Maternal body mass index is associated with an altered immunological profile at 28 weeks of gestation.

Healthy pregnancy is accompanied by various immunological and metabolic adaptations. Maternal obesity has been implicated in adverse pregnancy outcomes such as miscarriage, preeclampsia, and gestational diabetes mellitus (GDM), while posing a risk to the neonate. There is a lack of knowledge surrounding obesity and the maternal immune system. The objective of this study was to consider if immunological changes in pregnancy are influenced by maternal obesity. Peripheral blood was collected from fasted GDM-negative pregnant women at 26-28 weeks of gestation. Analysis was done using immunoassay, flow cytometry, bioenergetics analysis, and cell culture. The plasma profile was significantly altered with increasing BMI, specifically leptin (r = 0.7635), MCP-1 (r = 0.3024), and IL-6 (r = 0.4985). Circulating leukocyte populations were also affected with changes in the relative abundance of intermediate monocytes (r = -0.2394), CD4:CD8 T-cell ratios (r = 0.2789), and NKT cells (r = -0.2842). Monocytes analysed in more detail revealed elevated CCR2 expression and decreased mitochondrial content with increased BMI. However, LPS-stimulated cytokine production and bioenergetic profile of PBMCs were not affected by maternal BMI. The Th profile skews towards Th17 with increasing BMI; Th2 (r = -0.3202) and Th9 (r = -0.3205) cells were diminished in maternal obesity, and CytoStim™-stimulation exacerbates IL-6 (r = 0.4166), IL-17A (r = 0.2753), IL-17F (r = 0.2973), and IL-22 (r = 0.2257) production with BMI, while decreasing IL-4 (r = -0.2806). Maternal obesity during pregnancy creates an inflammatory microenvironment. Successful pregnancy requires Th2-biased responses yet increasing maternal BMI favours a Th17 response that could be detrimental to pregnancy. Further research should investigate key populations of cells identified here to further understand the immunological challenges that beset pregnant women with obesity.

Immunometabolic adaptation and immune plasticity in pregnancy and the bi-directional effects of obesity.

Mandatory maternal metabolic and immunological changes are essential to pregnancy success. Parallel changes in metabolism and immune function make immunometabolism an attractive mechanism to enable dynamic immune adaptation during pregnancy. Immunometabolism is a burgeoning field with the underlying principle being that cellular metabolism underpins immune cell function. With whole body changes to the metabolism of carbohydrates, protein and lipids well recognised to occur in pregnancy and our growing understanding of immunometabolism as a determinant of immunoinflammatory effector responses, it would seem reasonable to expect immune plasticity during pregnancy to be linked to changes in the availability and handling of multiple nutrient energy sources by immune cells. While studies of immunometabolism in pregnancy are only just beginning, the recognised bi-directional interaction between metabolism and immune function in the metabolic disorder obesity might provide some of the earliest insights into the role of immunometabolism in immune plasticity in pregnancy. Characterised by chronic low-grade inflammation including in pregnant women, obesity is associated with numerous adverse outcomes during pregnancy and beyond for both mother and child. Concurrent changes in metabolism and immunoinflammation are consistently described but any causative link is not well established. Here we provide an overview of the metabolic and immunological changes that occur in pregnancy and how these might contribute to healthy versus adverse pregnancy outcomes with special consideration of possible interactions with obesity.

A role for metabolism in determining neonatal immune function.

Immune responses of neonates differ markedly to those of adults, with skewed cytokine phenotypes, reduced inflammatory properties and drastically diminished memory function. Recent research efforts have started to unravel the role of cellular metabolism in determining immune cell fate and function. For studies in humans, much of the work on metabolic mechanisms underpinning innate and adaptive immune responses by different haematopoietic cell types is in adults. Studies investigating the contribution of metabolic adaptation in the unique setting of early life are just emerging, and much more work is needed to elucidate the contribution of metabolism to neonatal immune responses. Here, we discuss our current understanding of neonatal immune responses, examine some of the latest developments in neonatal immunometabolism and consider the possible role of altered metabolism to the distinctive immune phenotype of the neonate. Understanding the role of metabolism in regulating immune function at this critical stage in life has direct benefit for the child by affording opportunities to maximize immediate and long-term health. Additionally, gaining insight into the diversity of human immune function and naturally evolved immunometabolic strategies that modulate immune function could be harnessed for a wide range of opportunities including new therapeutic approaches.

Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning.

The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However, it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25-5.0 µg/mL) and/or carbendazim (0.8-1.6 µg/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the "DeepFlow" neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for 'mononucleates', 'binucleates', 'mononucleates with MN' and 'binucleates with MN', respectively. Successful classifications of 'trinucleates' (90%) and 'tetranucleates' (88%) in addition to 'other or unscorable' phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful (≤ 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent concentration regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks.

Jellyfish Collagen: A Biocompatible Collagen Source for 3D Scaffold Fabrication and Enhanced Chondrogenicity.

Osteoarthritis (OA) is a multifactorial disease leading to degeneration of articular cartilage, causing morbidity in approximately 8.5 million of the UK population. As the dense extracellular matrix of articular cartilage is primarily composed of collagen, cartilage repair strategies have exploited the biocompatibility and mechanical strength of bovine and porcine collagen to produce robust scaffolds for procedures such as matrix-induced chondrocyte implantation (MACI). However, mammalian sourced collagens pose safety risks such as bovine spongiform encephalopathy, transmissible spongiform encephalopathy and possible transmission of viral vectors. This study characterised a non-mammalian jellyfish (Rhizostoma pulmo) collagen as an alternative, safer source in scaffold production for clinical use. Jellyfish collagen demonstrated comparable scaffold structural properties and stability when compared to mammalian collagen. Jellyfish collagen also displayed comparable immunogenic responses (platelet and leukocyte activation/cell death) and cytokine release profile in comparison to mammalian collagen in vitro. Further histological analysis of jellyfish collagen revealed bovine chondroprogenitor cell invasion and proliferation in the scaffold structures, where the scaffold supported enhanced chondrogenesis in the presence of TGFß1. This study highlights the potential of jellyfish collagen as a safe and biocompatible biomaterial for both OA repair and further regenerative medicine applications.

Diffusion Mapping of Eosinophil-Activation State.

Eosinophils are granular leukocytes that play a role in mediating inflammatory responses linked to infection and allergic disease. Their activation during an immune response triggers spatial reorganization and eventual cargo release from intracellular granules. Understanding this process is important in diagnosing eosinophilic disorders and in assessing treatment efficacy; however, current protocols are limited to simply quantifying the number of eosinophils within a blood sample. Given that high optical absorption and scattering by the granular structure of these cells lead to marked image features, the physical changes that occur during activation should be trackable using image analysis. Here, we present a study in which imaging flow cytometry is used to quantify eosinophil activation state, based on the extraction of 85 distinct spatial features from dark-field images formed by light scattered orthogonally to the illuminating beam. We apply diffusion mapping, a time inference method that orders cells on a trajectory based on similar image features. Analysis of exogenous cell activation using eotaxin and endogenous activation in donor samples with elevated eosinophil counts shows that cell position along the diffusion-path line correlates with activation level (99% confidence level). Thus, the diffusion mapping provides an activation metric for each cell. Assessment of activated and control populations using both this spatial image-based, activation score and the integrated side-scatter intensity shows an improved Fisher discriminant ratio rd = 0.7 for the multivariate technique compared with an rd = 0.47 for the traditional whole-cell scatter metric. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

In Vitro Benchmarking Study of Ventricular Assist Devices in Current Clinical Use.

BACKGROUND: Left ventricular assist devices (LVADs) offer live-saving therapy to transplant-ineligible heart failure patients. A major limitation of the technology includes pump thrombosis, bleeding, and recurrent infection that prove difficult to predict from in vivo animal testing. Shear stress introduced by the LVAD affects more than just hemolysis because platelets, leukocytes, and plasma proteins all contribute to the propensity for complications. It is important to assess overall damage by a new device against a baseline as early as possible in the development process so that design iterations can be made if required. METHODS: Explanted VADs currently in clinical use (HeartMate 2 and HVAD) were carefully cleaned, inspected, and run at 5 L/min and pressure at 100 mmHg in a standard 500 mL mock circulatory loop using bovine blood. The CentriMag was used as a control pump because of its low blood damage profile. Samples were collected at regular intervals and the following were analyzed: complete cell counts, hemolysis, platelet activation, leukocyte-derived microparticles (LMPs), and von Willebrand factor (vWF) degradation. RESULTS: The HeartMate 2 had the highest levels of hemolysis and platelet activation after 6 hours compared with the HVAD and CentriMag. A decreased granulocyte count, high numbers of LMPs and CD11bBrightHLADR- LMPs, and decreased vWF collagen binding activity was most evident in the HVAD. CONCLUSIONS: The results indicate that it is possible to observe differences between different pump designs during in vitro testing that might translate to clinical performance. This study demonstrates the importance of developing standard in vitro total blood damage methods against which device developers could use to modify design to reduce complication risk long before implantation.

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.

Nanomaterials and Innate Immunity: A Perspective of the Current Status in Nanosafety.

Human exposure to engineered nanomaterials (ENMs) is inevitable due to the plethora of applications for which they are being manufactured and integrated within. ENMs demonstrate plentiful advantages in terms of industrial approaches as well as from a consumer perspective. However, despite such positives, doubts remain over the human health implications of ENM exposure. In light of the increased research focus upon the potential effects of ENM exposure to human health in recent decades, questions still remain regarding the safety of these highly advanced, precision-tuned physical entities. The risk of short-term, high-dose exposure to humans is considered relatively low, although this has formed the direction of the hazard-assessment community since the turn of the 21st century. However, the possibility of humans being exposed repeatedly over a long period of time to a low-dose of ENMs of varying physicochemical characteristics is of significant concern, and thus, industry, government, academic, and consumer agencies are only now beginning to consider this. Notably, when considering the human health implications of such low-dose, long-term, repeated exposure scenarios, the impact of ENMs upon the human immune system is of primary importance. However, there remains a real need to understand the impact of ENMs upon the human immune system, especially the innate immune system, at all stages of life, given exposure to nanosized particles begins before birth, that is, of the fetus. Therefore, the purpose of this perspective is to summarize what is currently known regarding ENM exposure of different components of the innate immune system and identify knowledge gaps that should be addressed if we are to fully deduce the impact of ENM exposure on innate immune function.

Label-Free Identification of White Blood Cells Using Machine Learning.

White blood cell (WBC) differential counting is an established clinical routine to assess patient immune system status. Fluorescent markers and a flow cytometer are required for the current state-of-the-art method for determining WBC differential counts. However, this process requires several sample preparation steps and may adversely disturb the cells. We present a novel label-free approach using an imaging flow cytometer and machine learning algorithms, where live, unstained WBCs were classified. It achieved an average F1-score of 97% and two subtypes of WBCs, B and T lymphocytes, were distinguished from each other with an average F1-score of 78%, a task previously considered impossible for unlabeled samples. We provide an open-source workflow to carry out the procedure. We validated the WBC analysis with unstained samples from 85 donors. The presented method enables robust and highly accurate identification of WBCs, minimizing the disturbance to the cells and leaving marker channels free to answer other biological questions. It also opens the door to employing machine learning for liquid biopsy, here, using the rich information in cell morphology for a wide range of diagnostics of primary blood. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Artificial shear stress effects on leukocytes at a biomaterial interface.

Medical devices, such as ventricular assist devices (VADs), introduce both foreign materials and artificial shear stress to the circulatory system. The effects these have on leukocytes and the immune response are not well understood. Understanding how these two elements combine to affect leukocytes may reveal why some patients are susceptible to recurrent device-related infections and provide insight into the development of pump thrombosis. Biomaterials-DLC: diamond-like carbon-coated stainless steel; Sap: single-crystal sapphire; and Ti: titanium alloy (Ti6 Al4 V) were attached to the parallel plates of a rheometer. Whole human blood was left between the two discs for 5 minutes at +37°C with or without the application of shear stress (0 s-1 or 1000 s-1 ). Blood was removed and used for complete blood cell counts, flow cytometry (leukocyte activation, cell death, microparticle generation, phagocytic ability, and reactive oxygen species [ROS] production), and the production of pro-inflammatory cytokines. L-selectin expression on monocytes was decreased when blood was exposed to the biomaterials both with and without shear. Applying shear stress to blood on a Sap and Ti surface led to activation of neutrophils shown as decreased L-selectin expression. Sap and Ti blunted the LPS-stimulated macrophage migration inhibitory factor (MIF) production, most notably when sheared on Ti. The biomaterials used here have been shown to activate leukocytes in a static environment. The introduction of shear appears to exacerbate this activation. Interestingly, a widely accepted biocompatible material (Ti) utilized in many different types of devices has the capacity for immune cell activation and inhibition of MIF secretion when combined with shear stress. These findings contribute to our understanding of the contribution of biomaterials and shear stress to recurrent infections and vulnerability to sepsis in some VAD patients as well as pump thrombosis.

Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro.

Use of imaging flow cytometry to assess induced DNA damage via the cytokinesis block micronucleus (CBMN) assay has thus far been limited to radiation dosimetry in human lymphocytes using high end, 'ImageStream X' series imaging cytometers. Its potential to enumerate chemically induced DNA damage using in vitro cell lines remains unexplored. In the present manuscript, we investigate the more affordable FlowSight® imaging cytometry platform to assess in vitro micronucleus (MN) induction in the human lymphoblastoid TK6 and metabolically competent MCL-5 cells treated with Methyl Methane Sulfonate (MMS) (0-5 µg/ml), Carbendazim (0-1.6 µg/ml), and Benzo[a]Pyrene (B[a]P) (0-6.3 µg/ml) for a period of 1.5-2 cell-cycles. Cells were fixed, and nuclei and MN were stained using the fluorescent nuclear dye DRAQ5™. Image acquisition was carried out using a 20X objective on a FlowSight® imaging cytometer (Amnis, part of Merck Millipore) equipped with a 488 nm laser. Populations of ∼20000 brightfield cell images, alongside DRAQ5™ stained nuclei/MN were rapidly collected (≤10 min). Single, in-focus cells suitable for scoring were then isolated using the IDEAS® software. An overlay of the brightfield cell outlines and the DRAQ5 nuclear fluorescence was used to facilitate scoring of mono-, bi-, tri-, and tetra-nucleated cells with or without MN events and in context of the cytoplasmic boundary of the parent cell.To establish the potential of the FlowSight® platform, and to establish 'ground truth' cell classification for the supervised machine learning based scoring algorithm that represents the next stage of our project, the captured images were scored manually. Alongside, MN frequencies were also derived using the 'gold standard' light microscopy and manual scoring. A minimum of 3000 bi-nucleated cells were assessed using both approaches. Using the benchmark dose approach, the comparability of genotoxic potency estimations for the different compounds and cell lines was assessed across the two scoring platforms as highly similar. This study therefore provides essential proof-of-concept that FlowSight® imaging cytometry is capable of reproducing the results of 'gold standard' manual scoring by light microscopy. We conclude that, with the right automated scoring algorithm, imaging flow cytometry could revolutionise the reportability and scoring throughput of the CBMN assay.

A high-throughput serum Raman spectroscopy platform and methodology for colorectal cancer diagnostics.

Vibrational spectroscopic techniques such as Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR) have huge potential for the analysis of biological specimens. The techniques allow the user to gain label-free, non-destructive biochemical information about a given sample. Previous studies using vibrational spectroscopy with the specific application of diagnosing colorectal diseases such as cancer have mainly focused on in vivo or in vitro studies of tissue specimens using microscopy or probe based techniques. There have been few studies of vibrational spectroscopic techniques based on the analysis of blood serum for the advancement of colorectal cancer diagnostics. With growing interest in the field of liquid biopsies, this study presents the development of a high-throughput (HT) serum Raman spectroscopy platform and methodology and compares dry and liquid data acquisition of serum samples. This work considers factors contributing to translatability of the methodologies such as HT design, inter-user variability and sample handling effects on diagnostic capability. The HT Raman methods were tested on a pilot dataset of serum from 30 cancer patients and 30 matched control patients using statistical analysis via cross-validated PLS-DA with a maximum achieved a sensitivity of 83% and specificity of 83% for detecting colorectal cancer.

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 multicolor 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 h in the CentriMag and regular samples analyzed for hemolysis, complete blood counts and LMP by flow cytometry during three different pump operating conditions (low flow, standard, high speed). The high speed condition caused significant increases in plasma-free hemoglobin; 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 characterize these two populations.

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

Inflammation is a key feature of preterm and term labor. Proinflammatory mediators are produced by gestation-associated tissues in response to pathogen-associated molecular patterns and damage-associated molecular patterns. Interleukin (IL)4, IL10, and IL13 are anti-inflammatory cytokines with potential as anti-inflammatory therapies to prevent preterm birth. The objective of this study was to determine if IL4 and IL13 exert anti-inflammatory effects on lipopolysaccharide (LPS)-stimulated production of proinflammatory cytokines produced by human term gestation-associated tissues (placenta, choriodecidua, and amnion). Both IL4 and IL13 reduced LPS-stimulated IL1B and macrophage inflammatory protein1A; this effect diminished with delay to exposure to either cytokine. There was no effect on LPS-stimulated prostaglandin production. Interleukin 4 receptor alpha (IL4RA) was expressed throughout the placenta, choriodecidua, and amnion, and the inhibitory effects of IL4 and IL13 were IL4RA dependent. Combined IL4 and IL13 did not enhance the anti-inflammatory potential of either cytokine; however, a combination of IL4 and IL10 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 IL4 and IL13, which could downregulate 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-1ß, IL-18, IL-33, IL-1Ra, IL-18BPa, ST2 and IL-1RAcP by placenta, choriodecidua and amnion. Caspase-dependent processing of IL-1α, IL-1ß, IL-18, and IL-33 and the ability of IL-1α, IL-1ß, 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-1ß. Caspases-1, 4 and 8 contributed to LPS-stimulated production of IL-1ß and IL-18, whereas calpain was required for IL-1α production. Exogenous administration of IL-1α, IL-1ß, 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-1ß 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.

Development of an in vitro PIG-A gene mutation assay in human cells.

Mutagens can be carcinogens, and traditionally, they have been identified in vitro using the Salmonella 'Ames' reverse mutation assay. However, prokaryotic DNA packaging, replication and repair systems are mechanistically very different to those in the humans we inevitably seek to protect. Therefore, for many years, mammalian cell line genotoxicity assays that can detect eukaryotic mutagens as well as clastogens and aneugens have been used. The apparent lack of specificity in these largely rodent systems, due partly to their mutant p53 status, has contributed to the use of animal studies to resolve data conflicts. Recently, silencing mutations at the PIG-A locus have been demonstrated to prevent glycophosphatidylinositol (GPI) anchor synthesis and consequentially result in loss of GPI-anchored proteins from the cell's extracellular surface. The successful exploitation of this mutant phenotype in animal studies has triggered interest in the development of an analogous in vitro PIG-A mutation screening assay. This article describes the development of a robust assay design using metabolically active human cells. The assay includes viability and cell membrane integrity assessment and conforms to the future ideas of the 21st-century toxicology testing.

Evaluation of the automated MicroFlow® and Metafer™ platforms for high-throughput micronucleus scoring and dose response analysis in human lymphoblastoid TK6 cells.

The use of manual microscopy for the scoring of chromosome damage in the in vitro micronucleus assay is often associated with user subjectivity. This level of subjectivity can be reduced by using automated platforms, which have added value of faster with high-throughput and multi-endpoint capabilities. However, there is a need to assess the reproducibility and sensitivity of these automated platforms compared with the gold standard of the manual scoring. The automated flow cytometry-based MicroFlow® and image analysis-based Metafer™ were used for dose response analyses in human lymphoblastoid TK6 cells exposed to the model clastogen, methyl methanesulfonate (MMS), aneugen, carbendazim, and the weak genotoxic carcinogen, ochratoxin A (OTA). Cells were treated for 4 or 30 h, with a 26- or 0-h recovery. Flow cytometry scoring parameters and the Metafer™ image classifier were investigated, to assess any potential differences in the micronucleus (MN) dose responses. Dose response data were assessed using the benchmark dose approach with chemical and scoring system set as covariate to assess reproducibility between endpoints. A clear increase in MN frequency was observed using the MicroFlow® approach on TK6 cells treated for 30 h with MMS, carbendazim and OTA. The MicroFlow®-based MN frequencies were comparable to those derived by using the Metafer™ and manual scoring platforms. However, there was a potential overscoring of MN with the MicroFlow® due to the cell lysis step and an underscoring with the Metafer™ system based on current image classifier settings. The findings clearly demonstrate that the MicroFlow® and Metafer™ MN scoring platforms are powerful tools for automated high-throughput MN scoring and dose response analysis.

Isolation of Mesenchymal Stromal Cells From Peripheral Blood of ST Elevation Myocardial Infarction Patients.

Bone marrow mesenchymal stromal cells (MSCs) have shown therapeutic potential in the treatment of myocardial infarction patients. However, bone marrow requires invasive harvesting techniques. Therefore, the aim was to carry out a feasibility study of using autologous peripheral blood (PB) as a source for MSCs and platelet lysate (PL), a potential novel therapeutic intervention in acute ST elevation myocardial infarction (STEMI) patients. Autologous PL and MSCs were prepared from STEMI patient and healthy control blood. MSCs were analyzed by trilineage differentiation and flow cytometry. PB MSCs were isolated from 83% of patients (n = 6) but not from controls. The use of PL was feasible in the first passage but not in subsequent ones due to volume. To conclude, PB is a promising alternative to bone marrow. It negates the need for invasive harvesting techniques, and reduces hemorrhagic risk in this patient population routinely managed with anticoagulant and antiplatelet agents.