Red Blood Cell Contamination of the Final Cell Product Impairs the Efficacy of Autologous Bone Marrow Mononuclear Cell Therapy

ObjectivesThe aim of this study was to identify an association between the quality and functional activity of bone marrow-derived progenitor cells (BMCs) used for cardiovascular regenerative therapies and contractile recovery in patients with acute myocardial infarction included in the placebo-controlled REPAIR-AMI (Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction) trial.BackgroundIsolation procedures of autologous BMCs might affect cell functionality and therapeutic efficacy.MethodsQuality of cell isolation was assessed by measuring the total number of isolated BMCs, CD34+ and CD133+ cells, their colony-forming unit (CFU) and invasion capacity, cell viability, and contamination of the final BMC preparation with thrombocytes and red blood cells (RBCs).ResultsThe number of RBCs contaminating the final cell product significantly correlated with reduced recovery of left ventricular ejection fraction 4 months after BMC therapy (p = 0.007). Higher numbers of RBCs in the BMC preparation were associated with reduced BMC viability (r = −0.23, p = 0.001), CFU capacity (r = −0.16, p = 0.03), and invasion capacity (r = −0.27, p < 0.001). To assess a causal role for RBC contamination, we coincubated isolated BMCs with RBCs for 24 h in vitro. The addition of RBCs dose-dependently abrogated migratory capacity (p = 0.003) and reduced CFU capacity (p < 0.05) of isolated BMCs. Neovascularization capacity was significantly impaired after infusion of BMCs contaminated with RBCs, compared with BMCs alone (p < 0.05). Mechanistically, the addition of RBCs was associated with a profound reduction in mitochondrial membrane potential of BMCs.ConclusionsContaminating RBCs affects the functionality of isolated BMCs and determines the extent of left ventricular ejection fraction recovery after intracoronary BMC infusion in patients with acute myocardial infarction. These results suggest a bioactivity response relationship very much like a dose–response relationship in drug trials. (Reinfusion of Enriched Progenitor cells and Infarct Remodeling in Acute Myocardial Infarction [REPAIR-AMI]; NCT00279175

Streptococcus pneumoniae Serotype 1 Capsular Polysaccharide Induces CD8+CD28− Regulatory T Lymphocytes by TCR Crosslinking

Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an α-helix configuration. In this paper we look at the immune response of CD8+ T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8+CD28− T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8+CD28− T lymphocytes. The Sp1-induced CD8+CD28− T lymphocytes are CD122lowCTLA-4+CD39+. They synthesize IL-10 and TGF-β. The Sp1-induced CD8+CD28− T cells exhibit immunosuppressive properties on CD4+ T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8+ T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8+ T cell activation by enhancing crosslinking of TCR. The expansion of CD8+CD28− T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8+CD28− T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-κB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8+CD28− population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8+CD28− T lymphocytes in vivo and in vitro. The underlying mechanism of CD8+ T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system

Nonstandard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty-nonstandard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for more reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

Non-Standard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty: Non-standard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for better reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

Dimethylfumarate induces apoptosis in human mast cells

Mast cells modulate autoimmune diseases such as psoriasis and multiple sclerosis. Fumaric acid esters (FAEs) are widely used for the treatment of psoriasis, and dimethylfumarate (DMF) has recently been approved for multiple sclerosis. In this study, we analysed the cytotoxic effect of FAEs on human mast cells. Specifically, cell death was analysed in the human mast cell line HMC-1 and in primary cord blood-derived mast cells (CBMCs) after incubation with fumaric acid (FA), monomethylfumarate (MMF), DMF and calcium bis(monomethylfumarate) (Ca-MF). Our data show that only DMF potently induces apoptotic cell death in HMC-1 cells and CBMCs. DMF-mediated apoptosis was associated with increased expression of Bax and Bak and activation of caspase-9 and caspase-6. Interestingly, DMF also enhanced the sensitivity of CBMCs towards TRAIL- and dexamethasone-induced apoptosis. These findings demonstrate for the first time that DMF induces apoptosis of human mast cells, primarily via the mitochondrial apoptotic pathway. Our study contributes to the understanding of the beneficial effects of FAEs in autoimmune diseases and provides a rationale for exploiting FAEs for other diseases associated with mast cells

Obesity Promotes Liver Carcinogenesis via Mcl-1 Stabilization Independent of IL-6Rα Signaling

Obesity increases the incidence of hepatocellular carcinoma (HCC) development in part through the activation of obesity-associated proinflammatory signaling. Here, we show that in lean mice, abrogation of IL-6Rα signaling protects against diethylnitrosamine (DEN)-induced HCC development. HCC protection occurs via Mcl-1 destabilization, thus promoting hepatocyte apoptosis. IL-6 regulates Mcl-1 stability via the inhibition of PP-1α expression, promoting GSK-3β inactivation. In addition, IL-6 suppresses expression of the Mcl-1 E3 ligase (Mule). Consequently, IL-6Rα deficiency activates PP-1α and Mule expression, resulting in increased Mcl-1 turnover and protection against HCC development. In contrast, in obesity, inhibition of PP-1α and Mule expression, leading to Mcl-1 stabilization, occurs independently of IL-6 signaling. Collectively, this study provides evidence that obesity inhibits hepatocyte apoptosis through Mcl-1 stabilization independent of IL-6 signaling, thus promoting liver carcinogenesis

Interleukin-6 is essential for zwitterionic polysaccharide-mediated abscess formation

Abscess formation associated with secondary peritonitis causes severe morbidity and can be fatal. Formation of abscesses requires the presence of CD4(+) T-cells. Zwitterionic polysaccharides (ZPSs) represent a novel class of immunomodulatory bacterial antigens that stimulate CD4(+) T-cells in a major histocompatibility complex (MHC) class II-dependent manner. The capsular polysaccharide Sp1 of Streptococcus pneumoniae serotype 1 possesses a zwitterionic charge with free amino groups and promotes T-cell-dependent abscess formation in an experimental mouse model. So far, nothing is known about the function of Interleukin (IL)-6 in intraperitoneal abscess formation. Here, we demonstrate that macrophages and dendritic cells (DCs), the most prevalent professional antigen-presenting cells involved in the formation of abscesses, secrete Interleukin (IL)-6 and are incorporated in the abscess capsule. Sp1 inhibits apoptosis of CD4(+) T-cells and causes IL-17 expression by CD4(+) T-cells in an IL-6-dependent manner. Abrogation of the Sp1-induced pleiotropic effects of IL-6 in IL-6-deficient mice and mice treated with an IL-6- specific neutralizing antibody results in significant inhibition of abscess formation. The data delineate the essential role of IL-6 in the linkage of innate and adaptive immunity in polysaccharide-mediated abscess formation

BAX/BAK-Induced Apoptosis Results in Caspase-8-Dependent IL-1β Maturation in Macrophages.

IL-1β is a cytokine of pivotal importance to the orchestration of inflammatory responses. Synthesized as an inactive pro-cytokine, IL-1β requires proteolytic maturation to gain biological activity. Here, we identify intrinsic apoptosis as a non-canonical trigger of IL-1β maturation. Guided by the discovery of the immunomodulatory activity of vioprolides, cyclic peptides isolated from myxobacteria, we observe IL-1β maturation independent of canonical inflammasome pathways, yet dependent on intrinsic apoptosis. Mechanistically, vioprolides inhibit MCL-1 and BCL2, which in turn triggers BAX/BAK-dependent mitochondrial outer membrane permeabilization (MOMP). Induction of MOMP results in the release of pro-apoptotic factors initiating intrinsic apoptosis, as well as the depletion of IAPs (inhibitors of apoptosis proteins). IAP depletion, in turn, operates upstream of ripoptosome complex formation, subsequently resulting in caspase-8-dependent IL-1β maturation. These results establish the ripoptosome/caspase-8 complex as a pro-inflammatory checkpoint that senses the perturbation of mitochondrial integrity