Cytokine changes in sickle-cell disease patients as markers predictive of the onset of delayed hemolytic transfusion reactions.

BACKGROUND: Changes in cytokine production are known to contribute to the pathogenesis of sickle-cell disease (SCD), particularly in painful acute complications (crises) and episodes of post-transfusion hemolysis. Little is known about cytokine profiles in patients with these complications. STUDY DESIGN AND METHODS: We investigated possible associations between cytokine profile and the onset of delayed hemolytic transfusion reactions (DHTRs), particularly during acute-phase episodes, to improve characterization of the biological parameters predictive of such events. We included SCD patients with severe acute symptoms (n = 36) or steady-state disease (n = 31), both possibly leading to a DHTR (n = 18) event. Luminex® technology was used to determine the plasma concentrations of 23 cytokines. RESULTS: Regardless of clinical context, the concentrations of interleukin (IL)-6, IL-10, inducible protein-10, and macrophage inflammatory protein-1ß were higher in plasma samples from SCD patients than in those from healthy controls. IL-6 and IL-10 concentrations were even higher in acute-phase plasma samples from SCD patients. In addition, IL-27 and TNFα levels were higher, and IL-6 and RANTES levels were lower in acute-phase SCD patients just before the onset of DHTR than in patients experiencing painful occlusive episodes. CONCLUSION: In addition to reporting the plasma cytokine profiles of SCD patients in various clinical phases of the disease, we provide the first evidence of a significant association between low plasma TNFα concentration, high plasma IP-10 concentration and the onset of DHTR in SCD patients.

Evidence of benefits from using fresh and cryopreserved blood to transfuse patients with acute sickle cell disease.

BACKGROUND: The transfusion of red blood cell (RBC) concentrates is the main treatment for acute vaso-occlusive symptoms in sickle cell disease (SCD). Units of packed RBCs (pRBCs) must retain optimal characteristics for transfusion throughout the storage period. Transfused RBCs interact with the plasma and the endothelium that lines blood vessels and may be the target of immune-hematologic conflict if the patient produces antibodies against RBCs. Questions remain concerning the benefit-risk balance of RBC transfusions, in particular about the shelf-life of the units. STUDY DESIGN AND METHODS: Plasma samples from 33 hemoglobin SS patients with SCD who had severe acute-phase symptoms or were in steady-state were put in contact with 10 fresh-stored and older stored samples from the same 10 RBC units. The factors affecting RBC survival (phosphatidylserine exposure, cytosolic calcium influx, cell size reduction) were analyzed. RESULTS: We show that the effects of plasma samples from patients with SCD on pRBCs depend on the clinical condition of the patients and the duration of red cell storage. Signs of RBC senescence were correlated with the clinical status of the patient from whom the plasma sample was obtained. A decrease in RBC size and an increase in phosphatidylserine exposure were correlated with the duration of RBC storage. The behavior of cryopreserved pRBCs was similar to that of fresh refrigerated RBCs when challenged with patient plasma samples. CONCLUSION: The key points of this study are that the clinical condition of patients with SCD can negatively affect the integrity of pRBCs for transfusion, and those effects increase with longer storage. Also, cryopreserved pRBCs behave similarly to fresh RBCs when challenged with plasma samples from patients with SCD in acute phase. Our data provide the first evidence that fresh RBCs stored for short periods may be of greater benefit to patients with SCD than RBCs that have been refrigerated for longer periods, particularly for those who have acute symptoms of SCD.

Electric pulses: a flexible tool to manipulate cytosolic calcium concentrations and generate spontaneous-like calcium oscillations in mesenchymal stem cells.

Human adipose mesenchymal stem cells (haMSCs) are multipotent adult stem cells of great interest in regenerative medicine or oncology. They present spontaneous calcium oscillations related to cell cycle progression or differentiation but the correlation between these events is still unclear. Indeed, it is difficult to mimic haMSCs spontaneous calcium oscillations with chemical means. Pulsed electric fields (PEFs) can permeabilise plasma and/or organelles membranes depending on the applied pulses and therefore generate cytosolic calcium peaks by recruiting calcium from the external medium or from internal stores. We show that it is possible to mimic haMSCs spontaneous calcium oscillations (same amplitude, duration and shape) using 100 µs PEFs or 10 ns PEFs. We propose a model that explains the experimental situations reported. PEFs can therefore be a flexible tool to manipulate cytosolic calcium concentrations. This tool, that can be switched on and off instantaneously, contrary to chemicals agents, can be very useful to investigate the role of calcium oscillations in cell physiology and/or to manipulate cell fate.

Robust, efficient, and practical electrogene transfer method for human mesenchymal stem cells using square electric pulses.

Mesenchymal stem cells (MSCs) are multipotent nonhematopoietic cells with the ability to differentiate into various specific cell types, thus holding great promise for regenerative medicine. Early clinical trials have proven that MSC-based therapy is safe, with possible efficacy in various diseased states. Moreover, genetic modification of MSCs to improve their function can be safely achieved using electrogene transfer. We previously achieved transfection efficiencies of up to 32% with preserved viability in rat MSCs. In this study, we further improved the transfection efficiency and transgene expression in human MSCs (hMSCs), while preserving the cells viability and ability to differentiate into osteoblasts and adipocytes by increasing the plasmid concentration and altering the osmotic pressure of the electrotransfer buffer. Using a square-wave electric pulse generator, we achieved a transfection efficiency of more than 80%, with around 70% viability and a detectable transgene expression of up to 30 days. Moreover, we demonstrated that this transfection efficiency can be reproduced reliably on two different sources of hMSCs: the bone marrow and adipose tissue. We also showed that there was no significant donor variability in terms of their transfection efficiency and viability. The cell confluency before electrotransfer had no significant effect on the transfection efficiency and viability. Cryopreservation of transfected cells maintained their transgene expression and viability upon thawing. In summary, we are reporting a robust, safe, and efficient protocol of electrotransfer for hMSCs with several practical suggestions for an optimal use of genetically engineered hMSCs for clinical application.

Effects of dimethyl sulfoxide in cholesterol-containing lipid membranes: a comparative study of experiments in silico and with cells.

Dimethyl sulfoxide (DMSO) has been known to enhance cell membrane permeability of drugs or DNA. Molecular dynamics (MD) simulations with single-component lipid bilayers predicted the existence of three regimes of action of DMSO: membrane loosening, pore formation and bilayer collapse. We show here that these modes of action are also reproduced in the presence of cholesterol in the bilayer, and we provide a description at the atomic detail of the DMSO-mediated process of pore formation in cholesterol-containing lipid membranes. We also successfully explore the applicability of DMSO to promote plasma membrane permeability to water, calcium ions (Ca(2+)) and Yo-Pro-1 iodide (Yo-Pro-1) in living cell membranes. The experimental results on cells in culture can be easily explained according to the three expected regimes: in the presence of low doses of DMSO, the membrane of the cells exhibits undulations but no permeability increase can be detected, while at intermediate DMSO concentrations cells are permeabilized to water and calcium but not to larger molecules as Yo-Pro-1. These two behaviors can be associated to the MD-predicted consequences of the effects of the DMSO at low and intermediate DMSO concentrations. At larger DMSO concentrations, permeabilization is larger, as even Yo-Pro-1 can enter the cells as predicted by the DMSO-induced membrane-destructuring effects described in the MD simulations.