ObjectivesFibrosis is a complex pathophysiological process involving interplay between multiple cell types. Experimental modelling of fibrosis is essential for the understanding of its pathogenesis and for testing of putative antifibrotic drugs. However, most current models employ either phylogenetically distant species or rely on human cells cultured in an artificial environment. Here we evaluated the potential of vascularised in vitro human skin equivalents as a novel model of skin fibrosis and a platform for the evaluation of antifibrotic drugs.MethodsSkin equivalents were assembled on a three-dimensional extracellular matrix by sequential seeding of endothelial cells, fibroblasts and keratinocytes. Fibrotic transformation on exposure to transforming growth factor-β (TGFβ) and response to treatment with nintedanib as an established antifibrotic agent were evaluated by quantitative polymerase chain reaction (qPCR), capillary Western immunoassay, immunostaining and histology.ResultsSkin equivalents perfused at a physiological pressure formed a mature, polarised epidermis, a stratified dermis and a functional vessel system. Exposure of these models to TGFβ recapitulated key features of SSc skin with activation of TGFβ pathways, fibroblast to myofibroblast transition, increased release of collagen and excessive deposition of extracellular matrix. Treatment with the antifibrotic agent nintedanib ameliorated this fibrotic transformation.ConclusionOur data provide evidence that vascularised skin equivalents can replicate key features of fibrotic skin and may serve as a platform for evaluation of antifibrotic drugs in a pathophysiologically relevant human setting.

Bone mass is maintained by the balance between osteoclast-induced bone resorption and osteoblast-triggered bone formation. In inflammatory arthritis such as rheumatoid arthritis (RA), however, increased osteoclast differentiation and activity skew this balance resulting in progressive bone loss. O-GlcNAcylation is a posttranslational modification with attachment of a single O-linked β-D-N-acetylglucosamine (O-GlcNAc) residue to serine or threonine residues of target proteins. Although O-GlcNAcylation is one of the most common protein modifications, its role in bone homeostasis has not been systematically investigated. We demonstrate that dynamic changes in O-GlcNAcylation are required for osteoclastogenesis. Increased O-GlcNAcylation promotes osteoclast differentiation during the early stages, whereas its downregulation is required for osteoclast maturation. At the molecular level, O-GlcNAcylation affects several pathways including oxidative phosphorylation and cell-cell fusion. TNFα fosters the dynamic regulation of O-GlcNAcylation to promote osteoclastogenesis in inflammatory arthritis. Targeted pharmaceutical or genetic inhibition of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) arrests osteoclast differentiation during early stages of differentiation and during later maturation, respectively, and ameliorates bone loss in experimental arthritis. Knockdown of NUP153, an O-GlcNAcylation target, has similar effects as OGT inhibition and inhibits osteoclastogenesis. These findings highlight an important role of O-GlcNAcylation in osteoclastogenesis and may offer the potential to therapeutically interfere with pathologic bone resorption.

Background:AX-202 is a monoclonal antibody that inhibits the bioactivity of S100A4. S100A4 is an alarm signal that is released from cells in response to stress or injury and functions as an amplifying mechanism of inflammation and fibrosis in the diseased tissue microenvironment. Previous in vitro studies have found that S100A4 induces fibroblast activation, sensitizes fibroblasts to the effects of TGFβ, drives epithelial-mesenchymal transition, and stimulates monocyte cytokine release (1-3). Moreover, S100A4-/- mice are protected from fibrosis in several animal models (1). In patients with systemic sclerosis (SSc), S100A4 is elevated both in lesional tissue and systemically and correlates with skin involvement, disease activity, and pulmonary function.Objectives:The aim of this study was to assess the antifibrotic effects of murine AX-202 in two pre-clinical models of SSs reflecting both inflammation-mediated and inflammation non-mediated fibrosis and confirm the in vivo activity of humanized AX-202.Methods:We first evaluated the effects of murine AX-202 in the bleomycin-induced skin fibrosis model and the tight-skin 1 (Tsk-1) model. In the bleomycin (BLM) model, fibrosis was induced by 3 weeks of BLM s.c. injections followed by 3 weeks of AX-202 treatment in parallel with continued BLM s.c. injections. The control groups included NaCl s.c. injections for 6 weeks, BLM s.c. injections for 6 weeks, or BLM s.c. injections for 3 weeks, followed by NaCl s.c. injections for 3 weeks. Three dosing regimens of AX-202 were tested: 3.75, 7.5, or 12.5 mg/kg i.p. every 3rd day. In the Tsk-1 model, treatment with 7.5 mg/kg i.p. every 3rd day was administered from week 5 until week 10. The control groups included pa mice, Tsk-1 mice, and Tsk-1 mice treated i.p. with isotype IgG. We subsequently evaluated the effects of humanized AX-202 in the model of BLM-induced skin fibrosis in a similar design as used for the murine AX-202 study. Three dosing regimens were tested: 8 mg/kg and 16 mg/kg i.p. every 3rd day and 24 mg/kg i.v. once weekly.Results:In the BLM model, murine AX-202 (7.5 mg/kg) was effective both in the prevention of progression of pre-established skin fibrosis and in the induction of regression of fibrosis as assessed by the dermal thickness (-55%, p<0.0001 vs BLM for 6 weeks, and -23%, p<0.0001 vs BLM for 3 weeks), myofibroblast count and hydroxyproline content. Murine AX-202 also ameliorated fibrosis in the Tsk-1 model as assessed by the hypodermal thickness (-24%, p=0.01 vs Tsk-1 isotype control), myofibroblast count, and hydroxyproline content. In both models, the antifibrotic effects were associated with a reduction in pSMAD3 expression. Humanized AX-202 was effective in the prevention of progression of pre-established skin fibrosis in all doses tested across all endpoints (dermal thickness, myofibroblast counts, hydroxyproline content). In the two groups treated with 16 mg/kg i.p. and 24 mg/kg i.v., humanized AX-202 also induced regression of fibrosis (-83%, p<0.001, and -61%, p<0.001 vs BLM for 3 weeks, respectively). Both murine and humanized AX-202 were well tolerated in all study groups in both models.Conclusion:We demonstrate that AX-202 confers potent antifibrotic effects in complementary models of SSc. These results confirm and expand previous data showing that inhibition of S100A4 by AX-202 is a promising potential therapeutic candidate for disease modification in SSc or other fibrotic conditions.References:[1]Tomcik M et al. S100A4 amplifies TGF-beta-induced fibroblast activation in systemic sclerosis. Ann Rheum Dis. 2015;74(9):1748-55.[2]Cerezo LA et al. The metastasis-associated protein S100A4 promotes the inflammatory response of mononuclear cells via the TLR4 signalling pathway in rheumatoid arthritis. Rheumatology (Oxford). 2014;53(8):1520-6.[3]Fei F, et al. Role of metastasis-induced protein S100A4 in human non-tumor pathophysiologies. Cell Biosci. 2017;7:64.Acknowledgements:The study was supported by Arxx Therapeutics and MHCR 023728.Disclosure of Interests:Michal Tomčík: None declared, Thuong Trinh-Minh: None declared, Cuong Tran Manh: None declared, Hana Štorkánová: None declared, Lenka Štorkánová: None declared, Ladislav Šenolt: None declared, Jörg Klingelhöfer Employee of: Arxx Therapeutics, Rizwan I Hussain Employee of: Arxx Therapeutics, Jonas Hallén Employee of: Arxx Therapeutics, Jörg H.W. Distler Shareholder of: the stock owner of 4D Science, Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, Grant/research support from: Anamar, Active Biotech, Array Biopharma, ARXX, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB

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In this paper,  we study the convergence for martingale sequences of  random bounded operators. The condition for the existence of such a infinite product is established

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