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Red blood cells (RBCs) have been shown to affect immune function and can induce inflammatory responses after transfusion. The transfusion of washed RBCs can significantly reduce adverse effects, however, the soluble factors that may mediate these effects have not been identified. Previous studies have identified, but not quantified, a small number of chemokines associated with RBCs. We isolated RBCs from healthy volunteers and quantified of a panel of 48 cytokines, chemokines, and growth factors in the lysate, cytosol, and conditioned media of these cells using Luminex ® technology. This analysis revealed that, after correcting for white blood cell and platelet contamination, 46 cytokines were detected in RBC lysates, and the median concentration in RBCs was 12-fold higher than in the plasma. In addition, extensive washing of RBCs, such as that performed in proteomics analyses or prior to some RBC transfusions, significantly attenuated the release of six cytokines following incubation at 37 °C. This supports the hypothesis that, alongside its gas exchange function, RBCs play a role in cytokine signalling. This discovery may help supplement disease biomarker research and may shed light on adverse inflammatory processes that can follow RBC transfusion.

Background Adipose tissue is an attractive source of cells for therapeutic purposes because of the ease of harvest and the high frequency of mesenchymal stem cells (MSCs). Whilst it is clear that MSCs have significant therapeutic potential via their ability to secrete immuno-modulatory and trophic cytokines, the therapeutic use of mixed cell populations from the adipose stromal vascular fraction (SVF) is becoming increasingly common. Methods In this study we have measured a panel of 27 cytokines and growth factors secreted by various combinations of human adipose-derived cell populations. These were 1. co-culture of freshly isolated SVF with adipocytes, 2. freshly isolated SVF cultured alone, 3. freshly isolated adipocytes alone and 4. adherent adipose-derived mesenchymal stem cells (ADSCs) at passage 2. In addition, we produced an ‘ in silico ’ dataset by combining the individual secretion profiles obtained from culturing the SVF with that of the adipocytes. This was compared to the secretion profile of co-cultured SVF and adipocytes. Two-tailed t-tests were performed on the secretion profiles obtained from the SVF, adipocytes, ADSCs and the ‘ in silico ’ dataset and compared to the secretion profiles obtained from the co-culture of the SVF with adipocytes. A p-value of < 0.05 was considered statistically different. To assess the overall changes that may occur as a result of co-culture we compared the proteomes of SVF and SVF co-cultured with adipocytes using iTRAQ quantitative mass spectrometry. Results A co-culture of SVF and adipocytes results in a distinct secretion profile when compared to all other adipose-derived cell populations studied. This illustrates that cellular crosstalk during co-culture of the SVF with adipocytes modulates the production of cytokines by one or more cell types. No biologically relevant differences were detected in the proteomes of SVF cultured alone or co-cultured with adipocytes. Conclusions The use of mixed adipose cell populations does not appear to induce cellular stress and results in enhanced secretion profiles. Given the importance of secreted cytokines in cell therapy, the use of a mixed cell population such as the SVF with adipocytes may be considered as an alternative to MSCs or fresh SVF alone.

Aims/hypothesis Adipose tissue (AT) distribution is a major determinant of mortality and morbidity in obesity. In mice, intra-abdominal transplantation of subcutaneous AT (SAT) protects against glucose intolerance and insulin resistance (IR), but the underlying mechanisms are not well understood. Methods We investigated changes in adipokines, tissue-specific glucose uptake, gene expression and systemic inflammation in male C57BL6/J mice implanted intra-abdominally with either inguinal SAT or epididymal visceral AT (VAT) and fed a high-fat diet (HFD) for up to 17 weeks. Results Glucose tolerance was improved in mice receiving SAT after 6 weeks, and this was not attributable to differences in adiposity, tissue-specific glucose uptake, or plasma leptin or adiponectin concentrations. Instead, SAT transplantation prevented HFD-induced hepatic triacylglycerol accumulation and normalised the expression of hepatic gluconeogenic enzymes. Grafted fat displayed a significant increase in glucose uptake and unexpectedly, an induction of skeletal muscle-specific gene expression. Mice receiving subcutaneous fat also displayed a marked reduction in the plasma concentrations of several proinflammatory cytokines (TNF-α, IL-17, IL-12p70, monocyte chemoattractant protein-1 [MCP-1] and macrophage inflammatory protein-1β [ΜIP-1β]), compared with sham-operated mice. Plasma IL-17 and MIP-1β concentrations were reduced from as early as 4 weeks after transplantation, and differences in plasma TNF-α and IL-17 concentrations predicted glucose tolerance and insulinaemia in the entire cohort of mice ( n  = 40). In contrast, mice receiving visceral fat transplants were glucose intolerant, with increased hepatic triacylglycerol content and elevated plasma IL-6 concentrations. Conclusions/interpretation Intra-abdominal transplantation of subcutaneous fat reverses HFD-induced glucose intolerance, hepatic triacylglycerol accumulation and systemic inflammation in mice.

It is now accepted that red blood cells (RBCs) from healthy individuals regulate T-cell activity through modulating cytokine interactions, and that stored RBCs or RBCs from inflammatory cohorts are dysfunctional. Our study aimed to investigate how changes in RBCs that have been intentionally modified can affect T-cell activity as a mechanistic test of this modification. Exposure to a cancer cell line in culture was used to alter the cytokine profile of intact RBCs and the effect of these modified RBCs (ccRBCs) on T-cells was evaluated using flow cytometry. We used RBCs from healthy volunteers and quantified cytokines in RBC lysates and conditioned media using Luminex technology. During in vitro cancer cell exposure, RBCs sequestered a variety of cytokines including IL-8, bFGF, and VEGF. Although unmodified RBCs (oRBCs) stimulated proliferation of T-cells (Jurkat cells and peripheral blood mononucleated cells), ccRBCs augmented this proliferative response (3.5-fold and 1.9-fold more respectively). Unlike oRBCs, T-cells stimulated with ccRBCs were no longer protected from phytohemagglutinin-P-driven overexpression of GATA-3 and T-bet and these T-cells were induced to secrete a variety of cytokines including IL-17 and MCP-3. This study supports the hypothesis that RBCs are capable of binding and releasing cytokines in blood, and that modification of these cells can then also affect the T-cell response.

Background. Biological therapeutics such as adipose-derived mesenchymal stem cell (MSC) therapy are gaining acceptance for knee-osteoarthritis (OA) treatment. Reports of OA-patients show reductions in cartilage defects and regeneration of hyaline-like-cartilage with MSC-therapy. Suspending MSCs in hyaluronan commonly occurs in animals and humans, usually without supporting data. Objective. To elucidate the effects of different concentrations of hyaluronan on MSC growth kinetics. Methods. Using a range of hyaluronan concentrations, we measured MSC adherence and proliferation on culture plastic surfaces and a novel cartilage-adhesion assay. We employed time-course and dispersion imaging to assess MSC binding to cartilage. Cytokine profiling was also conducted on the MSC-secretome. Results. Hyaluronan had dose-dependent effects on growth kinetics of MSCs at concentrations of entanglement point (1 mg/mL). At higher concentrations, viscosity effects outweighed benefits of additional hyaluronan. The cartilage-adhesion assay highlighted for the first time that hyaluronan-primed MSCs increased cell attachment to cartilage whilst the presence of hyaluronan did not. Our time-course suggested patients undergoing MSC-therapy for OA could benefit from joint-immobilisation for up to 8 hours. Hyaluronan also greatly affected dispersion of MSCs on cartilage. Conclusion. Our results should be considered in future trials with MSC-therapy using hyaluronan as a vehicle, for the treatment of OA.

Osteoarthritis (OA) can be a debilitating degenerative disease and is the most common form of arthritic disease. There is a general consensus that current nonsurgical therapies are insufficient for younger OA sufferers who are not candidates for knee arthroplasties. Adipose-derived mesenchymal stem cells (MSCs) therapy for the treatment of OA can slow disease progression and lead to neocartilage formation. The mechanism of action is secretion driven. Current clinical preparations from adipose tissue for the treatment of OA include autologous stromal vascular fraction (SVF), SVF plus mature adipocytes, and culture-purified MSCs. Herein we have combined these human adipose-derived preparations with Hyaluronan (Hylan G-F 20: Synvisc) in vitro and measured alterations in cytokine profile. SVF plus mature adipocytes showed the greatest decreased in the proinflammatory cytokines IL-1β, IFN-γ, and VEGF. MCP-1 and MIP-1α decreased substantially in the SVF preparations but not the purified MSCs. The purified MSC preparation was the only one to show increase in MIF. Overall the SVF plus mature adipocytes preparation may be most suited of all the preparations for combination with HA for the treatment of OA, based on the alterations of heavily implicated cytokines in OA disease progression. This will require further validation using in vivo models.

Background Alterations in gastrointestinal, pancreatic, and adipose hormone levels may have a greater role in weight loss than initially appreciated. The laparoscopic sleeve gastrectomy (LSG) operation is now the most frequently performed bariatric operation in many countries, but there are relatively few data regarding its molecular effects. We sought to characterize the effect of LSG on fasting plasma levels of selected hormones and on non-esterified fatty acids (NEFA), and to compare these to levels in non-obese control individuals. Materials and Methods The levels of nine plasma hormones were measured using a multiplex bead-based assay at baseline and at 3 months after operation in 11 obese patients undergoing LSG. NEFA levels were also measured. The levels were compared to those for 22 age- and sex-matched non-obese individuals. Results At baseline, obese patients showed significantly higher expression of C-peptide, insulin, and leptin and significantly lower ghrelin, glucose-dependent insulinotropic peptide (GIP), and resistin compared to non-obese controls ( p  < 0.05). LSG resulted in a reduction in BMI from 42.5 ± 6.47 kg/m 2 at operation to 35.2 ± 5.14 kg/m 2 at 3 months (42 % mean excess weight loss, p  < 0.001). LSG led to a significant decrease in ghrelin, glucagon-like peptide-1 (GLP-1), glucagon, leptin, plasminogen activator inhibitor-1 (PAI-1), and NEFA. Conclusion LSG induces marked early changes in the fasting levels of factors thought to be important regulators of obesity and metabolic health. These changes differ somewhat from the findings for operations with a malabsorptive component, suggesting that subtle differences exist in the mechanisms of weight loss between LSG and other bariatric operations.

60 years after the trials of the main German war criminals, the articles in this book attempt to assess the Nuremberg Trials from a historical and legal point of view, and to illustrate connections, contradictions and consequences. In view of constantly reoccurring reports of mass crimes from all over the world, we have only reached the halfway point in the quest for an effective system of international criminal justice. With the legacy of Nuremberg in mind, this volume is a contribution to the search for answers to questions of how the law can be applied effectively and those committing crimes against humanity be brought to justice for their actions.

Oxygen sensing is central to metazoan biology and has implications for human disease. Mammalian cells express multiple oxygen-dependent enzymes called 2-oxoglutarate (OG)-dependent dioxygenases (2-OGDDs), but they vary in their oxygen affinities and hence their ability to sense oxygen. The 2-OGDD histone demethylases control histone methylation. Hypoxia increases histone methylation, but whether this reflects direct effects on histone demethylases or indirect effects caused by the hypoxic induction of the HIF (hypoxia-inducible factor) transcription factor or the 2-OG antagonist 2-hydroxyglutarate (2-HG) is unclear. Here, we report that hypoxia promotes histone methylation in a HIF- and 2-HG–independent manner. We found that the H3K27 histone demethylase KDM6A/UTX, but not its paralog KDM6B, is oxygen sensitive. KDM6A loss, like hypoxia, prevented H3K27 demethylation and blocked cellular differentiation. Restoring H3K27 methylation homeostasis in hypoxic cells reversed these effects. Thus, oxygen directly affects chromatin regulators to control cell fate.

Alginates gel rapidly under ambient conditions and have widely documented potential to form protective matrices for sensitive bioactive cargo. Most commonly, alginate gelation occurs via calcium mediated electrostatic crosslinks between the linear polyuronic acid polymers. A recent breakthrough to form crosslinked alginate microcapsules (CLAMs) by in situ gelation during spray drying (“CLAMs process”) has demonstrated applications in protection and controlled delivery of bioactives in food, cosmetics, and agriculture. The extent of crosslinking of alginates in CLAMs impacts the effectiveness of its barrier properties. For example, higher crosslinking extents can improve oxidative stability and limit diffusion of the encapsulated cargo. Crosslinking in CLAMs can be controlled by varying the calcium to alginate ratio; however, the choice of alginates used in the process also influences the ultimate extent of crosslinking. To understand how to select alginates to target crosslinking in CLAMs, we examined the roles of alginate molecular properties. A surprise finding was the formation of alginic acid gelling in the CLAMs that is a consequence of simultaneous and rapid pH reduction and moisture removal that occurs during spray drying. Thus, spray dried CLAMs gelation is due to calcium crosslinking and alginic acid formation, and unlike external gelation methods, is insensitive to the molecular composition of the alginates. The ‘extent of gelation’ of spray dried CLAMs is influenced by the molecular weights of the alginates at saturating calcium concentrations. Alginate viscosity correlates with molecular weight; thus, viscosity is a convenient criterion for selecting commercial alginates to target gelation extent in CLAMs.