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Key Points TNF inhibitors are among the most effective protein-based drugs for reducing inflammation associated with several rheumatic diseases In addition to TNF, the TNF superfamily (TNFSF) comprises other ligand–receptor combinations that might participate in the pathogenesis of rheumatic disease TNFSF members initiate several processes, including immune activation, tissue inflammatory responses and cell death or suppression Many TNFSF proteins other than TNF are being evaluated in preclinical mouse or human studies as possible therapeutic targets in rheumatic diseases TNFSF members can be targeted to either restore tolerance in rheumatic diseases or to regulate tissue cell responses In this Review, the authors discuss the function of the TNF and TNF receptor superfamily, their role in rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus, and how current knowledge is being translated into potential disease therapies. TNF blockers are highly efficacious at dampening inflammation and reducing symptoms in rheumatic diseases such as rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis, and also in nonrheumatic syndromes such as inflammatory bowel disease. As TNF belongs to a superfamily of 19 structurally related proteins that have both proinflammatory and anti-inflammatory activity, reagents that disrupt the interaction between proinflammatory TNF family cytokines and their receptors, or agonize the anti-inflammatory receptors, are being considered for the treatment of rheumatic diseases. Biologic agents that block B cell activating factor (BAFF) and receptor activator of nuclear factor-κB ligand (RANKL) have been approved for the treatment of systemic lupus erythematosus and osteoporosis, respectively. In this Review, we focus on additional members of the TNF superfamily that could be relevant for the pathogenesis of rheumatic disease, including those that can strongly promote activity of immune cells or increase activity of tissue cells, as well as those that promote death pathways and might limit inflammation. We examine preclinical mouse and human data linking these molecules to the control of damage in the joints, muscle, bone or other tissues, and discuss their potential as targets for future therapy of rheumatic diseases.

IntroductionAngiopoietin-2 (Ang2) is a secreted ligand whose concentration is increased in several cardiovascular diseases, and which causes vascular inflammation, microvessel disintegration, cardiac fibrosis and myocardial damage. Ang2 binds the endothelial receptor Tie2, where it competes for binding with the protective ligand Ang1. In this study we aim to develop a ligand-trap to block Ang2 action. To do this we used directed protein evolution to change the binding specificity of the Tie2 ectodomain so that it specifically binds Ang2. The evolved ectodomain will then be used as a soluble trap for introduction into the circulation to bind and sequester Ang2 preventing it from exerting its effects on the endothelium.Methods and resultsTie2 ectodomain was evolved using a novel DT40 cell surface display and evolution system. Evolutions were performed for selective binding to Ang2 by iterative cycles of mutation and selection. Variants evolved for Ang2 binding were then sequenced revealing key residues in the Ang1/2 binding domain determining ligand specificity. Fusion proteins containing the evolved variants were expressed, purified and tested for their ability to selectively bind Ang2, rather than Ang1 and Ang4. These Ang2-selective ligand-traps were found to inhibit Ang2 action on endothelial cells, inhibit adhesion of platelet/leucocyte aggregates to endothelial monolayers and suppress LPS-induced oedema.Conclusions and implicationsUsing a novel method of cell surface display and directed evolution we have evolved Tie2 ectodomain to selectively bind Ang2. This has revealed the key amino acid residues determining ligand-binding specificity of Tie2. In addition we created a series of selective Ang2 ligand-traps. These traps are almost identical to the endogenous receptor ectodomain, differing in four or fewer amino acid residues from endogenous ectodomain, and are able to inhibit the actions of Ang2. These ligand-traps have the potential for development as therapeutics to block the pathogenic and inflammatory actions of Ang2 on the cardiovascular system.

Absorption of ultraviolet B (UVB) radiation by the B ring of 7-dehydrocholesterol (7-DHC) leads to the transformation of 7-DHC to previtamin D3, which after absorption of additional UVB isomerizes to tachysterol3 (T3) and lumisterol3 (L3). Previously we demonstrated that CYP11A1 can hydroxylate the side chain of vitamin D3 (D3), 7-DHC and L3. Similarly CYP27A1 can hydroxylate the side chain of L3 to biologically active hydroxyderivatives. In a continuation of these studies, we report that CYP11A1 and CYP27A1 hydroxylate T3 to 20S(OH)T3 and 25(OH)T3, respectively, plus minor unidentified hydroxyderivatives. Both 20S(OH)T3 and 25(OH)T3were detected in the human epidermis and serum. T3 was also detected in human serum and was present at a concentration of 7.3±2.5 ng/ml. 20S(OH)T3 and 25(OH)T3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti-oxidative genes in keratinocytes in a similar manner to 1,25-dihydroxyvitamin D3 . They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR-coupled GFP (VDR-GFP) from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay revealed marked activation of AhR by 20S(OH)T3, a smaller effect by 25(OH)T3 and only minimal activation by T3. The T3 hydroxyderivatives showed high affinity binding to the ligand binding domain (LBD) of the liver X receptor (LXR) α and β, and to the peroxisome proliferator-activated receptor γ (PPARg) in LanthaScreen TR-FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs and PPARγ revealed high docking scores for 20S(OH)T3 and 25(OH)T3, comparable to their previously characterized ligands. The scores for binding to the non-genomic site of the VDR were very low indicating a lack of interaction with hydroxy-T3 ligands. In conclusion, we have identified 20S(OH)T3 and 25(OH)T3 in the human epidermis and serum, identified CYP enzymes responsible for their production, demonstrated phenotypic effects on skin cells, and identified VDR, AhR, LXRs and PPARγ, and possibly RORs, as their genomic receptor targets. Thus CYP11A1, aside from its role in steroidogenesis, not only metabolizes vitamin D3, 7-DHC and L3 to biologically active metabolites, but also activates T3 to biologically active 20S(OH)T3. Similarly, CYP27A1, previously reported to act on L3, D3 and 7DHC, also activates T3 via hydroxylation at C25. We believe that these novel findings open new areas for future studies on the role of active forms of T3, not only in the skin, but also in systemic physiology and pathology. Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

BackgroundAutologous monocyte-derived mRNA co-electroporated dendritic cells with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively activated TLR4 (caTLR4) (referred to as TriMixDC-MEL) have anti-tumor activity in advanced melanoma patients. We investigated the safety and activity of adjuvant TriMixDC-MEL in stage III/IV melanoma patients.Materials and methodsForty-one patients were randomly assigned to treatment with TriMixDC-MEL (n = 21) and standard follow-up (n = 20). “Cross-over” was allowed at the time of non-salvageable recurrence. The primary endpoint was the percentage of patients alive and disease-free at 1-year. For a subset of patients, (formalin-fixed paraffin-embedded), tumor tissue samples were available for mRNA expression profiling and PD-L1 immunohistochemical staining.ResultsBaseline characteristics were well balanced. One-year after randomization, 71% of patients in the study arm were alive and free of disease compared to 35% in the control arm. After a median follow-up of 53 months (range 3–67), 23 patients experienced a non-salvageable melanoma recurrence (TriMixDC-Mel arm n = 9 and control arm n = 14).The median time to non-salvageable recurrence was superior in the TriMixDC-MEL arm (median 8 months (range 1–6) vs. not reached; log-rank p 0.044). TriMixDC-MEL-related adverse events (AE) consisted of transient local skin reactions, flu-like symptoms and post-infusion chills. No grade ≥ 3 AE’s occurred. The mRNA expression profiling revealed four genes (STAT2, TPSAB1, CD9 and CSF2) as potential predictive biomarkers.ConclusionTriMixDC-MEL id/iv as adjuvant therapy is tolerable and may improve the 1-year disease-free survival rate. Combination of optimized autologous monocyte-derived DC-formulations warrants further investigation in combination with currently approved adjuvant therapy options.

APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition.

In this trial involving patients with IgA nephropathy, sibeprenlimab, a humanized IgG2 monoclonal antibody that blocks a proliferation-inducing ligand, resulted in a greater decrease in proteinuria than placebo.