Harnessing the Secretome of Mesenchymal Stromal Cells for Traumatic Spinal Cord Injury: Multicell Comparison and Assessment of In Vivo Efficacy.

Cell therapy offers significant promise for traumatic spinal cord injury (SCI), which despite many medical advances, has limited treatment strategies. Able to address the multifactorial and dynamic pathophysiology of SCI, cells present various advantages over standard pharmacological approaches. However, the use of live cells is also severely hampered by logistical and practical considerations. These include specialized equipment and expertise, standardization of cell stocks, sustained cell viability post-thawing, and cryopreservation-induced delayed-onset cell death. For this reason, we suggest a novel and clinically translatable alternative to live-cell systemic infusion, which retains the efficacy of the latter while overcoming many of its limitations. This strategy involves the administration of concentrated cell secretome and exploits the trophic mechanism by which stromal cells function. In this study, we compare the efficacy of intravenously delivered concentrated conditioned media (CM) from human umbilical cord matrix cells (HUCMCs), bone marrow mesenchymal stromal cells, as well as newborn and adult fibroblasts in a rat model of moderately severe cervical clip compression/contusion injury (C7--T1, 35 g). This is further paired with a thorough profile of the CM cytokines, chemokines, and angiogenic factors. The HUCMC-derived CM was most effective at limiting acute (48 h post-SCI) vascular pathology, specifically lesion volume, and functional vascularity. Principle component analysis (PCA), hierarchical clustering, and interaction analysis of proteins highly expressed in the HUCMC secretome suggest involvement of the MAPK/ERK, JAK/STAT, and immune cell migratory pathways. This "secretotherapeutic" strategy represents a novel and minimally invasive method to target multiple organ systems and several pathologies shortly after traumatic SCI.

Peroxisome Elevation Induces Stem Cell Differentiation and Intestinal Epithelial Repair.

Epithelial-repair-dependent mucosal healing (MH) is associated with a more favorable prognosis for patients with inflammatory bowel disease (IBD). MH is accomplished via repair and regeneration of the intestinal epithelium. However, the mechanism underlying MH is ill defined. We found a striking upregulation of peroxisomes in the injured crypts of IBD patients. By increasing peroxisome levels in Drosophila midguts, we found that peroxisome elevation enhanced RAB7-dependent late endosome maturation, which then promoted stem and/or progenitor-cell differentiation via modulation of Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT)-SOX21A signaling. This in turn enhanced ISC-mediated regeneration. Importantly, RAB7 and SOX21 were upregulated in the crypts of IBD patients. Moreover, administration of drugs that increased peroxisome levels reversed the symptoms of dextran sulfate sodium (DSS)-induced colitis in mice. This study demonstrates a peroxisome-mediated epithelial repair mechanism, which opens a therapeutic avenue for the enhancement of MH in IBD patients.

Signalome-wide assessment of host cell response to hepatitis C virus.

Host cell signalling during infection with intracellular pathogens remains poorly understood. Here we report on the use of antibody microarray technology to detect variations in the expression levels and phosphorylation status of host cell signalling proteins during hepatitis C virus (HCV) replication. Following transfection with HCV RNA, the JNK and NF-κB pathways are suppressed, while the JAK/STAT5 pathway is activated; furthermore, components of the apoptosis and cell cycle control machineries are affected in the expression and/or phosphorylation status. RNAi-based hit validation identifies components of the JAK/STAT, NF-κB, MAPK and calcium-induced pathways as modulators of HCV replication. Selective chemical inhibition of one of the identified targets, the JNK activator kinase MAP4K2, does impair HCV replication. Thus this study provides a comprehensive picture of host cell pathway mobilization by HCV and uncovers potential therapeutic targets. The strategy of identifying targets for anti-infective intervention within the host cell signalome can be applied to any intracellular pathogen.

Deriving a Preference-Based Measure for Myelofibrosis from the EORTC QLQ-C30 and the MF-SAF.

BACKGROUND: Utility values are required for economic evaluation using cost-utility analyses. Often, generic measures such as the EuroQol five-dimensional questionnaire are used, but this may not appropriately reflect the health-related quality of life of patients with cancer including myelofibrosis. OBJECTIVE: To derive a condition-specific preference-based measure for myelofibrosis using appropriate existing measures, the Myelofibrosis-Symptom Assessment Form and the European Organisation for Research and Treatment of Cancer Quality of Life 30 Questionnaire. METHODS: Data from the Controlled Myelofibrosis Study with Oral JAK Inhibitor Treatment trial (n = 309) were used to derive the health state classification system. Psychometric and factor analyses were used to determine the dimensions of the classification system. Psychometric and Rasch analyses were then used to select an item to represent each dimension. Item selection was validated with experts. A selection of health states was valued by members of the general population using time trade-off. Finally, health state values were modeled using regression analysis to produce utility values for every state. RESULTS: The Myelofibrosis 8 dimensions has eight dimensions: physical functioning, emotional functioning, fatigue, itchiness, pain under ribs on the left side, abdominal discomfort, bone or muscle pain, and night sweats. Regression models were estimated using time trade-off data from 246 members of the general population valuing a total of 33 states. The best performing model was a random effects maximum likelihood model producing utility values ranging from 0.089 to 1. CONCLUSIONS: The Myelofibrosis 8 dimensions is a condition-specific preference-based measure for myelofibrosis. This measure can be used to generate utility values for myelofibrosis for any data set containing the Myelofibrosis-Symptom Assessment Form and the European Organisation for Research and Treatment of Cancer Quality of Life 30 Questionnaire data.

A Bayesian approach to targeted experiment design.

MOTIVATION: Systems biology employs mathematical modelling to further our understanding of biochemical pathways. Since the amount of experimental data on which the models are parameterized is often limited, these models exhibit large uncertainty in both parameters and predictions. Statistical methods can be used to select experiments that will reduce such uncertainty in an optimal manner. However, existing methods for optimal experiment design (OED) rely on assumptions that are inappropriate when data are scarce considering model complexity. RESULTS: We have developed a novel method to perform OED for models that cope with large parameter uncertainty. We employ a Bayesian approach involving importance sampling of the posterior predictive distribution to predict the efficacy of a new measurement at reducing the uncertainty of a selected prediction. We demonstrate the method by applying it to a case where we show that specific combinations of experiments result in more precise predictions. AVAILABILITY AND IMPLEMENTATION: Source code is available at: http://bmi.bmt.tue.nl/sysbio/software/pua.html.

Single cell network profiling (SCNP): mapping drug and target interactions.

Measuring target coverage of small molecule inhibitors is paramount-first, for selection of molecules to progress through the drug development process and second, once a candidate drug moves to clinical testing, for guiding dose/schedule selection. Single cell network profiling (SCNP) using multiparameter flow cytometry can measure compound effects on multiple signaling cascades in a cell-type-specific manner. We applied SCNP to a panel of compounds with reported inhibitory effects on Jak/Stat signaling using a novel system where modulation of multiple signaling cascades are simultaneously measured in discrete cell subsets in whole (ie, unfractionated) blood. Jak2 vs. Jak3 selectivity as well as "off-target" effects on other cell signaling pathways were measured using a combination of cytokines that target different white blood cell subsets, namely GM-CSF (monocytes/granulocytes), IL-2 (T cells), and CD40L (B cells). The compounds were then rank-ordered by potency and selectivity against the different pathways tested. Notably, SCNP performed in whole unfractionated blood compared to fractionated peripheral blood mononuclear cells (PBMC) from the same donors revealed potency loss for all compounds, with one exception. These studies show that SCNP can be used to efficiently measure a drug candidate's potency and selectivity in a physiologically relevant environment (eg, whole blood) and that robust IC(50) are attainable from rare subpopulations (<100 cells). The ability to generate in vitro IC(50) measurements in whole blood can be used not only for the preclinical selection of lead molecules, but also to determine the target plasma concentration for clinical studies and to measure target coverage after drug administration in early phase clinical trials. Knowledge of the compound plasma concentration necessary to achieve biochemical coverage permits rational design of clinical trials based on biologically active dose vs. the traditional maximum tolerated dose (MTD) design, which is better suited for cytotoxic, nontargeted drugs.