Examining the use of alternative light sources in medico-legal assessments of blunt-force trauma: a systematic review.

The ability to analyze blunt-force trauma is crucial for deciphering valuable clues concerning mechanisms of injury and as evidence for medico-legal investigations. The use of alternate light sources (ALS) has been studied over the past decade, and is proposed to outperform conventional white light (CWL) during bruise assessments. In response to the growing interest of the technology worldwide, a systematic review of the literature was conducted according to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) to address the ability of ALS to detect and visualize bruising. From an initial 4055 records identified, ten studies met the eligibly criteria and were selected for this review. Evaluation also included a novel framework, referred to as SPICOT, to further systematically assess both scientific evidence and risk of bias in forensic literature. Analysis reveals that narrowband wavelengths within in the infrared or ultraviolet spectral ranges do not significantly outperform CWL in visualizing or detecting bruising. However, wavelengths within the visible spectrum, particularly 415 nm combined with longpass or bandpass yellow filters, are more effective. However, the majority of selected studies only address the sensitivity of ALS, and therefore, results may only be considered valid when the location of a bruise is known. Further investigation is required to understand the specificity of ALS, in particular how the use of topical cosmetic products, previous wounds/scar-tissue, tattoos, moles and freckles may affect detection. The ethical concern regarding the interpretation of enhanced visualized trauma should also be considered in prospect discussions prior to implementing ALS into routine practice. Nevertheless, this review finds that narrowband ALS within the visible spectrum demonstrates potential for improved injury documentation, outperforming CWL in the detection and visualization of bruising.

Postmortem CT analysis of paranasal sinuses using an experimental model of drowning.

Fluid-filled paranasal sinuses are suggested to be a valuable tool to distinguish between drowning and non-drowning postmortem, yet the mechanisms governing fluid entry remains unknown. We investigate if fluid-filled paranasal sinuses are caused by a passive influx from submersion or an active aspiration mechanism during drowning. The ovine nasal cavity and maxillary sinuses are remarkably similar anatomically to humans, and have been used for endoscopic surgical training in recent decades. We submerged 15 decapitated ovine heads from agricultural waste at a depth of 2 m in flowing water for 1, 8, and 24 h and 7 days. Paranasal sinuses were CT imaged and compared pre- and post-submersion to non-submerged controls. Furthermore, we examined the paranasal sinuses of a single homicide case of a non-drowned submerged subject. Results demonstrate that fluid passively enters the maxillary sinus postmortem in the non-drowned ovine heads following 1 h of submersion. Fluid volume was independent of submersion time and influenced by time out of water as well as handling, since volume was reduced between consecutive CT scans. In contrast to our hypothesis, the filling of the paranasal sinuses is due to passive influx of fluid from submersion rather than an active aspiration during drowning. The observation that paranasal sinuses were fluid-filled in a single medico-legal case of postmortem submersion supports the finding of passive influx. Consequently, careful interpretation of fluid-filled paranasal sinuses is required when bodies are found in water, as the finding cannot distinguish between postmortem submersion and drowning.

A probability model for assessing age relative to the 18-year old threshold based on magnetic resonance imaging of the knee combined with radiography of third molars in the lower jaw.

OBJECTIVE: This study aims to generate a statistical model based on magnetic resonance imaging of the knee and radiography of third molars in the lower jaw, for assessing age relative to the 18-year old threshold. METHODS: In total, 58 studies correlating knee or tooth development to age were assessed, 5 studies for knee and 7 studies for tooth were included in the statistical model. The relation between the development of the anatomical site, based on a binary system, and age were estimated using logistic regression. Separate meta-populations for knee and tooth were generated from the individual based data for men and women. A weighted estimate of probabilities was made by combining the probability densities for knee and tooth. Margin of errors for males and females in different age groups and knee and tooth maturity were calculated within the larger framework of transition analysis using a logit model as a base. Evidentiary values for combinations of knee and tooth maturity were evaluated with likelihood ratios. RESULTS: For males, the sensitivity for the method was calculated to 0.78 (probability of correctly classifying adults), the specificity 0.90 (probability of correctly classifying minors), the negative predictive value 0.80 (proportion identified minors are minors) and the positive predictive value 0.89 (proportion identified adults are adults) indicating a model better at identifying minors than adults. The point at which half the female population has reached closed knee lies before the 18-year threshold, adding the knee as an indicator lowers specificity and increases sensitivity. The sensitivity when using tooth as an indicator for females is 0.24 and specificity 0.97, signifying few minors misclassified as adults but also a low probability of identifying adults. The negative predictive value for women when using tooth as the sole indicator is 0.56 and positive predictive value 0.88. Probabilities were calculated for males and females assuming a uniform age distribution between 15 and 21years. The calculated margin of error of minors classified as adults in a population between 15 and 21 years with the model was 11% for males and 12% for females. Further, the evidentiary value as well as margin of error vary for different combinations of knee and tooth maturity. CONCLUSION: The statistical model based on the combination of MRI knee and radiography of mandibular third molars is a valid method to assess age relative to the 18-year old threshold when applied on males and of limited value in females.

Fatal anaphylactic shock: A review of postmortem biomarkers and diagnostics.

Diagnosing anaphylactic shock postmortem is challenging since differential diagnoses exist and the forensic pathologist often faces subtle findings and lacks relevant information which prevents reaching an opinion of certainty. This review provides an overview of the literature covering research and existing recommendations on the postmortem diagnosis of anaphylactic shock. In order to harmonize the approach and provide guidance for diagnosing deaths from anaphylactic shock in the six forensic centers in Sweden, a guidance protocol aligned with the notion of a holistic view in the approach was devised. Areas in need of further studies include both immunohistological and biochemical investigations to stratify quantitative approaches based on condition and anaphylactic trigger and to lay the ground for and possibly establish alternative matrices.

Treatment of chronic GvHD with mesenchymal stromal cells induces durable responses: A phase II study.

Steroid-refractory chronic graft-vs-host disease (cGvHD) contributes to morbidity after allogeneic hematopoietic stem cell transplantation. Here, we report on 11 patients with severe, refractory cGvHD treated with repeated infusions of allogeneic bone marrow-derived mesenchymal stromal cells (MSC) over a 6- to 12-month period. Six patients responded to MSC treatment following National Institutes of Health response criteria, accompanied by improvement in GvHD-related symptoms and quality of life. This response was durable, with systemic immunosuppressive therapy withdrawn from two responders, and a further two free from steroids and tapering calcineurin inhibitors. All responders displayed a distinct immune phenotype characterized by higher levels of naïve T cells and B cells before treatment compared with the nonresponders, and a significantly higher fraction of CD31+ naïve CD4+ T cells. MSC treatment was associated with significant increases in naïve T cells, B cells, and Tregs 7 days after each infusion. Skin biopsies showed resolution of epidermal pathology. CXCL9 and CXCL10 showed differential responses in responder and nonresponder patients. Our data support the use of MSC infusions as treatment for steroid-refractory cGvHD with durable responses. We propose CXCL9 and CXCL10 as early biomarkers for responsiveness to MSC treatment. Our results highlight the importance of the MSC recipient immune phenotype in promoting treatment response. This trial was registered at www.ClinicalTrials.gov as #NCT01522716.

The Complement System Is Essential for the Phagocytosis of Mesenchymal Stromal Cells by Monocytes.

Mesenchymal stromal cell (MSC) therapy is a promising tool in the treatment of chronic inflammatory diseases. This has been ascribed to the capacity of MSC to release a large variety of immune-modulatory factors. However, all aspects of the mode of therapeutic MSC action in different diseases remain unresolved, mainly because most of the infused MSC are undetectable in the circulation within hours after infusion. The aim of this study was to elucidate the fate of MSC after contact with plasma. We found that upon contact with blood, complement proteins including C3b/iC3b are deposited on MSC. Importantly, we also found that complement bound to MSC enhanced their phagocytosis by classical and intermediate monocytes via a mechanism that involves C3 but not C5. Thus, we describe for the first time a mechanism which might explain, at least partly, why MSC are not found in the blood circulation after infusion. Our results indicate that MSC immune-modulatory effects could be mediated by monocytes that have phagocytosed them.

Pharmacokinetics of Total and Unbound Paclitaxel After Administration of Paclitaxel Micellar or Nab-Paclitaxel: An Open, Randomized, Cross-Over, Explorative Study in Breast Cancer Patients.

INTRODUCTION: Paclitaxel micellar is a novel formulation of paclitaxel in which retinoic acid derivates solubilize paclitaxel. The aim of the present study was to compare the unbound and total plasma pharmacokinetics of the new formulation with those of nanoparticle albumin-bound (nab)-paclitaxel and to further assess its safety. METHODS: In this open, randomized, cross-over study, 28 female patients with breast cancer were given paclitaxel micellar and nab-paclitaxel as a 1-h intravenous infusion at a dose of 260 mg/m2. Plasma samples were collected during 10 h, which were projected to cover at least 80% of the area to infinite time, AUCinf. Unbound paclitaxel was measured in ultrafiltrate of plasma. Total paclitaxel in plasma was measured after protein precipitation with acetonitrile. Both assays used ultra-performance liquid chromatography (UPLC) followed by MS/MS for drug quantification. The unbound fraction, fu, was calculated as the ratio between the unbound and the total concentration. RESULTS: No difference in fu of paclitaxel between the two formulations was observed. Statistical comparison of AUC0-10h and Cmax of unbound paclitaxel demonstrated that the two formulations met the criteria for bioequivalence. Regarding total paclitaxel levels, Cmax but not AUC0-10h met the criteria. This study supports a safe administration of paclitaxel micellar. CONCLUSION: The two formulations, paclitaxel micellar and nab-paclitaxel, behaved similarly following infusion. Probably, both formulations dissociate immediately in the blood, whereupon released paclitaxel rapidly distributes into tissue. Judged from the bioequivalence demonstrated for unbound paclitaxel, the two formulations are considered clinically equivalent. TRIAL REGISTRATION: EudraCT no.: 2010-019838-27. FUNDING: Oasmia Pharmaceutical AB.

Maximum Tolerated Dose and Pharmacokinetics of Paclitaxel Micellar in Patients with Recurrent Malignant Solid Tumours: A Dose-Escalation Study.

INTRODUCTION: A water-soluble Cremophor EL-free formulation of paclitaxel, in which retinoic acid derivates solubilize paclitaxel by forming micelles (paclitaxel micellar), was studied for the first time in man to establish the maximum tolerated dose (MTD) and to characterize the pharmacokinetics (PK). METHODS: This was an open-label, one-arm, dose-escalating study in patients with advanced solid malignant tumours, for which no standard therapy was available or had failed. Paclitaxel micellar was given as 1-h intravenous infusion every 21 days for 3 cycles, mainly without premedication. Plasma samples were collected during 24 h at the first cycle and paclitaxel concentrations were assayed by high-performance liquid chromatography. PK was evaluated using a two-compartment model. RESULTS: Thirty-four patients received paclitaxel micellar at doses ranging between 90 and 275 mg/m2. MTD was established as 250 mg/m2. Fatigue and neuropathy were the most frequent dose-limiting toxicities. No hypersensitivity reactions were observed. PK of paclitaxel was evaluated in 25 data sets. Paclitaxel micellar had a rapid initial distribution phase, mean half-life 0.55 h, estimated to be completed 3 h after dosing and a mean terminal half-life of 8.8 h. Mean clearance was 13.4 L/h/m2 with fivefold interindividual variability. The residual areas after 10 h and 24 h were 15.7 ± 8.6% and 5.7 ± 3.9% of the area under the plasma concentration-time curve to infinite time (AUCinf), respectively. CONCLUSION: No new side effects unknown for paclitaxel were observed. Maximum plasma concentration (Cmax) and AUCinf showed a tendency to increase linearly with dose within the 150-275 mg/m2 dose range. The possibility to administer paclitaxel micellar without steroid premedication makes it an attractive candidate for further studies in combination with immunotherapy. TRIAL REGISTRATION: EudraCT no: 2004-001821-54. FUNDING: Oasmia Pharmaceutical AB.

Mesenchymal Stromal Cell Secretion of Programmed Death-1 Ligands Regulates T Cell Mediated Immunosuppression.

Mesenchymal stromal cells (MSCs) exert broad immunosuppressive potential, modulating the activity of cells of innate and adaptive immune systems. As MSCs become accepted as a therapeutic option for the treatment of immunological disorders such as Graft versus Host Disease, our need to understand the intricate details by which they exert their effects is crucial. Programmed death-1 (PD-1) is an important regulator in T cell activation and homeostatic control. It has been reported that this pathway may be important in contact-dependent mediated immunomodulation by MSCs. The aim of this study was to establish whether MSCs, in addition to their cell-surface expression, are able to secrete PD-1 ligands (PD-L1 and PD-L2) and their potential importance in modulating contact-independent mechanisms of MSC immunosuppression. Here we report that MSCs express and secrete PD-L1 and PD-L2 and that this is regulated by exposure to interferon γ and tumor necrosis factor α. MSCs, via their secretion of PD-1 ligands, suppress the activation of CD4+ T cells, downregulate interleukin-2 secretion and induce irreversible hyporesponsiveness and cell death. Suppressed T cells demonstrated a reduction in AKT phosphorylation at T308 and a subsequent increase in FOXO3 expression that could be reversed with blockade of PD-L1. In conclusion, we demonstrate for the first time, that MSCs are able to secrete PD-1 ligands, with this being the first known report of a biological role for PD-L2 in MSCs. These soluble factors play an important role in modulating immunosuppressive effects of MSCs directly on T cell behavior and induction of peripheral tolerance. Stem Cells 2017;35:766-776.

Type 1 Diabetes Mellitus Donor Mesenchymal Stromal Cells Exhibit Comparable Potency to Healthy Controls In Vitro.

: Bone marrow mesenchymal stromal cells (BM-MSCs) have been characterized and used in many clinical studies based on their immunomodulatory and regenerative properties. We have recently reported the benefit of autologous MSC systemic therapy in the treatment of type 1 diabetes mellitus (T1D). Compared with allogeneic cells, use of autologous products reduces the risk of eliciting undesired complications in the recipient, including rejection, immunization, and transmission of viruses and prions; however, comparable potency of autologous cells is required for this treatment approach to remain feasible. To date, no analysis has been reported that phenotypically and functionally characterizes MSCs derived from newly diagnosed and late-stage T1D donors in vitro with respect to their suitability for systemic immunotherapy. In this study, we used gene array in combination with functional in vitro assays to address these questions. MSCs from T1D donors and healthy controls were expanded from BM aspirates. BM mononuclear cell counts and growth kinetics were comparable between the groups, with equivalent colony-forming unit-fibroblast capacity. Gene microarrays demonstrated differential gene expression between healthy and late-stage T1D donors in relation to cytokine secretion, immunomodulatory activity, and wound healing potential. Despite transcriptional differences, T1D MSCs did not demonstrate a significant difference from healthy controls in immunosuppressive activity, migratory capacity, or hemocompatibility. We conclude that despite differential gene expression, expanded MSCs from T1D donors are phenotypically and functionally similar to healthy control MSCs with regard to their immunomodulatory and migratory potential, indicating their suitability for use in autologous systemic therapy. SIGNIFICANCE: The potential for mesenchymal stromal cells (MSCs) as a cell-based therapy in the treatment of immunologic disorders has been well established. Recent studies reported the clinical potential for autologous MSCs as a systemic therapy in the treatment of type I diabetes mellitus (T1D). The current study compared the genotypic and phenotypic profiles of bone marrow-derived MSCs from T1D and healthy donors as autologous (compared with allogeneic) therapy provides distinct advantages, such as reduced risk of immune reaction and transmission of infectious agents. The findings of the current study demonstrate that despite moderate differences in T1D MSCs at the gene level, these cells can be expanded in culture to an extent corresponding to that of MSCs derived from healthy donors. No functional difference in terms of immunosuppressive activity, blood compatibility, or migratory capacity was evident between the groups. The study findings also show that autologous MSC therapy holds promise as a T1D treatment and should be evaluated further in clinical trials.

Autologous Peripheral Blood Mononuclear Cells as Treatment in Refractory Acute Respiratory Distress Syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a devastating disorder. Despite enormous efforts in clinical research, effective treatment options are lacking, and mortality rates remain unacceptably high. OBJECTIVES: A male patient with severe ARDS showed no clinical improvement with conventional therapies. Hence, an emergent experimental intervention was performed. METHODS: We performed intratracheal administration of autologous peripheral blood-derived mononuclear cells (PBMCs) and erythropoietin (EPO). RESULTS: We found that after 2 days of initial PBMC/EPO application, lung function improved and extracorporeal membrane oxygenation (ECMO) support was reduced. Bronchoscopy and serum inflammatory markers revealed reduced inflammation. Additionally, serum concentration of miR-449a, b, c and miR-34a, a transient upregulation of E-cadherin and associated chromatin marks in PBMCs indicated airway epithelial differentiation. Extracellular vesicles from PBMCs demonstrated anti-inflammatory capacity in a TNF-α-mediated nuclear factor-x03BA;B in vitro assay. Despite improving respiratory function, the patient died of multisystem organ failure on day 38 of ECMO treatment. CONCLUSIONS: This case report provides initial encouraging evidence to use locally instilled PBMC/EPO for treatment of severe refractory ARDS. The observed clinical improvement may partially be due to the anti-inflammatory effects of PBMC/EPO to promote tissue regeneration. Further studies are needed for more in-depth understanding of the underlying mechanisms of in vivo regeneration.

In Vivo Effects of Mesenchymal Stromal Cells in Two Patients With Severe Acute Respiratory Distress Syndrome.

UNLABELLED: Mesenchymal stromal cells (MSCs) have been investigated as a treatment for various inflammatory diseases because of their immunomodulatory and reparative properties. However, many basic questions concerning their mechanisms of action after systemic infusion remain unanswered. We performed a detailed analysis of the immunomodulatory properties and proteomic profile of MSCs systemically administered to two patients with severe refractory acute respiratory distress syndrome (ARDS) on a compassionate use basis and attempted to correlate these with in vivo anti-inflammatory actions. Both patients received 2×10(6) cells per kilogram, and each subsequently improved with resolution of respiratory, hemodynamic, and multiorgan failure. In parallel, a decrease was seen in multiple pulmonary and systemic markers of inflammation, including epithelial apoptosis, alveolar-capillary fluid leakage, and proinflammatory cytokines, microRNAs, and chemokines. In vitro studies of the MSCs demonstrated a broad anti-inflammatory capacity, including suppression of T-cell responses and induction of regulatory phenotypes in T cells, monocytes, and neutrophils. Some of these in vitro potency assessments correlated with, and were relevant to, the observed in vivo actions. These experiences highlight both the mechanistic information that can be gained from clinical experience and the value of correlating in vitro potency assessments with clinical effects. The findings also suggest, but do not prove, a beneficial effect of lung protective strategies using adoptively transferred MSCs in ARDS. Appropriate randomized clinical trials are required to further assess any potential clinical efficacy and investigate the effects on in vivo inflammation. SIGNIFICANCE: This article describes the cases of two patients with severe refractory adult respiratory syndrome (ARDS) who failed to improve after both standard life support measures, including mechanical ventilation, and additional measures, including extracorporeal ventilation (i.e., in a heart-lung machine). Unlike acute forms of ARDS (such in the current NIH-sponsored study of mesenchymal stromal cells in ARDS), recovery does not generally occur in such patients.

Therapeutic Potential of Multipotent Mesenchymal Stromal Cells and Their Extracellular Vesicles.

The therapeutic potential of mesenchymal stromal cells (MSCs) is evident by the number of new and ongoing trials targeting an impressive variety of conditions. In bone and cartilage repair, MSCs are expected to replace the damaged tissue, while in other therapies they modulate a therapeutic response by the secretion of bioactive molecules. MSCs possess a phenotypic plasticity and harbor an arsenal of bioactive molecules that can be released upon sensing signals in the local milieu either directly or packaged in extracellular vesicles (EVs). The reported paracrine effects comprise many of the important functions of MSCs, including supporting hematopoietic stem cells in the bone marrow, promoting angiogenesis, and modulating the immune system. The major drawback in MSC therapy is the incomplete understanding of cell fate following systemic administration as well as the mechanisms by which these cells correct disease. In this review we discuss what is known about MSC engraftment, hemocompatibility, and immunomodulation, as well as the potential of bringing the MSC-EV field toward a clinical translation.

Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting.

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs) derived from bone marrow, and labelled with a near-infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross-species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63-EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour-bearing mice and, after introduction of the rabies virus glycoprotein-targeting moiety, they were found more readily in acetylcholine-receptor-rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs.

Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?

We have recently reported that therapeutic mesenchymal stromal cells (MSCs) have low engraftment and trigger the instant blood mediated inflammatory reaction (IBMIR) after systemic delivery to patients, resulting in compromised cell function. In order to optimize the product, we compared the immunomodulatory, blood regulatory, and therapeutic properties of freeze-thawed and freshly harvested cells. We found that freeze-thawed MSCs, as opposed to cells harvested from continuous cultures, have impaired immunomodulatory and blood regulatory properties. Freeze-thawed MSCs demonstrated reduced responsiveness to proinflammatory stimuli, an impaired production of anti-inflammatory mediators, increased triggering of the IBMIR, and a strong activation of the complement cascade compared to fresh cells. This resulted in twice the efficiency in lysis of thawed MSCs after 1 hour of serum exposure. We found a 50% and 80% reduction in viable cells with freshly detached as opposed to thawed in vitro cells, indicating a small benefit for fresh cells. In evaluation of clinical response, we report a trend that fresh cells, and cells of low passage, demonstrate improved clinical outcome. Patients treated with freshly harvested cells in low passage had a 100% response rate, twice the response rate of 50% observed in a comparable group of patients treated with freeze-thawed cells at higher passage. We conclude that cryobanked MSCs have reduced immunomodulatory and blood regulatory properties directly after thawing, resulting in faster complement-mediated elimination after blood exposure. These changes seem to be paired by differences in therapeutic efficacy in treatment of immune ailments after hematopoietic stem cell transplantation.

Cracking the survival code: autophagy-related histone modifications.

Modifications of histones, the chief protein components of the chromatin, have emerged as critical regulators of life and death. While the "apoptotic histone code" came to light a few years ago, accumulating evidence indicates that autophagy, a cell survival pathway, is also heavily regulated by histone-modifying proteins. In this review we describe the emerging "autophagic histone code" and the role of histone modifications in the cellular life vs. death decision.

Do ABO blood group antigens hamper the therapeutic efficacy of mesenchymal stromal cells?

Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens - genetically governed antigenic determinants - at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome. We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products. We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals.

NCoR controls glioblastoma tumor cell characteristics.

BACKGROUND: We have previously shown that the transcriptional coregulator NCoR represses astrocytic differentiation of neural stem cells, suggesting that NCoR could be a plausible target for differentiation therapy of glioblastoma. METHODS: To study a putative role for NCoR in regulating glioblastoma cell characteristics, we used RNA-mediated knockdown followed by analysis of gene expression, proliferation and cell growth, autophagy, invasiveness in vitro, and tumor formation in vitro and in vivo. We further performed chromatin immunoprecipitation of NCoR followed by genome-wide sequencing in the human glioblastoma cell line U87 in order to reveal NCoR-occupied loci. RESULTS: RNA knockdown of NCoR resulted in a moderate increase in differentiation accompanied by a significant decrease in proliferation in adherent U87 human glioblastoma cells. chromatin immunoprecipitation sequencing approach revealed alternative mechanisms underlying the decrease in proliferation, as NCoR was enriched at promoters of genes associated with autophagy such as ULK3. Indeed, signs of an autophagy response in adherent glioblastoma cells included an increased expression of autophagy genes, such as Beclin1, and increased lipidation and nuclear puncta of LC3. Intriguingly, in parallel to the effects in the adherent cells, NCoR knockdown resulted in a significant increase in anchorage-independent growth, and this glioblastoma cell population showed dramatic increases in invasive properties in vitro and tumor formation capacity in vitro and in vivo along with an increased proliferation rate. CONCLUSION: Our results unveil unexpected aspects of NCoR regulation of tumor characteristics in glioblastoma cells and highlight the need for caution when transposing developmental concepts directly to cancer therapy.