Der Einfluß des BMP-Antagonisten Noggin auf die in vitro-Chondrogenese mesenchymaler Stammzellen

Sportlich und alters- bzw. degenerativ bedingter Verschleiß der hyalinen Knorpelkappen der Gelenke gehört heutzutage zu den häufigsten Erkrankungen weltweit, die sich hieraus ergebenden volkswirtschaftlichen Schäden sind enorm. Aufgrund seiner anatomischen Beschaffenheit und einer hiermit einhergehenden, im Vergleich zu anderen Gewebearten äußerst schlechten Regenerationsfähigkeit, ist einmal beschädigter Gelenkknorpel kaum mehr in der Lage, sich selbständig zu regenerieren, körpereigene Reparaturprozesse führen in der Regel lediglich zu minderwertigem Faserknorpel, der den mechanischen Anforderungen in den Gelenken nicht genügt. Auch die momentan angewendeten chirurgischen Maßnahmen bieten derzeit noch nicht die Möglichkeit, dem natürlichen Gelenkknorpel vergleichbare, funktionell hochwertige Ersatzgewebe zu generieren und den Patienten eine dauerhafte Beschwerdebesserung zu gewährleisten. Im Bereich des Tissue Engeneering haben sich in den letzten Jahren humane mesenchymale Stammzellen aufgrund ihrer relativ einfachen Isolierungsmöglichkeit sowie ihrer Fähigkeit, unter anderem chondrogen zu differenzieren, als vielversprechende Zellquelle für eine eventuelle Entwicklung derartiger Ersatzgewebe herausgestellt. Diese chondrogene Differenzierung kann in vitro in unterschiedlichen Chondrogenesemodellen induziert und untersucht werden, jedoch hat sich hier gezeigt, dass chondrogen differenzierende MSCs neben charakteristischen chondrogenen Markern wie Kollagen Typ II auch hypertrophieassoziierte Marker wie Kollagen Typ X und ALP exprimieren. Dies lässt vermuten, dass die chondrogene Differenzierung der MSCs in vitro vergleichbaren regulatorischen Mechanismen und Entwicklungsphasen folgt, wie sie in der endochondralen Ossifikation während der embryonalen Skelettentwicklung gefunden wird, in deren Verlauf die Chondrozyten hypertrophieren, schlussendlich apoptotisch werden und das Gewebe ossifiziert. Für die Herstellung von Knorpelersatzprodukten zur Behandlung von Knorpeldefekten ist diese Entwicklung jedoch höchst bedenklich, da die Voraussetzung hierfür die Stabilisierung des chondrogenen Phänotyps ist und hypertrophierende Zellen für derartige Ersatzgewbe ungeeignet sind. Insbesondere den BMPs (bone morphogenetic proteins) kommt dabei im komplexen Zusammenspiel einer Vielzahl unterschiedlicher Regelkreise eine entscheidende Rolle bei der Induktion der Hypertrophie zu. Ziel dieser Studie war die Untersuchung des BMP-Antagonisten Noggin, bzw. der Einfluß von Noggin auf die chondrogen differenzierenden MSCs in vitro und insbesondere inwieweit die Hypertrophie in diesen Zellen in Anwesenheit von Noggin im Kulturmedium beeinflusst bzw. unterdrückt werden kann. Hierzu wurden die MSCs in einem Pelletkulturmodell in einem definierten Medium mit u.a. TGF-ß und Dexamethason 14 Tage lang chondrogen vordifferenziert, im Anschluß wurde in einem Teil der Zellen durch Entzug von TGF-ß und Dexamethason sowie Zugabe des Schilddrüsenhormones T3 zum Medium die Hypertrophie angebahnt. Weiterhin wurden sowohl die in chondrogenem Medium belassenen, als auch die in hypertrophes Medium überführten Aggregate mit unterschiedlich hohen Dosen Noggin behandelt. Nach weiteren 14 Tagen wurden die Zellen dann histologisch untersucht, weiterhin wurde die Genexpression bestimmter Marker mittels PCR ermittelt. Die gewonnenen Ergebnisse zeigen deutlich, daß Noggin in der Lage ist, die T3-induzierte Hypertrophie in MSCs in vitro dosisabhängig zu inhibieren. Zusammenfassend weist dies darauf hin, daß die Beeinflußung des BMP-Signalweges mittels extrazellulärer BMP-Antagonisten wie Noggin ein möglicher Weg zur Verhinderung der Hypertrophie in der in vitro-Chondrogenese von MSCs und damit die Basis für die Herstellung MSC-basierter Knorpelersatzprodukte sein kann

Thyroid Hormone-Induced Hypertrophy in Mesenchymal Stem Cell Chondrogenesis Is Mediated by Bone Morphogenetic Protein-4

Chondrogenic differentiating mesenchymal stem cells (MSCs) express markers of hypertrophic growth plate chondrocytes. As hypertrophic cartilage undergoes ossification, this is a concern for the application of MSCs in articular cartilage tissue engineering. To identify mechanisms that elicit this phenomenon, we used an in vitro hypertrophy model of chondrifying MSCs for differential gene expression analysis and functional experiments with the focus on bone morphogenetic protein (BMP) signaling. Hypertrophy was induced in chondrogenic MSC pellet cultures by transforming growth factor β (TGFβ) and dexamethasone withdrawal and addition of triiodothyronine. Differential gene expression analysis of BMPs and their receptors was performed. Based on these results, the in vitro hypertrophy model was used to investigate the effect of recombinant BMP4 and the BMP inhibitor Noggin. The enhancement of hypertrophy could be shown clearly by an increased cell size, alkaline phosphatase activity, and collagen type X deposition. Upon induction of hypertrophy, BMP4 and the BMP receptor 1B were upregulated. Addition of BMP4 further enhanced hypertrophy in the absence, but not in the presence of TGFβ and dexamethasone. Thyroid hormone induced hypertrophy by upregulation of BMP4 and this induced enhancement of hypertrophy could be blocked by the BMP antagonist Noggin. BMP signaling is an important modulator of the late differentiation stages in MSC chondrogenesis and the thyroid hormone induces this pathway. As cartilage tissue engineering constructs will be exposed to this factor in vivo, this study provides important insight into the biology of MSC-based cartilage. Furthermore, the possibility to engineer hypertrophic cartilage may be helpful for critical bone defect repair

Aberrant plasma IL-7 and soluble IL-7 receptor levels indicate impaired T-cell response to IL-7 in human tuberculosis.

T-cell proliferation and generation of protective memory during chronic infections depend on Interleukin-7 (IL-7) availability and receptivity. Regulation of IL-7 receptor (IL-7R) expression and signalling are key for IL-7-modulated T-cell functions. Aberrant expression of soluble (s) and membrane-associated (m) IL-7R molecules is associated with development of autoimmunity and immune failure in acquired immune deficiency syndrome (AIDS) patients. Here we investigated the role of IL-7/IL-7R on T-cell immunity in human tuberculosis. We performed two independent case-control studies comparing tuberculosis patients and healthy contacts. This was combined with follow-up examinations for a subgroup of tuberculosis patients under therapy and recovery. Blood plasma and T cells were characterised for IL-7/sIL-7R and mIL-7R expression, respectively. IL-7-dependent T-cell functions were determined by analysing STAT5 phosphorylation, antigen-specific cytokine release and by analysing markers of T-cell exhaustion and inflammation. Tuberculosis patients had lower soluble IL-7R (p < 0.001) and higher IL-7 (p < 0.001) plasma concentrations as compared to healthy contacts. Both markers were largely independent and aberrant expression normalised during therapy and recovery. Furthermore, tuberculosis patients had lower levels of mIL-7R in T cells caused by post-transcriptional mechanisms. Functional in vitro tests indicated diminished IL-7-induced STAT5 phosphorylation and impaired IL-7-promoted cytokine release of Mycobacterium tuberculosis-specific CD4+ T cells from tuberculosis patients. Finally, we determined T-cell exhaustion markers PD-1 and SOCS3 and detected increased SOCS3 expression during therapy. Only moderate correlation of PD-1 and SOCS3 with IL-7 expression was observed. We conclude that diminished soluble IL-7R and increased IL-7 plasma concentrations, as well as decreased membrane-associated IL-7R expression in T cells, reflect impaired T-cell sensitivity to IL-7 in tuberculosis patients. These findings show similarities to pathognomonic features of impaired T-cell functions and immune failure described in AIDS patients

Liposome adhesion generates traction stress

International audienceMechanical forces generated by cells modulate global shape changes required for essential life processes, such as polarization, division and spreading. Although the contribution of the cytoskeleton to cellular force generation is widely recognized, the role of the membrane is considered to be restricted to passively transmitting forces. Therefore, the mechanisms by which the membrane can directly contribute to cell tension are overlooked and poorly understood. To address this, we directly measure the stresses generated during liposome adhesion. We find that liposome spreading generates large traction stresses on compliant substrates. These stresses can be understood as the equilibration of internal, hydrostatic pressures generated by the enhanced membrane tension built up during adhesion. These results underscore the role of membranes in the generation of mechanical stresses on cellular length scales and that the modulation of hydrostatic pressure due to membrane tension and adhesion can be channelled to perform mechanical work on the environment

Staging model in psychiatry: Review of the evolution of electroencephalography abnormalities in major psychiatric disorders

AIM: Clinical staging in psychiatry aims to classify patients according to the severity of their symptoms, from stage 0 (increased risk, asymptomatic) to stage 4 (severe illness), enabling adapted treatment at each stage of the illness. The staging model would gain specificity if one or more quantifiable biological markers could be identified. Several biomarkers reflecting possible causal mechanisms and/or consequences of the pathophysiology are candidates for integration into the clinical staging model of psychiatric illnesses. METHODS: This review covers the evolution (from stage 0 to stage 4) of the most important brain functioning impairments as measured with electroencephalography (EEG), in psychosis spectrum and in severe mood disorders. RESULTS: The present review of the literature demonstrates that it is currently not possible to draw any conclusion with regard to the state or trait character of any of the EEG impairments in both major depressive disorder and bipolar disorder. As for schizophrenia, the most promising markers of the stage of the illness are the pitch mismatch negativity as well as the p300 event-related potentials, as these components seem to deteriorate with increasing severity of the illness. CONCLUSIONS: Given the complexity of major psychiatric disorders, and that not a single impairment can be observed in all patients, future research should most likely consider combinations of markers in the quest for a better identification of the stages of the psychiatric illnesses