The superantigens staphylococcal enterotoxin A and E (SEA and SEE) can activate a large number of T-cells. SEA and SEE have approximately 80% sequence identity but show some differences in their biological function. Here, the two superantigens and analogues were characterized biophysically. SEE was shown to have a substantially higher thermal stability than SEA. Both SEA and SEE were thermally stabilized by 0.1 mM Zn(2+) compared with Zn(2+)-reduced conditions achieved using 1 mM EDTA or specific replacements that affect Zn(2+) coordination. The higher stability of SEE was only partly caused by the T-cell receptor (TCR) binding regions, whereas regions in the vicinity of the major histocompatibility complex class II binding sites affected the stability to a greater extent. SEE exhibited a biphasic denaturation between pH 5.0-6.5, influenced by residues in the TCR binding regions. Interestingly, enzyme-linked immunosorbent assay, isoelectric focusing, and circular dichroism analysis indicated that conformational changes had occurred in the SEA/E chimerical constructs relative to SEA and SEE. Thus, it is proposed that the Zn(2+) binding site is very important for the stability and potency of SEA and SEE, whereas residues in the TCR binding site have a substantial influence on the molecular conformation to control specificity and function.
Background:Serotonin or 5-hydroxytryptamine (5-HT) is well known as a stimulator of tissue fibrosis and a significant role of peripheral 5-HT2B receptors in fibrosis has been suggested with the receptor being upregulated in fibrotic tissues. In addition, agonism of the 5-HT2B receptor has been implicated in human tissue fibrosis caused by drugs known to activate the receptor. Pharmacologic inhibition of 5-HT2B receptor signalling consequently represents a promising treatment strategy for fibrotic disorders including systemic sclerosis. 5-HT is released from platelets activated upon vascular damage, one of the first pathological events in systemic sclerosis. The local 5-HT concentration is increased and leads to activation of 5-HT2B receptors on e.g. fibroblasts. The pro-fibrotic effects of 5-HT and the 5-HT2B receptor are believed to be mediated through activation of the TGF-β/Smad signaling pathway.Objectives:The objective of the present study was to evaluate a novel highly selective orally available 5-HT2B receptor antagonist, AM1476, for its ability to reduce pulmonary and dermal fibrosis in the sclerodermatous chronic graft-versus-host disease model and dermal fibrosis in the tight-skin-1 model of systemic sclerosis. Effects on the TGF-β/Smad signaling pathway were investigated in vivo. Exposure of AM1476 was measured to ensure proper target engagement.Methods:The murine sclerodermatous chronic graft-versus-host disease (cGvHD) model was used to evaluate anti-fibrotic effects after therapeutic dosing of the 5-HT2B receptor antagonist, AM1476. The compound was orally administered at 1, 10 and 30 mg/kg b.i.d. from day 21, several days after the first clinical signs of cGvHD, to day 49. Dermal thickness, myofibroblast counts and collagen production were used to evaluate dermal fibrosis. Effects on pulmonary fibrosis were measured using hydroxyproline content, Sirius Red staining and Ashcroft score. Plasma was collected for PK analysis at different timepoints in each treatment group using sparse sampling, on the last day of the experiment. The tight-skin-1 model was used to evaluate anti-fibrotic effects after therapeutic treatment. AM1476 was orally administered at 10 mg/kg, b.i.d. from week 5 to week 10. Hypodermal thickening, myofibroblast counts and hydroxyproline content in skin biopsies were evaluated at the end of the treatment period. The number of phosphorylated Smad3 (pSmad3) positive cells was used to evaluate inhibition of TGF-β signaling.Results:The 5-HT2B receptor antagonist AM1476, significantly reduced all measured dermal and pulmonary fibrosis readouts in the cGvHD model using an oral therapeutic treatment approach. Pharmacokinetic analysis of plasma samples supported 5-HT2B receptor engagement. Therapeutic treatment of dermal fibrosis in the tight-skin model effectively and significantly reduced hypodermal thickening, number of myofibroblast and hydroxyproline content. The number of pSmad3 positive cells was significantly reduced in skin samples isolated from treated animals.Conclusion:Inhibition...
Background: The microvascular damage is a pivotal event in the pathogenesis of Systemic Sclerosis (SSc) and, after injury, both endothelial cells (ECs) and pericytes might trans-differentiate toward myofibroblast, responsible of fibrosis. Platelet-derived growth factor B (PDGFB) and transforming growth factorβ (TGFβ) play a key role in SSc pathogenesis. PDGFB is a potent mitogen for myofibroblasts, while TGFβ stimulate myofibroblast activation, including alpha smooth muscle actin (αSMA) expression. A key regulator of PDGFB and TGFβ signaling may be the CD248, a trans-membrane receptor required for proliferation and migration of pericytes and fibroblasts. It has been showed that, in an animal model of kidney fibrosis, the genetic deletion of CD248 modulates the response of renal pericytes to injury, by reducing the differentiation of myofibroblasts. The expression of CD248 is required for TGFβ-induced αSMA expression in pericytes and CD248 enhances the PDGFB pathway, mediating the proliferation and migration of perivascular cells. Objectives: The aim of this work was to evaluate the expression of CD248, in SSc skin biopsies and its possible role in perivascular stromal cells proliferation, responsible to myofibroblast trans-differentiation, during SSc. Methods: After ethical approval, skin biopsies and bone marrow mesenchymal stem cells (MSCs) were collected from 20 diffuse SSc patients and 10 healthy control (HC). CD248 expression was investigated in the skin, and in isolated MSCs treated with TGFβ or PDGFB, by immunofluorescence, qRT-PCR and western. Furthermore, we silenced CD248 in SSc-MSCs, to confirm the role of this molecule in TGFβ-or PDGFB-signaling modulation. Results: CD248 expression in SSc skin was significantly higher when compared with HC skin. In particular, an increased expression of CD248 was found in ECs, stromal fibroblast and perivascular like stromal cells, co-expressing CD90, a marker of un-differentiated MSCs. Furthermore, in both, HC-and SSc-MSCs, TGFβ treatment induced a significant reduction of CD248 mRNA expression, in parallel with a significant increase of αSMA and a decrease of proliferation (ki67), when compared with untreated-(UT-) cells. Interestingly, the ability of TGFβ to inhibit CD248 expression in HC-MSCs was significantly higher than SSc-MSCs, suggesting that local environment in SSc patients affect TGFβ ability to suppress CD248 expression in SSc-MSCs. After treatment with PDGFB in both SSc-and HC-MSCs, CD248 expression was not affected, while significant reduction of αSMA and an increased expression of Ki67 was observed compared with UT-cells. After silencing of CD248 in SSc-MSCs, both TGFβ and PDGFB signaling were inhibited. Conclusions: CD248 over-expression may play an important role in tissue fibrosis by modulating the pericytes to myofibroblast trans-differentiation, via regulation of both PDGFB and TGFβ signaling, during SSc. References:
Background Serotonin (5-HT) is well known as a central neurotransmitter, but has important functions also in the periphery, e.g. as a regulator of vascular tone, platelet aggregation and cytokine expression. Objectives The aim of this study was to investigate the potential influence of selective 5-HT2B receptor antagonists on inflammation and inflammatory pain. For this purpose, AnaMar´s proprietary compounds were studied in vitro and in vivo. Methods In vitro, IL-6 production: Primary synoviocytes were isolated from pannus tissue obtained from female, Dark Agouti rats with antigen-induced arthritis and used for experiments after seven days in culture. The cells were stimulated with 5-HT and LPS, in the presence of the 5-HT2B receptor antagonists (0.1-10 µM). After three days incubation, supernatants were collected and the IL-6 content measured with ELISA. In vivo, systemic inflammation: Female BALB/c mice were pre-treated perorally with the 5-HT2B receptor antagonists (3-30 mg/kg). Thirty minutes later, LPS was administered systemically by intra-peritoneal injection. After 90 minutes, blood was collected and cytokines measured in plasma with ELISA. In vivo, inflammatory pain: Male Sprague-Dawley rats were pre-treated perorally with the 5-HT2B receptor antagonists (3-30 mg/kg). Sixty minutes later, formalin was injected subcutaneously into the dorsum of the right hind paw to induce pain. Pain responses were recorded by measuring the cumulative time of nociceptive behaviour per unit of time. Results AnaMar´s 5-HT2B receptor antagonists dose-dependently decreased the production of IL-6 from primary rat synoviocytes. Further, the compounds potently decreased the production of TNF-α triggered by systemic LPS injection in mice. In the inflammatory pain model, the compounds significantly reduced the pain response. Conclusions The 5-HT2B receptor antagonists reduced inflammatory cytokine production and pain responses. The results strongly support that approaches targeting 5-HT2B receptor signaling could have therapeutic potential in conditions associated with inflammation and inflammatory pain. It remains to be investigated whether the effect on inflammatory pain is mediated through a suppressed production of inflammatory cytokines, a direct effect on peripheral neurons, or a combination of these. References Mössner R, Lesch KP: Role of serotonin in the immune system and in neuroimmune interactions, Brain Behav Immun 1998, 12 (4): 249-271. Disclosure of Interest N. Palmqvist Employee of: AnaMar AB, C. Wenglén Employee of: AnaMar AB, A. Sjödin Employee of: AnaMar AB, A.-C. Ryde Employee of: AnaMar AB, M. Siller Employee of: AnaMar AB, H. Arozenius Employee of: AnaMar AB, A. Mathisson Employee of: AnaMar AB, C. Klint Employee of: AnaMar AB, A. Pramhed Employee of: AnaMar AB, L. Pettersson Employee of: AnaMar AB, G. Ekström Employee of: AnaMar AB
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