Spinal motor neurite outgrowth over glial scar inhibitors is enhanced by coculture with bone marrow stromal cells

Background context Transplantation of bone marrow cells into spinal cord lesions promotes functional recovery in animal models, and recent clinical trials suggest possible recovery also in humans. The mechanisms responsible for these improvements are still unclear. Purpose To characterize spinal cord motor neurite interactions with human bone marrow stromal cells (MSCs) in an in vitro model of spinal cord injury (SCI). Study design/setting Previously, we have reported that human MSCs promote the growth of extending sensory neurites from dorsal root ganglia (DRG), in the presence of some of the molecules present in the glial scar, which are attributed with inhibiting axonal regeneration after SCI. We have adapted and optimized this system replacing the DRG with a spinal cord culture to produce a central nervous system (CNS) model, which is more relevant to the SCI situation. Methods We have developed and characterized a novel spinal cord culture system. Human MSCs were cocultured with spinal motor neurites in substrate choice assays containing glial scar-associated inhibitors of nerve growth. In separate experiments, MSC-conditioned media were analyzed and added to spinal motor neurites in substrate choice assays. Results As has been reported previously with DRG, substrate-bound neurocan and Nogo-A repelled spinal neuronal adhesion and neurite outgrowth, but these inhibitory effects were abrogated in MSC/spinal cord cocultures. However, unlike DRG, spinal neuronal bodies and neurites showed no inhibition to substrates of myelin-associated glycoprotein. In addition, the MSC secretome contained numerous neurotrophic factors that stimulated spinal neurite outgrowth, but these were not sufficient stimuli to promote spinal neurite extension over inhibitory concentrations of neurocan or Nogo-A. Conclusions These findings provide novel insight into how MSC transplantation may promote regeneration and functional recovery in animal models of SCI and in the clinic, especially in the chronic situation in which glial scars (and associated neural inhibitors) are well established. In addition, we have confirmed that this CNS model predominantly comprises motor neurons via immunocytochemical characterization. We hope that this model may be used in future research to test various other potential interventions for spinal injury or disease states

Investigation of the blood proteome in response to spinal cord injury in rodent models

We would like to thank the Institute of Orthopaedics and the Midlands Centre for Spinal Cord Injury (MCSI) for funding this research. This work was also supported by the Wellcome Trust [grant number 094476/Z/10/Z] which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews.Study Design: Explanatory and mechanistic study. Objective: A better understanding of the 'whole-body' response following spinal cord injury (SCI) is needed to guide future research aimed at developing novel therapeutic interventions and identifying prognostic indicators for SCI. This study aimed to characterise the blood proteome following contusion or complete SCI compared to a sham injury in rat models. Setting: United Kingdom. Methods: Pooled blood samples from one and seven days after a contusion (serum; n = 5) or from 14 days and 112 days post-complete transection SCI (plasma; n = 8) and their sham-injured counterparts were subjected to independent iTRAQ nanoflow liquid chromatography tandem mass-spectrometry proteomic analyses. Pathway analyses of the proteins that were differentially abundant between SCI and their matched sham injured counterparts were completed to indicate biological pathways that may be changed in response to SCI. Results: Eleven and 42 proteins were differentially abundant (≥±2.0 FC; p ≤ 0.05) between the contusion SCI and sham injured animals at 24 h and seven days post-injury, respectively. Seven and tweleve proteins were differentially abundant between complete and sham injured rats at 14 and 112 days post-injury, respectively. Acute-phase response signalling and Liver X Receptor/Retinoic X Receptor activation were identified as differentially regulated pathways in both models of SCI. Conclusions: We have utilised longitudinal preclinical SCI models to provide an insight into the blood proteome changes that result following SCI and to highlight a number of biological pathways of interest for future studies.Publisher PDFPeer reviewe

Human mesenchymal stem cells stimulate EaHy926 endothelial cell migration:combined proteomic and in vitro analysis of the influence of donor-donor variability

Mesenchymal stem cells (MSCs) stimulate angiogenesis within a wound environment and this effect is mediated through paracrine interactions with the endothelial cells present. Here we report that human MSC-conditioned medium (n=3 donors) significantly increased EaHy-926 endothelial cell adhesion and cell migration, but that this stimulatory effect was markedly donor-dependent. MALDI-TOF/TOF mass spectrometry demonstrated that whilst collagen type I and fibronectin were secreted by all of the MSC cultures, the small leucine rich proteoglycan, decorin was secreted only by the MSC culture that was least effective upon EaHy-926 cells. These individual extracellular matrix components were then tested as culture substrata. EaHy-926 cell adherence was greatest on fibronectin-coated surfaces with least adherence on decorin-coated surfaces. Scratch wound assays were used to examine cell migration. EaHy-926 cell scratch wound closure was quickest on substrates of fibronectin and slowest on decorin. However, EaHy-926 cell migration was stimulated by the addition of MSC-conditioned medium irrespective of the types of culture substrates. These data suggest that whilst the MSC secretome may generally be considered angiogenic, the composition of the secretome is variable and this variation probably contributes to donor-donor differences in activity. Hence, screening and optimizing MSC secretomes will improve the clinical effectiveness of pro-angiogenic MSC-based therapies

A systematic review of the effectiveness and cost-effectiveness of peer education and peer support in prisons.

BACKGROUND: Prisoners experience significantly worse health than the general population. This review examines the effectiveness and cost-effectiveness of peer interventions in prison settings. METHODS: A mixed methods systematic review of effectiveness and cost-effectiveness studies, including qualitative and quantitative synthesis was conducted. In addition to grey literature identified and searches of websites, nineteen electronic databases were searched from 1985 to 2012. Study selection criteria were: Population: Prisoners resident in adult prisons and children resident in Young Offender Institutions (YOIs). INTERVENTION: Peer-based interventions Comparators: Review questions 3 and 4 compared peer and professionally led approaches. OUTCOMES: Prisoner health or determinants of health; organisational/ process outcomes; views of prison populations. STUDY DESIGNS: Quantitative, qualitative and mixed method evaluations. RESULTS: Fifty-seven studies were included in the effectiveness review and one study in the cost-effectiveness review; most were of poor methodological quality. Evidence suggested that peer education interventions are effective at reducing risky behaviours, and that peer support services are acceptable within the prison environment and have a positive effect on recipients, practically or emotionally. Consistent evidence from many, predominantly qualitative, studies, suggested that being a peer deliverer was associated with positive effects. There was little evidence on cost-effectiveness of peer-based interventions. CONCLUSIONS: There is consistent evidence from a large number of studies that being a peer worker is associated with positive health; peer support services are also an acceptable source of help within the prison environment and can have a positive effect on recipients. Research into cost-effectiveness is sparse. SYSTEMATIC REVIEW REGISTRATION: PROSPERO ref: CRD42012002349

The Up-Scale Manufacture of Chondrocytes for Allogeneic Cartilage Therapies

Background: Allogeneic chondrocyte therapies need to be developed to allow more individuals to be treated with a cell therapy for cartilage repair and to reduce the burden and cost of current two-stage autologous procedures. Up-scale manufacture of chondrocytes using a bioreactor could help provide an off-the-shelf allogeneic chondrocyte therapy with many doses being produced in a single manufacturing run. Here we assess a Good Manufacturing Practice compliant hollow-fibre bioreactor (Quantum®) for adult chondrocyte manufacture. Methods: Chondrocytes were isolated from knee arthroplasty derived cartilage (n=5) and expanded in media supplemented with 10% fetal bovine serum (FBS) or 5% human platelet lysate (hPL) on tissue culture plastic (TCP) for a single passage. hPL supplemented cultures were then expanded in the Quantum® bioreactor for a further passage. Matched, parallel cultures in hPL or FBS were maintained on TCP. Chondrocytes from all culture conditions were characterised in terms of growth kinetics, morphology, immunoprofile, chondrogenic potential (chondrocyte pellet assays) and single telomere length analysis. Results: Quantum® expansion of chondrocytes resulted in 86.4±38.5x106 cells in 8.4±1.5 days, following seeding of 10.2±3.6 x106 cells. This related to 3.0±1.0 population doublings in the Quantum® bioreactor, compared with 2.1±0.6 and 1.3±1.0 on TCP in hPL and FBS supplemented media, respectively. Quantum® and TCP expanded cultures retained equivalent chondropotency and mesenchymal stromal cell markers immunoprofiles, with only integrin marker, CD49a, decreasing following Quantum® expansion. Quantum® expanded chondrocytes demonstrated equivalent chondrogenic potential (as assessed by ability to form and maintain chondrogenic pellets) with matched hPL TCP populations. hPL manufacture however, led to reduced chondrogenic potential and increased cell surface positivity of integrins CD49b, CD49c and CD51/61 compared with FBS cultures. Quantum® expansion of chondrocytes did not result in shortened 17p telomere length when compared with matched TCP cultures. Discussion: This study demonstrates that large numbers of adult chondrocytes can be manufactured in the Quantum® hollow-fibre bioreactor. This rapid, up-scale expansion, does not alter chondrocyte phenotype when compared with matched TCP expansion. Therefore, the Quantum® provides an attractive method of manufacturing chondrocytes for clinical use. Media supplementation with hPL for chondrocyte expansion may, however, be unfavourable in terms of retaining chondrogenic capacity

Two independent proteomic approaches provide a comprehensive analysis of the synovial fluid proteome response to Autologous Chondrocyte Implantation

Background: Autologous chondrocyte implantation (ACI) has a failure rate of approximately 20%, but it is yet to be fully understood why. Biomarkers are needed that can pre-operatively predict in which patients it is likely to fail, so that alternative or individualised therapies can be offered. We previously used label-free quantitation (LF) with a dynamic range compression proteomic approach to assess the synovial fluid (SF) of ACI responders and non-responders. However, we were able to identify only a few differentially abundant proteins at baseline. In the present study, we built upon these previous findings by assessing higher-abundance proteins within this SF, providing a more global proteomic analysis on the basis of which more of the biology underlying ACI success or failure can be understood. Methods: Isobaric tagging for relative and absolute quantitation (iTRAQ) proteomic analysis was used to assess SF from ACI responders (mean Lysholm improvement of 33; n = 14) and non-responders (mean Lysholm decrease of 14; n = 13) at the two stages of surgery (cartilage harvest and chondrocyte implantation). Differentially abundant proteins in iTRAQ and combined iTRAQ and LF datasets were investigated using pathway and network analyses. Results: iTRAQ proteomic analysis confirmed our previous finding that there is a marked proteomic shift in response to cartilage harvest (70 and 54 proteins demonstrating ≥ 2.0-fold change and p < 0.05 between stages I and II in responders and non-responders, respectively). Further, it highlighted 28 proteins that were differentially abundant between responders and non-responders to ACI, which were not found in the LF study, 16 of which were altered at baseline. The differential expression of two proteins (complement C1s subcomponent and matrix metalloproteinase 3) was confirmed biochemically. Combination of the iTRAQ and LF proteomic datasets generated in-depth SF proteome information that was used to generate interactome networks representing ACI success or failure. Functional pathways that are dysregulated in ACI non-responders were identified, including acute-phase response signalling. Conclusions: Several candidate biomarkers for baseline prediction of ACI outcome were identified. A holistic overview of the SF proteome in responders and non-responders to ACI  has been profiled, providing a better understanding of the biological pathways underlying clinical outcome, particularly the differential response to cartilage harvest in non-responders

MIDIS. JWST NIRCam and MIRI unveil the stellar population properties of Lyα\alpha-emitters and Lyman-Break galaxies at z ~ 3-7

We study the stellar population properties of 182 spectroscopically-confirmed (MUSE/VLT) Lyman-$\alpha$ emitters (LAEs) and 450 photometrically-selected Lyman-Break galaxies (LBGs) at z = 2.8 - 6.7 in the Hubble eXtreme Deep Field (XDF). Leveraging the combined power of HST and JWST NIRCam and MIRI observations, we analyse their rest-frame UV-through-near-IR spectral energy distributions (SEDs) with MIRI playing a crucial role in robustly assessing the LAE's stellar mass and ages. Our LAEs are low-mass objects (log$_{10}$(M$_\star$[M$_\odot$]) ~ 7.5), with little or no dust extinction (E(B - V) ~ 0.1) and a blue UV continuum slope ($\beta$ ~ -2.2). While 75% of our LAEs are young (< 100 Myr), the remaining 25% have significantly older stellar populations (> 100 Myr). These old LAEs are statistically more massive, less extinct and have lower specific star formation rate (sSFR) compared to young LAEs. Besides, they populate the M$_\star$ - SFR plane along the main-sequence (MS) of star-forming galaxies, while young LAEs populate the starburst region. The comparison between the LAEs properties to those of a stellar-mass matched sample of LBGs shows no statistical difference between these objects, except for the LBGs redder UV continuum slope and marginally larger E(B - V) values. Interestingly, 48% of the LBGs have ages < 10 Myr and are classified as starbursts, but lack detectable Ly$\alpha$ emission. This is likely due to HI resonant scattering and/or selective dust extinction. Overall, we find that JWST observations are crucial in determining the properties of LAEs and shedding light on the properties and similarities between LAEs and LBGs.Comment: 19 pages, 15 figures, 5 tables. Submitted to AP

Identification and relative quantification of tyrosine nitration in a model peptide using two-dimensional infrared spectroscopy

Nitration of tyrosine in proteins and peptides is a post-translational modification that occurs under conditions of oxidative stress. It is implicated in a variety of medical conditions, including neurodegenerative and cardiovascular diseases. However, monitoring tyrosine nitration and understanding its role in modifying biological function remains a major challenge. In this work, we investigate the use of electron-vibration-vibration (EVV) two-dimensional infrared (2DIR) spectroscopy for the study of tyrosine nitration in model peptides. We demonstrate the ability of EVV 2DIR spectroscopy to differentiate between the neutral and deprotonated states of 3-nitrotyrosine, and we characterize their spectral signatures using information obtained from quantum chemistry calculations and simulated EVV 2DIR spectra. To test the sensitivity of the technique, we use mixed-peptide samples containing various levels of tyrosine nitration, and we use mass spectrometry to independently verify the level of nitration. We conclude that EVV 2DIR spectroscopy is able to provide detailed spectroscopic information on peptide side-chain modifications and to detect nitration levels down to 1%. We further propose that lower nitration levels could be detected by introducing a resonant Raman probe step to increase the detection sensitivity of EVV 2DIR spectroscopy. (Graph Presented)

Autologous chondrocyte implantation-derived synovial fluids display distinct responder and non-responder proteomic profiles

Hulme, Charlotte H. & Wilson, Emma L. - Equal contributorsBackground Autologous chondrocyte implantation (ACI) can be used in the treatment of focal cartilage injuries to prevent the onset of osteoarthritis (OA). However, we are yet to understand fully why some individuals do not respond well to this intervention. Identification of a reliable and accurate biomarker panel that can predict which patients are likely to respond well to ACI is needed in order to assign the patient to the most appropriate therapy. This study aimed to compare the baseline and mid-treatment proteomic profiles of synovial fluids (SFs) obtained from responders and non-responders to ACI. Methods SFs were derived from 14 ACI responders (mean Lysholm improvement of 33 (17–54)) and 13 non-responders (mean Lysholm decrease of 14 (4–46)) at the two stages of surgery (cartilage harvest and chondrocyte implantation). Label-free proteome profiling of dynamically compressed SFs was used to identify predictive markers of ACI success or failure and to investigate the biological pathways involved in the clinical response to ACI. Results Only 1 protein displayed a ≥2.0-fold differential abundance in the preclinical SF of ACI responders versus non-responders. However, there is a marked difference between these two groups with regard to their proteome shift in response to cartilage harvest, with 24 and 92 proteins showing ≥2.0-fold differential abundance between Stages I and II in responders and non-responders, respectively. Proteomic data has been uploaded to ProteomeXchange (identifier: PXD005220). We have validated two biologically relevant protein changes associated with this response, demonstrating that matrix metalloproteinase 1 was prominently elevated and S100 calcium binding protein A13 was reduced in response to cartilage harvest in non-responders. Conclusions The differential proteomic response to cartilage harvest noted in responders versus non-responders is completely novel. Our analyses suggest several pathways which appear to be altered in non-responders that are worthy of further investigation to elucidate the mechanisms of ACI failure. These protein changes highlight many putative biomarkers that may have potential for prediction of ACI treatment success