78 research outputs found

    Bioimprint aided cell recognition and depletion of human leukemic HL60 cells from peripheral blood

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    We report a large scale preparation of bioimprints of layers of cultured human leukemic HL60 cells which can perform cell shape and size recognition from a mixture with peripheral blood mononuclear cells (PBMCs). We demonstrate that the bioimprint-cell attraction combined with surface modification and flow rate control allows depletion of the HL60 cells from peripheral blood which can be used for development of alternative therapies of acute myeloid leukaemia (AML).AML is a clonal malignant proliferation of transformed, bone-marrow derived myeloid precursors. The disease is characterised by the rapid proliferation of the neoplastic cells (myeloblasts) resulting in failure of normal haematopoiesis with consequential bone marrow failure rapidly resulting in death if untreated.1–3 In the UK, overall survival is 16% 5 years from diagnosis. The prognosis is significantly worse in the elderly which is especially relevant as the majority of patients present over the age of 60 years.1,4–7 Therapy relies on 2–3 cycles of myeloablative chemotherapy followed by allogeneic stem cell transplants for a relatively small number of fit patients with poor prognostic features.8,9 This is accompanied by significant discomfort, and long therapy for AML is also associated with prolonged inpatient stays, considerable morbidity related to anaemia, sepsis and bleeding with an attributable mortality of 5–10%. The majority of patients relapse following induction of chemotherapy for AML and subsequent therapy is associated with a low probability of cure. Outcomes for AML patients have improved marginally over the past few decades, largely due to improvements in supportive care rather than dramatic improvements in the chemotherapeutic regimen's efficacy.10Bioimprinting is a promising area of materials chemistry aimed at mimicking and exploiting the lock-and-key interactions seen ubiquitously in nature.11–14 Cell recognition systems are relatively cheap and simple to produce with few stipulations on storage and shelf life when compared with biological interventions. The scope for possible targets is also much greater, being able to target polysaccharides, enzymes, aptamers, DNA sequences, antibodies and whole cells.12,15,16,21–24 Bioimprints of whole cells were first reported by Dickert et al.17 who imprinted yeast into a sol–gel matrix. When incubated with several strains of yeast, the substrates showed a high affinity to the template yeast strain. This effect was attributed to the large contact surface areas between the cells and the imprinted cavities. Other cell bioimprinting studies have progressed to cover a range of micro-organisms and human cells. Hayden et al.18 functionalised polyurethane with erythrocyte imprints, capable of discriminating between ABO blood groups. Though all cell targets possessed the same geometrical shape and size, imprints were able to discriminate on account of varied surface antigen expression. Subsequent studies were further able to discriminate cells with identical antibodies in different quantities to separate blood groups A1 and A2.19 Recent cell bioimprint studies largely focus on biosensor applications20,26 and are hindered by the small overall size of imprinted areas that can be produced which limits their applications for large scale extraction of targeted cells from cell mixtures. This research area is undergoing a rapid expansion towards using molecularly imprinted polymers as receptor mimics for selective cell recognition and sensing, and a recent review of size and shape targeting of cancer found no evidence so far of the use of cancer cell bioimprints in a therapeutic setting.11Here we utilised for the first time AML cell bioimprints on a large scale as a vehicle to selectively target myeloblasts due to the inherent size and morphological discrepancies compared to normal peripheral blood mononuclear cells (PBMCs) (see Fig. S1, ESI†). We explore AML cells bioimprinting to develop a new method for depletion of myeloblasts from peripheral blood cells by introducing selectivity via bespoke cell size and shape discrimination aided by myeloblast-bioimprint interactions. Our idea is based on incorporating AML cells-imprinted substrates into a flow-through type of device which offers an alternative method for removal of the leukemic burden directly from patient blood. Successful leukophoresis can potentially be used more frequently in the extraction of myeloblasts from peripheral blood which is critical in stabilizing AML patients with leukostasis associated with hyperleuocytosis. By reducing the number of circulating tumour cells, the likelihood of early relapse is also diminished.25HL60 is an immortalized human cell line derived from peripheral blood lymphocytes of a patient suffering from acute promyleocytic leukaemia. HL60 was used as a very good proxy for primary (patient derived) myeloblast cells throughout our study due to their availability and ease of culture. Here we show how the desired HL60 cell bioimprints were produced from HL60 cell layers. We also discuss the integration of the produced myeloblast imprint in a PDMS-based flow-through cell, in which its selectivity towards HL60 cells over PBMCs is investigated (Fig. 1). We fabricated bioimprints by impressing a layer of cultured HL60 cells with a curable polymer, which captures information on the cell shape, size and morphology. These were further casted with another polymer to create a “positive imprint” whose surface matches the original cell layer. Using roll-to-roll printing from the positive replica we produced a very large area of HL60 cell imprints. We engineered the surface of the bioimprint to have a weak attraction with the cells, which is strongly amplified when there is a shape and size match between the individual cells and the imprinted surface. Due to inherent size and morphology differences between myeloblasts and normal blood cells, this resulted in much higher retention of the former on the bioimprint. This allows their selective trapping from peripheral blood based on cell shape and size recognition, much cheaper than using surface functionalisation with a combination of specific antibodies for myeloblasts. We tested the bioimprints selectivity in a device for depleting cultured HL60 cells from healthy white blood cells. This cell recognition technology can potentially deplete myeloblasts from the blood of AML patients and provide an alternative route for inducing minimal residual disease, which is associated with reduced relapses and improved patient outcomes

    Removal of Human Leukemic Cells from Peripheral Blood Mononuclear Cells by Cell Recognition Chromatography with Size Matched Particle Imprints

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    We report a cell recognition chromatography approach for blood cancer cell separation from healthy peripheral blood mononuclear cells (PBMCs) based on sizematched functionalized particle imprints. Negative imprints were prepared from layers of 15 μm polymeric microbeads closely matching the size of cultured human leukemic cells (HL60). We replicated these imprints on a large scale with UV curable polyurethane resin using nanoimprinting lithography. The imprints were functionalized with branched polyethylene imine (bPEI) and passivated by Poloxamer 407 to promote a weak attraction toward cells. When a matching cell fits into an imprint cavity, its contact area with the imprint is maximized, which amplifies the attraction and the binding selectivity. We tested these imprints specificity for depleting myeloblasts from a mixture with healthy human PBMCs in a cell recognition chromatography setup hosting the imprint. The mixture of fixed HL60/PBMCs ratio was circulated over the imprint and at each step the selectivity toward HL60 was assessed by flow cytometry. The role of the imprint length, flow rate, channel depth, and the bPEI coating concentration were examined. The results show that HL60 cells, closely matching the imprint cavities, get trapped on the imprint, while the smaller PBMCs are carried away by the drag force of the flow. Lower flow rates, longer imprints, and interim channel depth favor HL60 specific retention. The bPEI concentration higher than 1 wt % on the imprint made it less selective toward the HL60 because of indiscriminate attraction with all cells. Particle imprint based cell recognition chromatography was able to achieve selective myeloblast depletion from initial 11.7% HL60 (88.3% PBMC) to less than 1.3% HL60 for 3 h of circulation. The cell recognition chromatography with size-matched microbead imprints can be employed as an efficient cell separation technique and potentially lead to alternative therapies for myeloblasts removal from peripheral blood of patients with acute myeloid leukemia

    Targeted removal of blood cancer cells from mixed cell populations by cell recognition with matching particle imprints

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    We report a new approach for separation of blood cancer cells from healthy white blood cells based on cell recognition by surface functionalised particle imprints. We prepared polymeric particle imprints from a layer of suspension of monodisperse PMMA microbeads which closely match the size of in vitro cultured human leukaemia cells (HL60). The imprints were replicated on a large scale with UV curable polyurethane resin using nanoimprinting lithography and surface functionalized with a cationic polymer, a branched polyethylene imine (bPEI), and a Pluronic surfactant, Poloxamer 407, to engineer a weak attraction towards the cells. The latter is amplified several orders of magnitude when a cell of a closely matching size and shape fits into the imprint cavity which multiplies the contact area between the cell surface and the imprint. The particle imprints were optimised for their specificity toward blood cancer cells by treatment with oxygen plasma and then subsequent coatings with bPEI and Poloxamer 407 with various functionalisation concentrations. We tested the surface functionalised imprints for their specificity in retaining in vitro cultured human leukaemic cells (HL60) over healthy human peripheral blood mononuclear cells (PBMCs) in a flow through chamber. The effect of the flushing flow rate of the mixed cell suspension over the particle imprint and the imprint length were also investigated. At each step the selectivity towards HL60 was assessed. Selective isolation of an increased amount of HL60 tumour cells over PBMC was ultimately achieved as a function of the cell seeding ratio on the particle imprint. The effect is attributed to the substantial size difference between the HL60 cell and the PBMCs. The data presented show that relatively inexpensive PMMA microbeads imprints can be utilised as a cell separation technique which could ultimately lead to novel therapies for removal of neoplastic cells from the peripheral blood of acute myeloid leukaemia patients

    Oesophageal atresia: prevalence, prenatal diagnosis and associated anomalies in 23 European regions

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    Objective To describe prevalence, prenatal diagnosis and epidemiological data on oesophageal atresia from 23 well-defined European regions and compare the prevalence between these regions. Design Population-based study using data from a large European database for surveillance of congenital anomalies (EUROCAT) for two decades (1987-2006). Settings Twenty-three participating registries based on multiple sources of information including information about live births, fetal deaths with gestational age &#8805;20 weeks and terminations of pregnancy. Patients 1222 cases of oesophageal atresia in a population of 5 019 804 births. Results The overall prevalence was 2.43 cases per 10 000 births (95% CI 2.30 to 2.57). There were regional differences in prevalence ranging from 1.27 to 4.55. Prenatal detection rates varied by registry from >50% of cases to <10% of cases. A total of 546 cases (44.7%) had an isolated oesophageal anomaly, 386 (31.6%) were multiple malformed and 290 (23.7%) had an association or a syndrome. There were 1084 live born cases (88.7%), 43 cases were fetal deaths and 95 cases were terminations of pregnancy. One-week survival for live births was 86.9% and 99.2% if the gestational age was &#8805;38 weeks and isolated oesophageal atresia was present. Males accounted for 57.3% of all cases and 38.5% of live born cases were born with gestational age <37 weeks. Conclusion There were regional differences in prevalence of oesophageal atresia in Europe. Half of all cases had associated anomalies. Prenatal detection rate increased from 26% to 36.5% over the two decades. Survival in infants with isolated oesophageal atresia born at term is hig

    Potent Inhibitor of Human Trypsins from the Aeruginosin Family of Natural Products

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    Serine proteases regulate many physiological processes and play a key role in a variety of cancers. Aeruginosins are a family of natural products produced by cyanobacteria that exhibit pronounced structural diversity and potent serine protease inhibition. Here, we sequenced the complete genome of Nodularia sphaerocarpa UHCC 0038 and identified the 43.7 kb suomilide biosynthetic gene cluster. Bioinformatic analysis demonstrated that suomilide belongs to the aeruginosin family of natural products. We identified 103 complete aeruginosin biosynthetic gene clusters from 12 cyanobacterial genera and showed that they encode an unexpected chemical diversity. Surprisingly, purified suomilide inhibited human trypsin-2 and -3, with IC50 values of 4.7 and 11.5 nM, respectively, while trypsin-1 was inhibited with an IC50 of 104 nM. Molecular dynamics simulations suggested that suomilide has a long residence time when bound to trypsins. This was confirmed experimentally for trypsin-1 and -3 (residence times of 1.5 and 57 min, respectively). Suomilide also inhibited the invasion of aggressive and metastatic PC-3M prostate cancer cells without affecting cell proliferation. The potent inhibition of trypsin-3, together with a long residence time and the ability to inhibit prostate cancer cell invasion, makes suomilide an attractive drug lead for targeting cancers that overexpress trypsin-3. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and suggest that aeruginosins may be a source of selective inhibitors of human serine proteases.</p

    Oxytocin Receptor Genotype Modulates Ventral Striatal Activity to Social Cues and Response to Stressful Life Events

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    Background Common variants in the oxytocin receptor gene (OXTR) have been shown to influence social and affective behavior and to moderate the effect of adverse experiences on risk for social-affective problems. However, the intermediate neurobiological mechanisms are not fully understood. Although human functional neuroimaging studies have reported that oxytocin effects on social behavior and emotional states are mediated by amygdala function, animal models indicate that oxytocin receptors in the ventral striatum (VS) modulate sensitivity to social reinforcers. This study aimed to comprehensively investigate OXTR-dependent brain mechanisms associated with social-affective problems. Methods In a sample of 1445 adolescents we tested the effect of 23-tagging single nucleotide polymorphisms across the OXTR region and stressful life events (SLEs) on functional magnetic resonance imaging blood oxygen level-dependent activity in the VS and amygdala to animated angry faces. Single nucleotide polymorphisms for which gene-wide significant effects on brain function were found were then carried forward to examine associations with social-affective problems. Results A gene-wide significant effect of rs237915 showed that adolescents with minor CC-genotype had significantly lower VS activity than CT/TT-carriers. Significant or nominally significant gene × environment effects on emotional problems (in girls) and peer problems (in boys) revealed a strong increase in clinical symptoms as a function of SLEs in CT/TT-carriers but not CC-homozygotes. However, in low-SLE environments, CC-homozygotes had more emotional problems (girls) and peer problems (boys). Moreover, among CC-homozygotes, reduced VS activity was related to more peer problems. Conclusions These findings suggest that a common OXTR-variant affects brain responsiveness to negative social cues and that in "risk- carriers" reduced sensitivity is simultaneously associated with more social-affective problems in "favorable environments" and greater resilience against stressful experiences. © 2014 Society of Biological Psychiatry

    Polygenic risk of psychosis and ventral striatal activation during reward processing in healthy adolescents

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    Importance: Psychotic disorders are characterized by attenuated activity in the brain’s valuation system in key reward processing areas, such as the ventral striatum (VS), as measured with functional magnetic resonance imaging. Objective: To examine whether common risk variants for psychosis are associated with individual variation in the VS. Design, setting, and participants: A cross-sectional study of a large cohort of adolescents from the IMAGEN study (a European multicenter study of reinforcement sensitivity in adolescents) was performed from March 1, 2008, through December 31, 2011. Data analysis was conducted from October 1, 2015, to January 9, 2016. Polygenic risk profile scores (RPSs) for psychosis were generated for 1841 healthy adolescents. Sample size and characteristics varied across regression analyses, depending on mutual information available (N = 1524-1836). Main outcomes and measures: Reward-related brain function was assessed with blood oxygen level dependency (BOLD) in the VS using the monetary incentive delay (MID) task, distinguishing reward anticipation and receipt. Behavioral impulsivity, IQ, MID task performance, and VS BOLD were regressed against psychosis RPS at 4 progressive P thresholds (P < .01, P < .05, P < .10, and P < .50 for RPS models 1-4, respectively). Results: In a sample of 1841 healthy adolescents (mean age, 14.5 years; 906 boys and 935 girls), we replicated an association between increasing psychosis RPS and reduced IQ (matrix reasoning: corrected P = .003 for RPS model 2, 0.4%variance explained), supporting the validity of the psychosis RPS models. We also found a nominally significant association between increased psychosis RPS and reduced MID task performance (uncorrected P = .03 for RPS model 4, 0.2%variance explained). Our main finding was a positive association between psychosis RPS and VS BOLD during reward anticipation at all 4 psychosis RPS models and for 2 P thresholds for reward receipt (RPS models 1 and 3), correcting for the familywise error rate (0.8%-1.9%variance explained). Conclusions and relevance: These findings support an association between psychosis RPS and VS BOLD in adolescents. Genetic risk for psychosis may shape an individual’s response to rewarding stimuli

    An Analysis of the Myocardial Transcriptome in a Mouse Model of Cardiac Dysfunction with Decreased Cholinergic Neurotransmission

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    Autonomic dysfunction is observed in many cardiovascular diseases and contributes to cardiac remodeling and heart disease. We previously reported that a decrease in the expression levels of the vesicular acetylcholine transporter (VAChT) in genetically-modified homozygous mice (VAChT KDHOM) leads to decreased cholinergic tone, autonomic imbalance and a phenotype resembling cardiac dysfunction. In order to further understand the molecular changes resulting from chronic long-term decrease in parasympathetic tone, we undertook a transcriptome-based, microarray-driven approach to analyze gene expression changes in ventricular tissue from VAChT KDHOM mice. We demonstrate that a decrease in cholinergic tone is associated with alterations in gene expression in mutant hearts, which might contribute to increased ROS levels observed in these cardiomyocytes. In contrast, in another model of cardiac remodeling and autonomic imbalance, induced through chronic isoproterenol treatment to increase sympathetic drive, these genes did not appear to be altered in a pattern similar to that observed in VAChT KDHOM hearts. These data suggest the importance of maintaining a fine balance between the two branches of the autonomic nervous system and the significance of absolute levels of cholinergic tone in proper cardiac function

    Epigenome-wide meta-analysis of blood DNA methylation and its association with subcortical volumes:findings from the ENIGMA Epigenetics Working Group

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    DNA methylation, which is modulated by both genetic factors and environmental exposures, may offer a unique opportunity to discover novel biomarkers of disease-related brain phenotypes, even when measured in other tissues than brain, such as blood. A few studies of small sample sizes have revealed associations between blood DNA methylation and neuropsychopathology, however, large-scale epigenome-wide association studies (EWAS) are needed to investigate the utility of DNA methylation profiling as a peripheral marker for the brain. Here, in an analysis of eleven international cohorts, totalling 3337 individuals, we report epigenome-wide meta-analyses of blood DNA methylation with volumes of the hippocampus, thalamus and nucleus accumbens (NAcc)-three subcortical regions selected for their associations with disease and heritability and volumetric variability. Analyses of individual CpGs revealed genome-wide significant associations with hippocampal volume at two loci. No significant associations were found for analyses of thalamus and nucleus accumbens volumes. Cluster-based analyses revealed additional differentially methylated regions (DMRs) associated with hippocampal volume. DNA methylation at these loci affected expression of proximal genes involved in learning and memory, stem cell maintenance and differentiation, fatty acid metabolism and type-2 diabetes. These DNA methylation marks, their interaction with genetic variants and their impact on gene expression offer new insights into the relationship between epigenetic variation and brain structure and may provide the basis for biomarker discovery in neurodegeneration and neuropsychiatric conditions
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