit beyond traditional radiographic-based response criteria. In an exploratory analysis, early on-treatment reduction in circulating tumour DNA was strongly associated with overall survival, even in patients with radiographic progression. Our findings indicate that tebentafusp has promising clinical activity with an acceptable safety profile in patients with previously treated metastatic uveal melanoma, and data suggesting ctDNA as an early indicator of clinical benefit from tebentafusp need confirmation in a randomized trial.