BMP2 and mechanical loading cooperatively regulate immediate early signalling events in the BMP pathway

<p>Abstract</p> <p>Background</p> <p>Efficient osteogenic differentiation is highly dependent on coordinated signals arising from growth factor signalling and mechanical forces. Bone morphogenetic proteins (BMPs) are secreted proteins that trigger Smad and non-Smad pathways and thereby influence transcriptional and non-transcriptional differentiation cues. Crosstalk at multiple levels allows for promotion or attenuation of signalling intensity and specificity. Similar to BMPs, mechanical stimulation enhances bone formation. However, the molecular mechanism by which mechanical forces crosstalk to biochemical signals is still unclear.</p> <p>Results</p> <p>Here, we use a three-dimensional bioreactor system to describe how mechanical forces are integrated into the BMP pathway. Time-dependent phosphorylation of Smad, mitogen-activated protein kinases and Akt in human fetal osteoblasts was investigated under loading and/or BMP2 stimulation conditions. The phosphorylation of R-Smads is increased both in intensity and duration under BMP2 stimulation with concurrent mechanical loading. Interestingly, the synergistic effect of both stimuli on immediate early Smad phosphorylation is reflected in the transcription of only a subset of BMP target genes, while others are differently affected. Together this results in a cooperative regulation of osteogenesis that is guided by both signalling pathways.</p> <p>Conclusions</p> <p>Mechanical signals are integrated into the BMP signalling pathway by enhancing immediate early steps within the Smad pathway, independent of autocrine ligand secretion. This suggests a direct crosstalk of both mechanotransduction and BMP signalling, most likely at the level of the cell surface receptors. Furthermore, the crosstalk of both pathways over longer time periods might occur on several signalling levels.</p

Portal Hypertension Promotes Bacterial Translocation in Rats Mono- and Non Mono-Associated with Escherichia Coli C25

The basis for the high incidence of infectious complications in portal hypertension (PHT) remains unclear. The hypothesis that PHT induces bacterial translocation (BT) was tested in a rat model with or without mono-association with streptomycin resistant Escherichia coli C25 and with or without hypovolemic shock. PHT was achieved by partial portal vein ligation and three weeks later hypovolemic shock (HS) was induced. Blood, liver, spleen and mesenteric lymph nodes cultures were performed twenty-four hours later

Load‐induced osteogenic differentiation of mesenchymal stromal cells is caused by mechano‐regulated autocrine signaling

Mechanical boundary conditions critically influence the bone healing process. In this context, previous in vitro studies have demonstrated that cyclic mechanical compression alters migration and triggers osteogenesis of mesenchymal stromal cells (MSC), both processes being relevant to healing. However, it remains unclear whether this mechanosensitivity is a direct consequence of cyclic compression, an indirect effect of altered supply or a specific modulation of autocrine bone morphogenetic protein (BMP) signaling. Here, we investigate the influence of cyclic mechanical compression (ε = 5% and 10%, f = 1 Hz) on human bone marrow MSC (hBMSC) migration and osteogenic differentiation in a 3D biomaterial scaffold, an in vitro system mimicking the mechanical environment of the early bone healing phase. The open-porous architecture of the scaffold ensured sufficient supply even without cyclic compression, minimizing load-associated supply alterations. Furthermore, a large culture medium volume in relation to the cell number diminished autocrine signaling. Migration of hBMSCs was significantly downregulated under cyclic compression. Surprisingly, a decrease in migration was not associated with increased osteogenic differentiation of hBMSCs, as the expression of RUNX2 and osteocalcin decreased. In contrast, BMP2 expression was significantly upregulated. Enabling autocrine stimulation by increasing the cell-to-medium ratio in the bioreactor finally resulted in a significant upregulation of RUNX2 in response to cyclic compression, which could be reversed by rhNoggin treatment. The results indicate that osteogenesis is promoted by cyclic compression when cells condition their environment with BMP. Our findings highlight the importance of mutual interactions between mechanical forces and BMP signaling in controlling osteogenic differentiation

Pharmacodynamics of interferon beta in multiple sclerosis patients with or without serum neutralizing antibodies

Abstract : To analyze the in vivo biological effect of anti-interferon beta (IFN-beta) neutralizing antibodies (NABs), blood concentrations of neopterin, beta2microglobulin (Beta2-MG), mRNA-dependent myxovirusresistant protein A (MxA) and dsRNA-dependent protein kinase (PKR) were measured before (predose) and 24 hours after (postdose) IFN-beta administration in 49 patients with multiple sclerosis (MS) with (n = 25) and without (n = 24) NABs. The results indicated that predose levels of MxA-mRNA and PKR-mRNA were highly variable [coefficient of variation (CV) > 100%] among patients. A lower inter-individual variability was observed for pre-dose levels of Beta2-MG and neopterin (CVs of 29% and 44%, respectively). Significantly lower pre- and post-dose blood levels of IFN induced markers, except for postdose PKR-mRNA (p = 0.09), were seen in NAB+ compared with NAB-patients and between patients with high (> 200 t1/10) and low (£ 200 t1/10) NAB titers. A significant inverse correlation between NAB titer and pre-dose levels of the above IFN-induced markers was found. In summary, our findings confirm that NABs affect absolute concentrations of IFN-beta induced markers and suggest that such an effect occurs in a titer-dependent manne

Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo-expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank (n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics

Intramuscular and intratendinous placenta‐derived mesenchymal stromal‐like cell treatment of a chronic quadriceps tendon rupture

Background: Quadriceps tendon ruptures (QTRs) are rare but debilitating injuries, often associated with chronic metabolic conditions or long-term steroid treatment. While the surgical treatment for acute QTRs is described thoroughly, no common strategy exists for the often frustrating treatment of chronic, reoccurring QTRs. The pro-angiogenic and immunomodulatory properties of placenta-derived adherent mesenchymal stromal-like (PLX-PAD) cells have been described to protect musculoskeletal tissues from inflammation and catabolic cytokine migration, yet little is known about the regenerative potential of PLX-PAD cells in repetitively damaged tendon tissue. Case: We report the case of an 80-year-old male patient with a chronic three-time QTR of his right knee. The quadriceps tendon was reconstructed applying a conventional suture anchor repair procedure combined with a synthetic mesh augmentation and additional intramuscular and intratendineous PLX-PAD cell injections as an individualized treatment approach. No adverse events were reported, and excellent radiological and functional outcomes with a passive range of motion of 0/0/120 degrees knee extension-flexion were observed at the 12 month follow-up. Gait analysis confirmed restoration of joint motion, including gait speed, deficit in step length, and knee extensor muscle strength (pre-surgery: 0.98 m/s, 40 cm, 42.4 +/- 12.4 N; 9 months post-surgery: 1.07 m/s, 0 cm, 10.4 +/- 18.9 N) as well as hyperextension throughout stance and late swing phases (pre-surgery: -11.2 +/- 0.9 degrees; 9 months post-surgery: -2.7 +/- 1.6 degrees). Postoperative lymphocyte and cytokine analyses from the patient's peripheral blood serum suggested a systemic short-term immunoregulatory reaction with postoperatively increased interleukin (IL)-6 (pre-surgery: 0.79 pg/mL; day 1: 139.97 pg/mL; day 5: 5.58 pg/mL; 9 months: 1.76 pg/mL) and IL-10 (pre-surgery: 0.9 pg/mL; day 1: 1.21 pg/ mL; day 5: 0.3 pg/mL; 9 months: 0.34 pg/mL) levels that decreased again over time. Conclusions: Herein, we demonstrate a successfully treated chronic QTR with a synergistic surgical and biological reconstructive treatment approach. This local add-on treatment with PLX-PAD cells may be considered in specific cases of chronic QTRs, not susceptible to traditional suture anchor procedures and which exhibit a high risk of treatment failure. Further scientific engagement is warranted to explore underlying immunomodulatory mechanisms of action behind PLX-PAD cell treatment for tendon injuries

MS disease activity in RESTORE: a randomized 24-week natalizumab treatment interruption study

Objective: RESTORE was a randomized, partially placebo-controlled exploratory study evaluating multiple sclerosis (MS) disease activity during a 24-week interruption of natalizumab. Methods: eligible patients were relapse-free through the prior year on natalizumab and had no gadolinium-enhancing lesions on screening brain MRI. Patients were randomized 1:1:2 to continue natalizumab, to switch to placebo, or to receive alternative immunomodulatory therapy (other therapies: IM interferon β-1a [IM IFN-β-1a], glatiramer acetate [GA], or methylprednisolone [MP]). During the 24-week randomized treatment period, patients underwent clinical and MRI assessments every 4 weeks. Results: patients (n = 175) were randomized to natalizumab (n = 45), placebo (n = 42), or other therapies (n = 88: IM IFN-β-1a, n = 17; GA, n = 17; MP, n = 54). Of 167 patients evaluable for efficacy, 49 (29%) had MRI disease activity recurrence: 0/45 (0%) natalizumab, 19/41 (46%) placebo, 1/14 (7%) IM IFN-β-1a, 8/15 (53%) GA, and 21/52 (40%) MP. Relapse occurred in 4% of natalizumab patients and in 15%-29% of patients in the other treatment arms. MRI disease activity recurred starting at 12 weeks (n = 3 at week 12) while relapses were reported as early as 4-8 weeks (n = 2 in weeks 4-8) after the last natalizumab dose. Overall, 50/167 patients (30%), all in placebo or other-therapies groups, restarted natalizumab early because of disease activity. Conclusions: MRI and clinical disease activity recurred in some patients during natalizumab interruption, despite use of other therapies. Classification of evidence: this study provides Class II evidence that for patients with MS taking natalizumab who are relapse-free for 1 year, stopping natalizumab increases the risk of MS relapse or MRI disease activity as compared with continuing natalizumab

The Influence of Recovery and Training Phases on Body Composition, Peripheral Vascular Function and Immune System of Professional Soccer Players

Professional soccer players have a lengthy playing season, throughout which high levels of physical stress are maintained. The following recuperation period, before starting the next pre-season training phase, is generally considered short but sufficient to allow a decrease in these stress levels and therefore a reduction in the propensity for injury or musculoskeletal tissue damage. We hypothesised that these physical extremes influence the body composition, blood flow, and endothelial/immune function, but that the recuperation may be insufficient to allow a reduction of tissue stress damage. Ten professional football players were examined at the end of the playing season, at the end of the season intermission, and after the next pre-season endurance training. Peripheral blood flow and body composition were assessed using venous occlusion plethysmography and DEXA scanning respectively. In addition, selected inflammatory and immune parameters were analysed from blood samples. Following the recuperation period a significant decrease of lean body mass from 74.4±4.2 kg to 72.2±3.9 kg was observed, but an increase of fat mass from 10.3±5.6 kg to 11.1±5.4 kg, almost completely reversed the changes seen in the pre-season training phase. Remarkably, both resting and post-ischemic blood flow (7.3±3.4 and 26.0±6.3 ml/100 ml/min) respectively, were strongly reduced during the playing and training stress phases, but both parameters increased to normal levels (9.0±2.7 and 33.9±7.6 ml/100 ml/min) during the season intermission. Recovery was also characterized by rising levels of serum creatinine, granulocytes count, total IL-8, serum nitrate, ferritin, and bilirubin. These data suggest a compensated hypo-perfusion of muscle during the playing season, followed by an intramuscular ischemia/reperfusion syndrome during the recovery phase that is associated with muscle protein turnover and inflammatory endothelial reaction, as demonstrated by iNOS and HO-1 activation, as well as IL-8 release. The data provided from this study suggest that the immune system is not able to function fully during periods of high physical stress. The implications of this study are that recuperation should be carefully monitored in athletes who undergo intensive training over extended periods, but that these parameters may also prove useful for determining an individual's risk of tissue stress and possibly their susceptibility to progressive tissue damage or injury

Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation

IntroductionThe complement system is a key component of the innate immune system, and its aberrant activation underlies the pathophysiology of various diseases. Zilucoplan is a macrocyclic peptide that binds and inhibits the cleavage/activation of human complement component 5 (C5). We present in vitro and ex vivo data on the mechanism of action of zilucoplan for the inhibition of C5 activation, including two clinically relevant C5 polymorphisms at R885.MethodsThe interaction of zilucoplan with C5, including for clinical C5 R885 variants, was investigated using surface plasmon resonance (SPR), hemolysis assays, and ELISA. The interference of C5b6 formation by zilucoplan was investigated by native gel analysis and hemolysis assay. The permeability of zilucoplan in a reconstituted basement membrane was assessed by the partition of zilucoplan on Matrigel-coated transwell chambers.ResultsZilucoplan specifically bound human complement C5 with high affinity, competitively inhibited the binding of C5 to C3b, and blocked C5 cleavage by C5 convertases and the assembly of the cytolytic membrane attack complex (MAC, or C5b9). Zilucoplan fully prevented the in vitro activation of C5 clinical variants at R885 that have been previously reported to respond poorly to eculizumab treatment. Zilucoplan was further demonstrated to interfere with the formation of C5b6 and inhibit red blood cell (RBC) hemolysis induced by plasmin-mediated non-canonical C5 activation. Zilucoplan demonstrated greater permeability than a monoclonal C5 antibody in a reconstituted basement membrane model, providing a rationale for the rapid onset of action of zilucoplan observed in clinical studies.ConclusionOur findings demonstrate that zilucoplan uses a dual mode of action to potently inhibit the activation of C5 and terminal complement pathway including wild-type and clinical R885 variants that do not respond to eculizumab treatment. These data may be relevant to the clinically demonstrated benefits of zilucoplan