Multidimensional Flow Cytometry for Testing Blood-Handling Medical Devices

Blood handling devices such as left ventricular assist devices and total artificial hearts offer life-saving treatments for patients suffering from severe heart failure. Current devices have clinically proven that heart assist pumps are a safe and effective therapy, and indeed in many cases they are the only available method of treatment. However, current devices cause side effects including stroke, bleeding, infection, and thrombosis, preventing the technology from reaching its full potential. If the side effects could be reduced, then more patients could benefit from these devices. The complications are related to damage to blood cells and proteins as a result of contact with foreign materials and mechanical stress. There is a need for better devices with minimal blood impact to enable more patients to be safely treated; better tools, especially flow cytometry, could support the device development life cycle. In this chapter we review the clinical, in vivo, and in vitro flow cytometry data available for ventricular assist devices, conduct a gap analysis, and identify areas of future possibilities for device developers to establish new flow cytometry-based methodologies

The Spring 1985 high precision baseline test of the JPL GPS-based geodetic system

The Spring 1985 High Precision Baseline Test (HPBT) was conducted. The HPBT was designed to meet a number of objectives. Foremost among these was the demonstration of a level of accuracy of 1 to 2:10 to the 7th power, or better, for baselines ranging in length up to several hundred kilometers. These objectives were all met with a high degree of success, with respect to the demonstration of system accuracy in particular. The results from six baselines ranging in length from 70 to 729 km were examined for repeatability and, in the case of three baselines, were compared to results from colocated VLBI systems. Repeatability was found to be 5:10 to the 8th power (RMS) for the north baseline coordinate, independent of baseline length, while for the east coordinate RMS repeatability was found to be larger than this by factors of 2 to 4. The GPS-based results were found to be in agreement with those from colocated VLBI measurements, when corrected for the physical separations of the VLBI and CPG antennas, at the level of 1 to 2:10 to the 7th power in all coordinates, independent of baseline length. The results for baseline repeatability are consistent with the current GPA error budget, but the GPS-VLBI intercomparisons disagree at a somewhat larger level than expected. It is hypothesized that these differences may result from errors in the local survey measurements used to correct for the separations of the GPS and VLBI antenna reference centers

Interleukin‐5 drives glycolysis and reactive oxygen species‐dependent citric acid cycling by eosinophils

Introduction Eosinophils have been long implicated in antiparasite immunity and allergic diseases and, more recently, in regulating adipose tissue homeostasis. The metabolic processes that govern eosinophils, particularly upon activation, are unknown. Methods Peripheral blood eosinophils were isolated for the analysis of metabolic processes using extracellular flux analysis and individual metabolites by stable isotope tracer analysis coupled to gas chromatography‐mass spectrometry following treatment with IL‐3, IL‐5 or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Eosinophil metabolism was elucidated using pharmacological inhibitors. Results Human eosinophils engage a largely glycolytic metabolism but also employ mitochondrial metabolism. Cytokine stimulation generates citric acid cycle (TCA) intermediates from both glucose and glutamine revealing this previously unknown role for mitochondria upon eosinophil activation. We further show that the metabolic programme driven by IL‐5 is dependent on the STAT5/PI3K/Akt signalling axis and that nicotinamide adenine dinucleotide phosphate oxidase (NOX)‐dependent ROS production might be a driver of mitochondrial metabolism upon eosinophil activation. Conclusion We demonstrate for the first time that eosinophils are capable of metabolic plasticity, evidenced by increased glucose‐derived lactate production upon ROS inhibition. Collectively, this study reveals a role for both glycolysis and mitochondrial metabolism in cytokine‐stimulated eosinophils. Selective targeting of eosinophil metabolism may be of therapeutic benefit in eosinophil‐mediated diseases and regulation of tissue homeostasis

Ovine leukocyte microparticles generated by the CentriMag ventricular assist device in vitro

Ventricular assist devices (VADs) are a life-saving form of mechanical circulatory support in heart failure patients. However, VADs have not yet reached their full potential due to the associated side effects (thrombosis, bleeding, infection) related to the activation and damage of blood cells and proteins caused by mechanical stress and foreign materials. Studies of the effects of VADs on leukocytes are limited, yet leukocyte activation and damage including microparticle generation can influence both thrombosis and infection rates. Therefore, the aim was to develop a multicolour flow cytometry assessment of leukocyte microparticles (LMPs) using ovine blood and the CentriMag VAD as a model for shear stress. Ovine blood was pumped for 6 hours in the CentriMag and regular samples analysed for haemolysis, complete blood counts and leukocyte microparticles by flow cytometry during three different pump operating conditions (low flow, standard, high speed). The high speed condition caused significant increases in plasma-free haemoglobin; decreases in total leukocytes, granulocytes, monocytes and platelets; increases in CD45+ LMPs as well as two novel LMP populations: CD11bbright/HLA-DR- and CD11bdull/HLA-DR+, both of which were CD14-/CD21-. CD11bbright/HLA-DR- LMPs appeared to respond to an increase in shear magnitude whereas the CD11bdull/HLA-DR+ LMPs significantly increased in all pumping conditions. We propose that these two populations are released from granulocytes and T cells, respectively, but further research is needed to better characterise these two populations

Interleukin 4 and interleukin 13 downregulate the lipopolysaccharide-mediated inflammatory response by human gestation-associated tissues†

Local and systemic inflammation is a key feature of preterm and term labour and delivery. Pro-inflammatory mediators are produced by gestation-associated tissues in response to a broad range of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). Interleukin (IL)-4, IL-10 and IL-13 are well recognised anti-inflammatory cytokines and might have potential as anti-inflammatory therapies to prevent preterm birth. The objective of this study was to determine if IL-4 and IL-13 exert anti-inflammatory effects on lipopolysaccharide (LPS)-stimulated production of pro-inflammatory cytokines produced by human term gestation-associated tissues (placenta, choriodecidua and amnion). Both IL-4 and IL-13 reduced LPS-stimulated IL-1β and macrophage inflammatory protein (MIP)-1α with this effect diminishing with delay to exposure to IL-4 or IL-13; there was no effect on LPS-stimulated prostaglandin production. IL-4 receptor α (IL-4Rα) was expressed throughout the placenta, choriodecidua and amnion, and the inhibitory effects of IL-4 and IL-13 were IL-4Rα-dependent. A combination of IL-4 and IL-13 did not enhance the anti-inflammatory potential of either cytokine; however, a combination of IL-4 and IL-10 had a greater anti-inflammatory effect than either cytokine alone. These findings demonstrate that human term gestation-associated tissues are responsive to the anti-inflammatory cytokines IL-4 and IL-13, which could down-regulate LPS induced cytokine production in these tissues. Anti-inflammatory cytokines might offer an adjunct to existing therapeutics to prevent adverse obstetric outcome

Production and regulation of interleukin-1 family cytokines at the materno-fetal interface

IL-1 family members regulate innate immune responses, are produced by gestation-associated tissues, and have a role in healthy and adverse pregnancy outcomes. To better understand their role at the materno-fetal interface we used a human tissue explant model to map lipopolysaccharide (LPS)-stimulated production of IL-1α, IL-1β, IL-18, IL-33, IL-1Ra, IL-18BPa, ST2 and IL-1RAcP by placenta, choriodecidua and amnion. Caspase-dependent processing of IL-1α, IL-1β, IL-18, and IL-33 and the ability of IL-1α, IL-1β, IL-18, and IL-33 to regulate the production of IL-1RA, IL-18BPa, ST2 and IL-1RAcP was also determined. LPS acted as a potent inducer of IL-1 family member expression especially in the placenta and choriodecidua with the response by the amnion restricted to IL-1β. Caspases-1, 4 and 8 contributed to LPS-stimulated production of IL-1 and IL-18, whereas calpain was required for IL-1 production. Exogenous administration of IL-1α, IL-1β, IL-18, and IL-33 lead to differential expression of IL-1Ra, IL-18BPa, ST2 and IL-1RAcP across all tissues examined. Most notable were the counter-regulatory effect of LPS on IL-1 and IL-1Ra in the amnion and the broad responsiveness of the amnion to IL-1 family cytokines for increased production of immunomodulatory peptides and soluble receptors. The placenta and membranes vary not only in their output of various IL-1 family members but also in their counter-regulatory mechanisms through endogenous inhibitory peptides, processing enzymes and soluble decoy receptors. This interactive network of inflammatory mediators likely contributes to innate defence mechanisms at the materno-fetal interface to limit, in particular, the detrimental effects of microbial invasion

Expression and function of NOD-like receptors by human term gestation-associated tissues

Introduction: Nucleotide-binding oligomerization domain (NOD)-like receptors or NOD-like receptors (NLRs) have been implicated in several disease pathologies associated with inflammation. Since local and systemic inflammation is a hallmark of both term and preterm labour, a role for NLRs at the materno-fetal interface has been postulated. Methods: Gene expression and immunolocalisation of NLR family members in human placenta, choriodecidua, and amnion were examined. Tissue explants were used to examine the response to activators of NOD1 (Tri-DAP), NOD2 (MDP) and NLRP3 (nigericin). Cell/tissue-free supernatants were examined for the production of interleukin (IL)-1, IL-6, IL-8 and IL-10 using specific ELISAs. Results: Expression of transcripts for NOD1, NOD2, NLRP3, NLRC4, NLRX1, NLRP1 and NAIP and protein expression of NOD1, NOD2 and NLRP3 were a broad feature of all term gestation-associated tissues. Production of cytokines was increased significantly in response to all ligands in placenta and choriodecidua, except for MDP-induced IL-10. Similarly, there was a significant increase in cytokine production in the amnion except for MDP induced IL-1β and IL-10 response to either agonist. IL-1β production was dependent on caspase-1 regardless of agonist used or tissue examined.Discussion: Term human gestation-associated tissues express functional NLRs which likely play a role in both sterile and pathogen-driven inflammatory responses at the materno-fetal interface

Molecular Mechanisms of Neonatal Brain Injury

Fetal/neonatal brain injury is an important cause of neurological disability. Hypoxia-ischemia and excitotoxicity are considered important insults, and, in spite of their acute nature, brain injury develops over a protracted time period during the primary, secondary, and tertiary phases. The concept that most of the injury develops with a delay after the insult makes it possible to provide effective neuroprotective treatment after the insult. Indeed, hypothermia applied within 6 hours after birth in neonatal encephalopathy reduces neurological disability in clinical trials. In order to develop the next generation of treatment, we need to know more about the pathophysiological mechanism during the secondary and tertiary phases of injury. We review some of the critical molecular events related to mitochondrial dysfunction and apoptosis during the secondary phase and report some recent evidence that intervention may be feasible also days-weeks after the insult