Development of Ewing's sarcoma from primary bone marrow-derived mesenchymal stem cells

SUMMARY : Ewing's sarcoma is a member of Ewing's family tumors (ESPY) and the second most common solid bone and soft tissue malignancy of children and young adults. It is associated in 85% of cases with the t(11;22)(q24:q12) chromosomal translocation that generates fusion of the 5' segment of the EWSR1 gene with the 3' segment of the ETS family gene FLI-1. The EWSR1-FLI-1 fusion protein behaves as an aberrant transcriptional activator and is believed to contribute to ESFT development. However, EWSR1-FLI-1 induces growth arrest and apoptosis in normal fibroblasts, and primary cells that are pemissive for its putative oncogenic properties have not been discovered, hampering basic understanding of ESFT biology. Here, we show that EWSR1-FLI-1 alone can transform mouse primary bone marrow-derived mesenchymal progenitor cells and generate tumors that display hallmarks of Ewing's sarcoma, including a small round cell phenotype, expression of ESFT-associated markers, insulin like growth factor-I dependence, and induction or repression of numerous EWSR1-FLI-1 target genes. Consistent with this finding, we tested the possibility that human mesenchymal stem cells (hMSC) might also provide a permissive cellular environment for EWSR1-FLI-1, and could represent the first adequate primary human cellular background for the oncogenic properties of the fusion protein. Indeed, expression of EWSR1-FLI-1 in human mesenchymal stem cells (hMSC) was not only stably maintained without inhibiting proliferation, but induced a gene expression profile bearing striking similarity to that of ESFT, including genes that are among the highest ESFT discriminators. Expression of EWSR1-FLI-1 in hMSCs may recapitulate the initial steps of Ewing's sarcoma development, allowing identification of genes that play an important role early in its pathogenesis. Among relevant candidate transcripts induced by EWSR1-FL/-1 in hMSC we found the polycomb group gene EZH2 which we show to play a critical role in Ewing's sarcoma growth. These observations provide the first identification of candidate primary cells from which ESFTs originate and suggest that EWSR1-FLI-1 expression may constitute the initiating event in ESFT pathogenesis. Le sarcome d' Ewing est un membre de la famille des tumeurs Ewing (ESFT) et représente la deuxième tumeur maligne solide de l'os et des tissus mous chez les enfants et les jeunes adultes. Cette tumeur est associée dans 85% des cas avec la translocation chromosomique t(11;22)(g24:g12), qui génère la fusion entre le segment 5' du gène EWSR1 avec le segment 3' du gène FLI-1, appartenant à la famille des facteurs de transcription ETS. La protéine de fusion EWSR1-FLI-1 qui en dérive joue le rSle d'un facteur de transcription aberrant, et est supposée contribuer de manière décisive au processus de développement des ESFTs. Néanmoins, l'expression de EWSR1-FLI-1 dans des fibroblastes normaux induit un arrêt de croissance et leur apoptose, et les cellules primaires permissives pour les propriétés oncogéniques attribuées à la translocation n'ont pas encore été identifiées, empêchant la compréhension de la biologie de base du sarcome d'Ewing. Dans ce travail on montre que l'expression de EWSR1-FLI-1 uniquement est capable de transformer des cellules souches mésenchymateuses dérivées de la moelle osseuse de la souris, pour générer des tumeurs qui présentent les caractéristiques du sarcome d' Ewing humain, et notamment une morphologie de petites cellules bleues et rondes, l'expression de marqueurs associés aux ESFTs, une dépendance du facteur de croissance IGF-1, et l'induction ou la répression de nombreux gènes cibles connus de EWSR1-FLI-1. Sur la base de ces observations, on a testé la possibilité que les cellules souches mésenchymateuses humaines (hMSCs) puissent aussi fournir un environnement cellulaire permissif pour EWSR1-FLI-1 ; et représenter le premier background cellulaire humain adéquat pour la manifestation du pouvoir oncogénique de la protéine de fusion. En effet, l'expression de EWSR1-FLI-1 dans des cellules souches mésenchymateuses humaines s'est révélée non seulement maintenue, mais elle a induit un profil d'expression génétique étonnamment similaire à celui des ESFTs humains, incluant les gènes qui ont été rapportés comme étant les plus discriminatifs pour ces tumeurs. L'expression de EWSR1-FLI-1 dans les hMSCs pourrait récapituler les étapes initiales du développement du sarcome d' Ewing, et de ce fait consentir à identifier les gènes qui jouent un rôle crucial dans sa pathogenèse précoce. Parmi les transcrits relevant indults par EWSR1-FL/-9 dans les hMSCs nous avons découvert le gène du groupe des polycomb EZH2, que nous avons par la suite démontré jouer un rôle essentiel dans la croissance du sarcome de Ewing. Ces observations apportent pour la première fois l'identification d'une cellule primaire candidate pour représenter la cellule d'origine des ESFTs, et en même temps suggèrent que l'expression de EWSR1-FLI-1 peut constituer l'événement initial dans la pathogenèse du sarcome d' Ewing.

Metallic renal artery MR imaging stent: artifact-free lumen visualization with projection and standard renal MR angiography.

A cardiac-triggered free-breathing three-dimensional (3D) balanced fast field-echo projection renal magnetic resonance (MR) angiographic sequence was investigated for in-stent lumen visualization of a dedicated metallic renal artery stent. Fourteen prototype stents were deployed in the renal arteries of six pigs (in two pigs, three stents were deployed). Projection renal MR angiography was compared with standard contrast material-enhanced 3D breath-hold MR angiography. Artifact-free in-stent lumen visualization was achieved with both projection MR angiography and contrast-enhanced MR angiography. These promising results warrant further studies for visualization of in-stent restenosis.

Propositions pratiques d'utilisation des ultrasons quantitatifs osseux dans le management de l'ostéoporose [Practical proposals for the use of quantitative bone ultrasound in the management of osteoporosis]

As an emerging alternative to DXA, there is a growing interest in the use of quantitative ultrasound (QUS) measurements for the non invasive assessment of fracture risk in the management of osteoporosis. While the potential of QUS in the management of osteoporosis have been highly recognized by the scientific community and granted by the majority of the international bone disease organizations, it becomes important to develop strategies how to use ultrasound clinically. Our paper is highlighting Swiss operational clinical propositions for 2 QUS devices sold in Switzerland, on how to use the QUS in the management of osteoporosis.

Methicillin-susceptible Staphylococcus aureus clonal complex 398: high prevalence and geographical heterogeneity in bone and joint infection and nasal carriage.

The prevalence of clonal complex (CC) 398 methicillin-susceptible Staphylococcus aureus (MSSA) was unexpectedly high among bone and joint infections (BJIs) and nasal-colonizing isolates in France, with surprising geographical heterogeneity. With none of the major, most-known staphylococcal virulence genes, MSSA CC398 BJI was associated with lower biological inflammatory syndrome and lower treatment failure rates.

Investigation of Postmortem Gases: A Forensic Imaging and Analytical Chemistry Approach

Background: Distinguishing postmortem gas accumulations in the body due to natural decomposition and other phenomena such as gas embolism can prove a difficult task using purely Multi-Detector Computed Tomography (MDCT). The Radiological Alteration Index (RAI) was created with the intention to be able to identify bodies undergoing the putrefaction process based on the quantity of gas detected within the body. The flaw in this approach is the inability to absolutely determine putrefaction as the origin of gas volumes in cases of moderate alteration. The aim of the current study is to identify percentage compositions of O2, N2, CO2 and the presence of gases such as H2 and H2S within these sampling sites in order to resolve this complication. Materials and methods: All cases investigated in our University Center of Legal Medicine are undergoing a Post-Mortem Computed Tomography (PMCT)-scan before external examination or autopsy as a routine investigation. In the obtained images, areas of gas were characterized as 0, I, II or III based on the amount of gas present according to the RAI (1). The criteria for these characterizations were dependent of the site of gas, for example thoracic and abdominal cavities were graded as I (1 - 3cm gas), II (3 - 5cm gas) and III (>5cm gas). Cases showing gaseous sites with grade II or III were selected for this study. The sampling was performed under CT-guidance to target the regions to be punctured. Luer-lock PTFE syringes equipped with a three-way valve and needles were used to sample the gas directly (2). Gaseous samples were then analysed using gas chromatography coupled to a thermal conductivity detector (GC-TCD). The components present in the samples were expressed as a percentage of the overall gas present. Results: Up to now, we have investigated more than 40 cases using our standardized procedure for sampling and analysis of gas. O2, N2 and CO2 were present in most samples. The following distributions were found to correlate to gas origins of gas embolism/scuba diving accidents, trauma and putrefaction: ? Putrefaction → O2 = 1 - 5%; CO2 > 15%; N2 = 10 - 70%; H2 / H2S / CH4 variable presence ? Gas embolism/Scuba diving accidents → O2 and N2= varying percentages; CO2 > 20% ? Trauma → O2 = small percentage; CO2 < 15%; N2 > 65% H2 and H2S indicated levels of putrefaction along with methane which can also gauge environmental conditions or conditions of body storage/burial. Many cases showing large RAI values (advanced alteration) did reveal a radiological diagnosis which was in concordance with the interpretation of the gas composition. However, in certain cases (gas embolism, scuba divers) radiological interpretation was not possible and only chemical gas analysis was found to lead to the correct diagnosis, meaning that it provided complementary information to the radiological diagnosis. Conclusion: Investigation of postmortem gases is a useful tool to determine origin of gas generation which can aid the diagnosis of the cause of death. Levels of gas can provide information on stage of putrefaction and help to perform essential medico-legal diagnosis such as vital gas embolism.

Anti-tumor necrosis factor drug survival in axial spondyloarthritis is independent of the classification criteria.

To compare the impact of meeting specific classification criteria [modified New York (mNY), European Spondyloarthropathy Study Group (ESSG), and Assessment of SpondyloArthritis international Society (ASAS) criteria] on anti-tumor necrosis factor (anti-TNF) drug retention, and to determine predictive factors of better drug survival. All patients fulfilling the ESSG criteria for axial spondyloarthritis (SpA) with available data on the axial ASAS and mNY criteria, and who had received at least one anti-TNF treatment were retrospectively retrieved in a single academic institution in Switzerland. Drug retention was computed using survival analysis (Kaplan-Meier), adjusted for potential confounders. Of the 137 patients classified as having axial SpA using the ESSG criteria, 112 also met the ASAS axial SpA criteria, and 77 fulfilled the mNY criteria. Drug retention rates at 12 and 24 months for the first biologic therapy were not significantly different between the diagnostic groups. Only the small ASAS non-classified axial SpA group (25 patients) showed a nonsignificant trend toward shorter drug survival. Elevated CRP level, but not the presence of bone marrow edema on magnetic resonance imaging (MRI) scans, was associated with significantly better drug retention (OR 7.9, ICR 4-14). In this cohort, anti-TNF drug survival was independent of the classification criteria. Elevated CRP level, but not positive MRI, was associated with better drug retention.

Biofilm formation by staphylococci on fresh, fresh-frozen and processed human and bovine bone grafts.

Biofilm formation is a multi-step process influenced by surface properties. We investigated early and mature biofilm of Staphylococcus aureus on 4 different biological calcium phosphate (CaP) bone grafts used for filling bone defects. We investigated standardised cylinders of fresh and fresh-frozen human bone grafts were harvested from femoral heads; processed humanand bovine bone grafts were obtained preformed. Biofilm formation was done in tryptic soy broth (TSB) using S. aureus (ATCC 29213) with static conditions. Biofilm density after 3 h (early biofilm) and 24 h (mature biofilm) was investigated by sonication and microcalorimetry. After 3 h, bacterial density was highest on fresh-frozenandfresh bone grafts. After 24 h, biofilm density was lowest on freshbone grafts (p < 0.001) compared to the other 3 materials, which did not differ quantitatively (p > 0.05). The lowest increase in bacterial density was detected on fresh bone grafts (p < 0.001). Despite normal shaped colonies, we found additional small colonies on the surface of the fresh and fresh-frozen samples by sonication. This was also apparent in microcalorimetric heat-flow curves. The four investigated CaP bone grafts showed minor structural differences in architecture but marked differences concerning serum coverage and the content of bone marrow, fibrous tissue and bone cells. These variations resulted in a decreased biofilm density on freshand fresh-frozenbone grafts after 24 h, despite an increased early biofilm formation and might also be responsible for the variations in colony morphology (small colonies). Detection of small colony variants by microcalorimetry might be a new approach to improve the understanding of biofilm formation.

Diffusion-weighted MR imaging of the spine at 3-T: feasibility, optimization of b-value and utility to differentiate benign from pathological vertebral compression fractures

Purpose: To evaluate the feasibility, determine the optimal b-value, and assess the utility of 3-T diffusion-weighted MR imaging (DWI) of the spine in differentiating benign from pathologic vertebral compression fractures.Methods and Materials: Twenty patients with 38 vertebral compression fractures (24 benign, 14 pathologic) and 20 controls (total: 23 men, 17 women, mean age 56.2years) were included from December 2010 to May 2011 in this IRB-approved prospective study. MR imaging of the spine was performed on a 3-T unit with T1-w, fat-suppressed T2-w, gadolinium-enhanced fat-suppressed T1-w and zoomed-EPI (2D RF excitation pulse combined with reduced field-of-view single-shot echo-planar readout) diffusion-w (b-values: 0, 300, 500 and 700s/mm2) sequences. Two radiologists independently assessed zoomed-EPI image quality in random order using a 4-point scale: 1=excellent to 4=poor. They subsequently measured apparent diffusion coefficients (ADCs) in normal vertebral bodies and compression fractures, in consensus.Results: Lower b-values correlated with better image quality scores, with significant differences between b=300 (mean±SD=2.6±0.8), b=500 (3.0±0.7) and b=700 (3.6±0.6) (all p<0.001). Mean ADCs of normal vertebral bodies (n=162) were 0.23, 0.17 and 0.11×10-3mm2/s with b=300, 500 and 700s/mm2, respectively. In contrast, mean ADCs were 0.89, 0.70 and 0.59×10-3mm2/s for benign vertebral compression fractures and 0.79, 0.66 and 0.51×10-3mm2/s for pathologic fractures with b=300, 500 and 700s/mm2, respectively. No significant difference was found between ADCs of benign and pathologic fractures.Conclusion: 3-T DWI of the spine is feasible and lower b-values (300s/mm2) are recommended. However, our preliminary results show no advantage of DWI in differentiating benign from pathologic vertebral compression fractures.

Magnetic resonance coronary lumen and vessel wall imaging.

Coronary magnetic resonance angiography (MRA) is a technique aimed at establishing a noninvasive test for the assessment of significant coronary stenoses. There are certain boundary conditions that have hampered the clinical success of coronary MRA and coronary vessel wall imaging. Recent advances in hardware and software allow for consistent visualization of the proximal and mid portions of the native coronary arteries. Current research focuses on the use of intravascular MR contrast agents and black blood coronary angiography. One common goal is to create a noninvasive test which might allow for screening for major proximal and mid coronary artery disease. These novel approaches will represent a major step forward in diagnostic cardiology.

Interaction between calcium intake and menarcheal age on bone mass gain: an eight-year follow-up study from prepuberty to postmenarche.

Both late menarcheal age and low calcium intake (Ca intake) during growth are risk factors for osteoporosis, probably by impairing peak bone mass. We investigated whether lasting gain in areal bone mineral density (aBMD) in response to increased Ca intake varies according to menarcheal age and, conversely, whether Ca intake could influence menarcheal age. In an initial study, 144 prepubertal girls were randomized in a double-blind controlled trial to receive either a Ca supplement (Ca-suppl.) of 850 mg/d or placebo from age 7.9-8.9 yr. Mean aBMD gain determined by dual energy x-ray absorptiometry at six sites (radius metaphysis, radius diaphysis, femoral neck, trochanter, femoral diaphysis, and L2-L4) was significantly (P = 0.004) greater in the Ca-suppl. than in the placebo group (27 vs. 21 mg/cm(2)). In 122 girls followed up, menarcheal age was recorded, and aBMD was determined at 16.4 yr of age. Menarcheal age was lower in the Ca-suppl. than in the placebo group (P = 0.048). Menarcheal age and Ca intake were negatively correlated (r = -0.35; P < 0.001), as were aBMD gains from age 7.9-16.4 yr and menarcheal age at all skeletal sites (range: r = -0.41 to r = -0.22; P < 0.001 to P = 0.016). The positive effect of Ca-suppl. on the mean aBMD gain from baseline remained significantly greater in girls below, but not in those above, the median of menarcheal age (13.0 yr). Early menarcheal age (12.1 +/- 0.5 yr): placebo, 286 +/- 36 mg/cm(2); Ca-suppl., 317 +/- 46 (P = 0.009); late menarcheal age (13.9 +/- 0.5 yr): placebo, 284 +/- 58; Ca-suppl., 276 +/- 50 (P > 0.05). The level of Ca intake during prepuberty may influence the timing of menarche, which, in turn, could influence long-term bone mass gain in response to Ca supplementation. Thus, both determinants of early menarcheal age and high Ca intake may positively interact on bone mineral mass accrual.

Current Molecular Imaging of Spinal Tumors in Clinical Practice.

Energy metabolism measurements in spinal cord tumors, as well as in osseous spinal tumors/metastasis in vivo, are rarely performed only with molecular imaging (MI) by positron emission tomography (PET). This imaging modality developed from a small number of basic clinical science investigations followed by subsequent work that influenced and enhanced the research of others. Apart from precise anatomical localization by coregistration of morphological imaging and quantification, the most intriguing advantage of this imaging is the opportunity to investigate the time course (dynamics) of disease-specific molecular events in the intact organism. Most importantly, MI represents one of the key technologies in translational molecular neuroscience research, helping to develop experimental protocols that may later be applied to human patients. PET may help monitor a patient at the vertebral level after surgery and during adjuvant treatment for recurrent or progressive disease. Common clinical indications for MI of primary or secondary CNS spinal tumors are: (i) tumor diagnosis, (ii) identification of the metabolically active tumor compartments (differentiation of viable tumor tissue from necrosis) and (iii) prediction of treatment response by measurement of tumor perfusion or ischemia. While spinal PET has been used under specific circumstances, a question remains as to whether the magnitude of biochemical alterations observed by MI in CNS tumors in general (specifically spinal tumors) can reveal any prognostic value with respect to survival. MI may be able to better identify early disease and to differentiate benign from malignant lesions than more traditional methods. Moreover, an adequate identification of treatment effectiveness may influence patient management. MI probes could be developed to image the function of targets without disturbing them or as treatment to modify the target's function. MI therefore closes the gap between in vitro and in vivo integrative biology of disease. At the spinal level, MI may help to detect progression or recurrence of metastatic disease after surgical treatment. In cases of nonsurgical treatments such as chemo-, hormone- or radiotherapy, it may better assess biological efficiency than conventional imaging modalities coupled with blood tumor markers. In fact, PET provides a unique possibility to correlate topography and specific metabolic activity, but it requires additional clinical and experimental experience and research to find new indications for primary or secondary spinal tumors.

New Horizons in Forensic Imaging: Post Mortem CT Angiography - Benefits and Practical Application.

Puropse/Aim: To learn about the developement of post mortem CT angiography, its indications, benefits, pitfalls and practical application. Content Organization: A. Developement of post mortem CT angiography B. Technical prerequisites C. Practical application of post mortem CT angiography (preparation of the body, injection of contrast agent, examination protocol) D. Indications and benefits (including a comparison with conventional autopsy) E. Interpretation of imaging data (with case demonstrations) F. Artifacts, pitfalls and limitations G. Current and potential future use. Summary: This exhibit demonstrates the developement, application and interpretation of post mortem CT angiography. Teaching points: 1. post mortem CT angiography is feasible and useful for identification of the cause of death 2. depending on the indication it can be superior to autopsy 3. limitations and artifacts need to be known for interpreta

Imaging of experience-dependent structural plasticity in the mouse neocortex in vivo

The functionality of adult neocortical circuits can be altered by novel experiences or learning. This functional plasticity appears to rely on changes in the strength of neuronal connections that were established during development. Here we will describe some of our studies in which we have addressed whether structural changes, including the remodeling of axons and dendrites with synapse formation and elimination, could underlie experience-dependent plasticity in the adult neocortex. Using 2-photon laser-scanning microscopes and transgenic mice expressing GFP in a subset of pyramidal cells, we have observed that a small subset of dendritic spines continuously appear and disappear on a daily basis, whereas the majority of spines persists for months. Axonal boutons from different neuronal classes displayed similar behavior, although the extent of remodeling varied. Under baseline conditions, new spines in the barrel cortex were mostly transient and rarely survived for more than a week. However, when every other whisker was trimmed, the generation and loss of persistent spines was enhanced. Ultrastructural reconstruction of previously imaged spines and boutons showed that new spines slowly form synapses. New spines persisting for a few days always had synapses, whereas very young spines often lacked synapses. New synapses were predominantly found on large, multi-synapse boutons, suggesting that spine growth is followed by synapse formation, preferentially on existing boutons. Altogether our data indicate that novel sensory experience drives the stabilization of new spines on subclasses of cortical neurons and promotes the formation of new synapses. These synaptic changes likely underlie experience-dependent functional remodeling of specific neocortical circuits.