Role of IL-33 and ST2 signalling pathway in multiple sclerosis: expression by oligodendrocytes and inhibition of myelination in central nervous system

Recent research findings have provided convincing evidence indicating a role for Interleukin-33 (IL-33) signalling pathway in a number of central nervous system (CNS) diseases including multiple sclerosis (MS) and Alzheimer’s disease. However, the exact function of IL-33 molecule within the CNS under normal and pathological conditions is currently unknown. In this study, we have mapped cellular expression of IL-33 and its receptor ST2 by immunohistochemistry in the brain tissues of MS patients and appropriate controls; and investigated the functional significance of these findings in vitro using a myelinating culture system. Our results demonstrate that IL-33 is expressed by neurons, astrocytes and microglia as well as oligodendrocytes, while ST2 is expressed in the lesions by oligodendrocytes and within and around axons. Furthermore, the expression levels and patterns of IL-33 and ST2 in the lesions of acute and chronic MS patient brain samples are enhanced compared with the healthy brain tissues. Finally, our data using rat myelinating co-cultures suggest that IL-33 may play an important role in MS development by inhibiting CNS myelination

Fibroblast growth factor signalling in multiple sclerosis: inhibition of myelination and induction of pro-inflammatory environment by FGF9

The failure of remyelination in multiple sclerosis is largely unexplained. Lindner et al. report that glial cells in demyelinating lesions show increased expression of fibroblast growth factor 9 (FGF9). This induces astrocyte-dependent responses that inhibit remyelination and stimulate expression of pro-inflammatory chemokines, supporting a feedback loop that amplifies disease activit

Fibroblast growth factor signalling in multiple sclerosis:inhibition of myelination and induction of pro-inflammatory environment by FGF9

Remyelination failure plays an important role in the pathophysiology of multiple sclerosis, but the underlying cellular and molecular mechanisms remain poorly understood. We now report actively demyelinating lesions in patients with multiple sclerosis are associated with increased glial expression of fibroblast growth factor 9 (FGF9), which we demonstrate inhibits myelination and remyelination in vitro. This inhibitory activity is associated with the appearance of multi-branched ‘pre-myelinating’ MBP+/PLP+ oligodendrocytes that interact with axons but fail to assemble myelin sheaths; an oligodendrocyte phenotype described previously in chronically demyelinated multiple sclerosis lesions. This inhibitory activity is not due to a direct effect of FGF9 on cells of the oligodendrocyte lineage but is mediated by factors secreted by astrocytes. Transcriptional profiling and functional validation studies demonstrate that these include effects dependent on increased expression of tissue inhibitor of metalloproteinase-sensitive proteases, enzymes more commonly associated with extracellular matrix remodelling. Further, we found that FGF9 induces expression of Ccl2 and Ccl7, two pro-inflammatory chemokines that contribute to recruitment of microglia and macrophages into multiple sclerosis lesions. These data indicate glial expression of FGF9 can initiate a complex astrocyte-dependent response that contributes to two distinct pathogenic pathways involved in the development of multiple sclerosis lesions. Namely, induction of a pro-inflammatory environment and failure of remyelination; a combination of effects predicted to exacerbate axonal injury and loss in patients

Oxidative tissue injury in experimental disease models of multiple sclerosis

Multiple Sklerose (MS) ist eine chronische durch Demyelinisierung gekennzeichnete entzündliche Erkrankung des zentralen Nervensystems (ZNS). Große Fortschritte konnten in der Aufklärung der Entzündung und Neurodegeneration zugrunde liegenden Mechanismen erzielt werden. Eine Vielzahl von verschiedenen Tiermodellen trug zu einem besseren Verständnis der MS-Pathologie und fundamentalen immunologischen Konzepten bei. Außerdem ermöglichten experimentelle Modelle die Entwicklung von immunmodulatorischen Therapien für MS Patienten. Die Forschung der letzten Jahre erbrachte wichtige Hinweise dafür, dass oxidativer Schaden eine wichtige Rolle im Hinblick auf die Neurodegeneration in der MS spielt. Daher war unser Ziel, die Beschaffenheit und das Ausmaß von oxidativem Schaden in verschiedenen Tiermodellen zu ermitteln und direkt mit MS Läsionen zu vergleichen. Hierbei konzentrierten wir uns auf die Expression von freien Radikale-produzierenden Enzymen (die essentielle Untereinheit der NADPH Oxidase p22phox und die induzierbare Stickstoffmonoxid Synthetase, iNOS), die Anreicherung von oxidierten Phospholipiden und Eisen, das oxidativen Schaden verstärken kann. Die Erkrankungen in den verschiedenen Tiermodellen wurden durch unterschiedliche inflammatorische Mechanismen induziert, die jeweils andere Aspekte der MS Pathologie repräsentieren. Das einzige Modell, in dem wir annähernd so viel oxidativen Schaden wie in MS Läsionen detektierten, war die durch Coronavirus-Infektion hervorgerufene demyelinisierende Encephalomyelitis. In der MS als auch im Coronavirus-Modell war der oxidative Schaden mit einer massiven Aktivierung der Mikroglia und Makrophagen verbunden. Diese zeichnete sich durch ausgeprägte Expression von p22phox und geringes Vorkommen von iNOS aus. Das zweite in unsere Studie eingeschlossene Virusmodell war eine chronische Demyelinisierung, die durch Theilers Virus (TMEV) verursacht wurde. In diesem Fall erreichte jedoch die Expression von p22phox in aktivierten Mikroglia und Makrophagen nicht das Ausmaß des Coronavirus-Modells. Außerdem konnten wir im TMEV-Modell oxidative Schaden zwar detektieren, aber zu einem geringeren Grad als im Coronavirus-Modell. Die iNOS Expression erschien in beiden Modellen ähnlich gering. Läsionen, die durch den passiven Transfer von T-Zellen hervorgerufenen wurden, zeigten massive p22phox und iNOS Expression in der akuten Phase der Entzündung. Ähnlich dazu fanden wir p22phox exprimierende Makrophagen in Tieren mit chronischen Läsionen, die durch T-Zellen und Makrophagen hervorgerufen wurden, aber im Gegensatz dazu wenige iNOS positive Zellen. Chronische Läsionen von Antikörpern mediierter Demyelinisierung enthielten phagozytisch aktive Makrophagen, die aber keine Expression von p22phox oder iNOS zeigten. In akuten Läsionen, die durch den Transfer von CD8+ T-Zellen oder Mechanismen des angeborenen Immunsystems hervorgerufen wurden, detektieren wir die Expression von p22phox in Mikroglia und Makrophagen. Im Gegensatz dazu war iNOS nur geringfügig zu finden. Zusätzlich zum oxidativem Schaden und den verschiedenen Expressionsmustern von p22phox und iNOS, untersuchten wir auch den Einfluss von Eisen in den jeweiligen experimentellen Modellen. Die alters-abhängige Eisenanreicherung in Oligodendrozyten und dessen Freisetzung, die man in MS Läsionen findet, werden nur wenig in Tiermodellen widergespiegelt. Wir versuchten die Rolle von Eisen in der Neurodegeneration mittels Zellkultur zu studieren, konnten eine Eisen Anreicherung aber nur in Mikroglia und nicht in Oligodendrozyten hervorrufen. Unsere Studie zeigt, dass in den verschiedenen Tiermodellen der MS auch ein unterschiedliches Muster von oxidativem Schaden zu finden ist, dessen Ausmaß aber in den wenigsten Fällen jenes der MS erreicht. Dies liegt möglicherweise daran, dass den Tiermodellen mögliche Amplifizierungsmechanismen fehlen, die in der MS Pathogenese eine wichtige Rolle spielen.Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) characterised by inflammation and demyelination. Major advances have been made in unravelling the mechanisms of inflammatory and neurodegenerative processes underlying the disease. A number of different animal models of MS contributed not only to a better understanding of MS pathology but also explained fundamental immunological concepts. Additionally, experimental models enabled the development of immune-modulatory therapies for MS patients. In recent years, growing evidence for a major role of oxidative injury in demyelination and neurodegeneration in MS has been emerging. Hence, we aimed to characterise the nature and the extent of oxidative damage in different experimental models in comparison to MS. For this purpose, we emphasised the expression of enzymes involved in reactive species production (the essential NADPH oxidase subunit p22phox and inducible nitric oxide synthase; iNOS), the accumulation of oxidised phospholipids and iron, which is a potential amplifier of oxidative damage. The different animal models were triggered by diverse inflammatory mechanisms, each representing distinct aspects of MS pathology. The only experimental model accumulating oxidised phospholipids to a comparable extent as MS lesions was the coronavirus-triggered demyelinating encephalomyelitis. In MS as well as in the coronavirus-model, oxidative injury was associated with a profound activation of microglia and macrophages. This was characterised by the pronounced expression of p22phox but only scattered expression of iNOS. The second virus model in our study, Theilers murine encephalomyelitis virus-induced chronic demyelination, did not reach the level of microglia and macrophage activation, regarding p22phox expression, observed in the coronavirus-model. Further, a staining for oxidised phospholipids was detectable but only to a lesser extent compared with the coronavirus-induced disease. In both virus models, iNOS expression was minor. In contrast, animals suffering from passive T cell transfer experimental autoimmune encephalomyelitis (EAE) exhibited profound p22phox and iNOS expression at the peak of the disease. Similarly, we found p22phox-expressing macrophages in animals suffering from chronic T cell and macrophage-induced disease, but few iNOS+ cells. In contrast, chronic lesions of animals affected by antibody-mediated demyelination contained phagocytosing macrophages lacking p22phox and iNOS expression. Further, acute lesions induced by CD8+ T cell transfer or by mechanisms of the innate immune system showed p22phox expression in activated microglia and macrophages, but iNOS reactivity was marginal. Additionally to oxidative damage and activation patterns in microglia and macrophages concerning iNOS and p22phox, we assessed the impact of iron in experimental demyelination. Age-dependent iron accumulation and subsequent lesion-associated cellular iron release, as observed in the human brain, was only poorly reflected in the central nervous system of rodents. Therefore, we used a complex cell culture-based approach to study a possible amplification of neurodegeneration by iron accumulation in oligodendrocytes. Unfortunately, we were only able to induce iron accumulation in microglia but not in any other cell population. The presented study reports a diverging extent of oxidative injury and underlying mechanisms in different models for inflammatory demyelination. We conclude that rodent models seem to lack amplification processes that have been suggested to play a role in MS pathogenesis. Therefore, established experimental models appear to reflect the aspect of oxidative injury in the human disease only to a minor extent.submitted by Cornelia SchuhAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2014OeBB(VLID)171388

Targeted proteolysis sustains calcineurin activation.

BACKGROUND: Calcineurin (CnA) is important in the regulation of myocardial hypertrophy. We demonstrated that targeted proteolysis of the CnA autoinhibitory domain under pathological myocardial workload leads to increased CnA activity in human myocardium. Here, we investigated the proteolytic mechanism leading to activation of CnA. METHODS AND RESULTS: In patients with diseased myocardium, we found strong nuclear translocation of CnA. In contrast, in normal human myocardium, there was a cytosolic distribution of CnA. Stimulation of rat cardiomyocytes with angiotensin (Ang) II increased calpain activity significantly (433+/-11%; P<0.01; n=6) and caused proteolysis of the autoinhibitory domain of CnA. Inhibition of calpain by a membrane-permeable calpain inhibitor prevented proteolysis. We identified the cleavage site of calpain in the human CnA sequence at amino acid 424. CnA activity was increased after Ang II stimulation (310+/-29%; P<0.01; n=6) and remained high after removal of Ang II (214+/-17%; P<0.01; n=6). Addition of a calpain inhibitor to the medium decreased CnA activity (110+/-19%; P=NS; n=6) after removal of Ang II. Ang II stimulation of cardiomyocytes also translocated CnA into the nucleus as demonstrated by immunohistochemical staining and transfection assays with GFP-tagged CnA. Calpain inhibition and therefore suppression of calpain-mediated proteolysis of CnA enabled CnA exit from the nucleus. CONCLUSIONS: Ang II stimulation of cardiomyocytes increased calpain activity, leading to proteolysis of the autoinhibitory domain of CnA. This causes an increase in CnA activity and results in nuclear translocation of CnA. Loss of the autoinhibitory domain renders CnA constitutively nuclear and active, even after removal of the hypertrophic stimulus

A Multimodal Stress-Prevention Program Supplemented by Telephone-Coaching Sessions to Reduce Perceived Stress among German Farmers: Results from a Randomized Controlled Trial

This study compared the effectiveness of a 12-day stress-prevention program (SGS) supplemented by individualized, structured, four-session telephone-coaching to that of an SGS without telephone-coaching in entrepreneurs from the green professions presenting with increased stress levels. All participants went through the SGS before being randomized either to the telephone-coaching group (TC) or to the control group without telephone-coaching (noTC). SGS included four key therapeutic elements: stress-management intervention, relaxation, physical exercise, and balneotherapy. The primary outcome was the current degree of subjectively experienced stress assessed with the Perceived Stress Questionnaire (PSQ) at a 9-month follow-up. Secondary outcomes included burnout symptoms, well-being, health status, sleep disorders, expectation of self-efficacy, depression, anxiety, ability to work, pain, and days of sick leave. Assessments were conducted at baseline, 12 days (end of program), and 1 (start telephone-coaching), 3, 6 (end of telephone-coaching), and 9 months. Data from 103 adults (TC = 51; noTC = 52), mostly fulltime farmers, were available for analysis (mean age: 55.3; 49.1% female). Participants experienced significant immediate improvement in all outcome measurements, which declined somewhat during the first three months after the end of SGS and then remained stable for at least another six months. While within-group changes from baseline to 9 months showed significant improvements at medium to large effect sizes for all target variables (PSQ-total, TC: -13.38 (+/- 14.98); 95%-CI: (-17.68; -9.07); noTC: -11.09 (+/- 14.15); 95%-CI: (-15.11; -7.07)), no statistically significant differences were found between the groups at any time and for any target variable (between-group ANCOVA for PSQ-total at 9 months, parameter estimator for the group: -1.58; 95%-CI: (-7.29; 4.13)). The stress-prevention program SGS is a feasible, effective, and practical way to reduce perceived stress and improve participants' resources. Four subsequent telephone-coaching sessions do not seem to contribute to a further improvement in the results

Influence of upstream disturbances on the primary and secondary instabilities in a supersonic separated flow over a backward-facing step

The development of primary and secondary instabilities is investigated numerically for a supersonic transitional flow over a backward-facing step at Ma = 1.7 and Reδ0 = 13 718. Oblique Tollmien-Schlichting (T-S) waves with properties according to linear stability theory (LST) are introduced at the domain inlet with zero, low, or high amplitude (cases ZA, LA, and HA). A well-resolved large eddy simulation (LES) is carried out for the three cases to characterize the transition process from laminar to turbulent flow. The results for the HA case show a rapid transition due to the high initial disturbance level such that the non-linear interactions already occur upstream of the step, before the Kelvin-Helmholtz (K-H) instability could get involved. In contrast, cases ZA and LA share a similar transition road map in which transition occurs in the separated shear flow behind the step. Case LA is analyzed in detail based on the results from LST and LES to scrutinize the evolution of T-S, K-H, and secondary instabilities, as well as their interactions. Upstream of the step, the linear growth of the oblique T-S waves is the prevailing instability. Both T-S and K-H modes act as the primary mode within a short distance behind the step and undergo a quasilinear growth with a weak coupling. Upon pairing of the large K-H vortices, subharmonic waves are produced, and secondary instabilities begin to dominate the transition. Simultaneously, the growth of T-S waves is retarded by the slow resonance between subharmonic K-H and secondary instabilities. The vortex breakdown and reattachment downstream further contribute to the development of the turbulent boundary layer.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.Aerodynamic