Comparative rheology of the adhesion of platelets and leukocytes from flowing blood: why are platelets so small?

We investigated rheological adaptation of leukocytes and platelets for their adhesive functions in inflammation and hemostasis, respectively. Adhesion and margination of leukocytes or platelets were quantified for blood perfused through capillaries coated with P-selectin or collagen, when flow rate, suspending phase viscosity, red cell aggregation, or rigidity was modified. Independent variation of shear rate and shear stress indicated that the ability of platelets to attach at higher levels than leukocytes was largely attributable to their smaller size, reducing their velocity before attachment, and, especially, drag after attachment. Increasing red cell aggregation increased the number of marginated and adhering leukocytes but inhibited platelet adhesion without effect on the number marginated. Increasing red cell rigidity tended to inhibit leukocyte adhesion but promote platelet adhesion. The effects on platelets may be explained by changes in the depth of the near-wall, red cell-depleted layer; broadening (or narrowing) this layer to greater (or less) than the platelet diameter would decrease (or increase) the normal force applied by red blood cells and make attachment less (or more) efficient. Thus different adhesive capabilities of leukocytes and platelets may arise from their differences in size, both directly because of influence on cell velocity and force experienced at the wall and indirectly through effects of size on margination in the bloodstream and interaction with the cell-free layer. In addition, red cell aggregation (of hitherto uncertain physiological significance) may be useful in promoting leukocyte adhesion in inflamed venules but inhibiting unwanted platelet deposition in veins.

Variability of gene expression profiles in human blood and lymphoblastoid cell lines.

BACKGROUND: Readily accessible samples such as peripheral blood or cell lines are increasingly being used in large cohorts to characterise gene expression differences between a patient group and healthy controls. However, cell and RNA isolation procedures and the variety of cell types that make up whole blood can affect gene expression measurements. We therefore systematically investigated global gene expression profiles in peripheral blood from six individuals collected during two visits by comparing five of the following cell and RNA isolation methods: whole blood (PAXgene), peripheral blood mononuclear cells (PBMCs), lymphoblastoid cell lines (LCLs), CD19 and CD20 specific B-cell subsets. RESULTS: Gene expression measurements were clearly discriminated by isolation method although the reproducibility was high for all methods (range rho = 0.90-1.00). The PAXgene samples showed a decrease in the number of expressed genes (P < 1*10(-16)) with higher variability (P < 1*10(-16)) compared to the other methods. Differentially expressed probes between PAXgene and PBMCs were correlated with the number of monocytes, lymphocytes, neutrophils or erythrocytes. The correlations (rho = 0.83; rho = 0.79) of the expression levels of detected probes between LCLs and B-cell subsets were much lower compared to the two B-cell isolation methods (rho = 0.98). Gene ontology analysis of detected genes showed that genes involved in inflammatory responses are enriched in B-cells CD19 and CD20 whereas genes involved in alcohol metabolic process and the cell cycle were enriched in LCLs. CONCLUSION: Gene expression profiles in blood-based samples are strongly dependent on the predominant constituent cell type(s) and RNA isolation method. It is crucial to understand the differences and variability of gene expression measurements between cell and RNA isolation procedures, and their relevance to disease processes, before application in large clinical studies.

All the right moves: why in utero transfer is both important for the baby and difficult to achieve and new strategies for change.

The best way to ensure that preterm infants benefit from relevant neonatal expertise as soon as they are born is to transfer the mother and baby to an appropriately specialised neonatal facility before birth (" in utero"). This review explores the evidence surrounding the importance of being born in the right unit, the advantages of in utero transfers compared to ex utero transfers, and how to accurately assess which women are at most risk of delivering early and the challenges of in utero transfers. Accurate identification of the women most at risk of preterm birth is key to prioritising who to transfer antenatally, but the administrative burden and pathway variation of in utero transfer in the UK are likely to compromise optimal clinical care. Women reported the impact that in utero transfers have on them, including the emotional and financial burdens of being transferred and the anxiety surrounding domestic and logistical concerns related to being away from home. The final section of the review explores new approaches to reforming the in utero transfer process, including learning from outside the UK and changing policy and guidelines. Examples of collaborative regional guidance include the recent Pan-London guidance on in utero transfers. Reforming the transfer process can also be aided through technology, such as utilising the CotFinder app. In utero transfer is an unavoidable aspect of maternity and neonatal care, and the burden will increase if preterm birth rates continue to rise in association with increased rates of multiple pregnancy, advancing maternal age, assisted reproductive technologies, and obstetric interventions. As funding and capacity pressures on health services increase because of the COVID-19 pandemic, better prioritisation and sustained multi-disciplinary commitment are essential to maximise better outcomes for babies born too soon.

Red cell aggregation as a factor influencing margination and adhesion of leukocytes and platelets.

Leukocytes and platelets must adhere to the wall of blood vessels to carry out their protective functions. Rheological factors influencing these processes are the delivery of the cells to the wall, referred to as margination, and the local shear rates and stresses at the wall. Margination requires leukocytes and platelets to be excluded from the central flow of the much more numerous red blood cells. This exclusion may be influenced by red cell aggregation. Red cell aggregation also influences development of plug flow in small vessels, which in turn modifies the wall shear rate and stress from those expected in ideal Poiseuille flow. Promotion of aggregation by added agents such as high molecular weight dextrans or by reduction in shear rate, increases margination of leukocytes and efficiency of attachment to the vessel wall. Interestingly, however, fewer studies exist for platelets, and these suggest that margination is actually promoted by increasing shear rate. Direct studies of the effects of red cell aggregation on platelets are required, but it appears that aggregation has different effects on delivery of platelets compared to leukocytes. These differences may represent adaptations for efficient adhesion of leukocytes and platelets in different regions of the circulation.

Effects of vessel size, cell sedimentation and haematocrit on the adhesion of leukocytes and platelets from flowing blood.

BACKGROUND: Leukocytes and platelets typically fulfil their functions through adhesion to the walls of vessels with different size, haematocrit and shear rate. OBJECTIVE: We aimed to investigate differential effects of these variables on leukocyte and platelet adhesion. METHODS: Blood with varying haematocrit was perfused at a range of wall shear rates through capillaries of depth 100 or 300 µm coated with P-selectin or collagen. RESULTS: Adhesion of leukocytes was much more efficient in the smaller capillaries, but was equal on the upper and lower surfaces and showed nearly identical shear rate dependence for either size of vessel. Platelets also adhered more efficiently in the smaller vessels (although the effect of size was not so great), and equally on upper and lower surfaces, but their adhesion was much less sensitive to increasing shear rate. In previous studies using vertically-orientated capillaries, leukocyte adhesion increased with increasing haematocrit (Am. J. Physiol.285 (2003), H229-H240). Here, in horizontal 100 µm capillaries, leukocyte adhesion was highly efficient at haematocrit of 10% but restricted to the lower surface. Adhesion decreased initially as haematocrit was increased to 30% and then increased slightly again at 40% haematocrit. Increasing haematocrit supported a monotonic increase in platelet adhesion in the horizontal capillaries. CONCLUSIONS: Platelets adhere efficiently over a wider range of sizes and shear rates, and at high haematocrit. Leukocytes adhere better in smaller vessels and at low haematocrit in horizontal vessels. The different behaviours may represent 'rheological adaptation' to functions in inflammation vs. haemostasis.

The use of genome-wide eQTL associations in lymphoblastoid cell lines to identify novel genetic pathways involved in complex traits.

The integrated analysis of genotypic and expression data for association with complex traits could identify novel genetic pathways involved in complex traits. We profiled 19,573 expression probes in Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) from 299 twins and correlated these with 44 quantitative traits (QTs). For 939 expressed probes correlating with more than one QT, we investigated the presence of eQTL associations in three datasets of 57 CEU HapMap founders and 86 unrelated twins. Genome-wide association analysis of these probes with 2.2 m SNPs revealed 131 potential eQTLs (1,989 eQTL SNPs) overlapping between the HapMap datasets, five of which were in cis (58 eQTL SNPs). We then tested 535 SNPs tagging the eQTL SNPs, for association with the relevant QT in 2,905 twins. We identified nine potential SNP-QT associations (P<0.01) but none significantly replicated in five large consortia of 1,097-16,129 subjects. We also failed to replicate previous reported eQTL associations with body mass index, plasma low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides levels derived from lymphocytes, adipose and liver tissue. Our results and additional power calculations suggest that proponents may have been overoptimistic in the power of LCLs in eQTL approaches to elucidate regulatory genetic effects on complex traits using the small datasets generated to date. Nevertheless, larger tissue-specific expression data sets relevant to specific traits are becoming available, and should enable the adoption of similar integrated analyses in the near future.

Prospective, observational study of outcomes in neonates with severe thrombocytopenia.

OBJECTIVE: A cross-sectional, observational study of outcomes for neonates with severe neonatal thrombocytopenia (SNT; platelet count of <60 x 10(9) platelets per L) was performed to examine hemorrhage and use of platelet transfusions. METHODS: Neonates who were admitted to 7 NICUs and developed SNT were enrolled for daily data collection. RESULTS: Among 3652 neonatal admissions, 194 neonates (5%) developed SNT. The median gestational age of 169 enrolled neonates was 27 weeks (interquartile range [IQR]: 24-32 weeks), and the median birth weight was 822 g (IQR: 670-1300 g). Platelet count nadirs were <20 x 10(9), 20 to 39 x 10(9), and 40 to 59 x 10(9) platelets per L for 58 (34%), 64 (39%), and 47 (28%) of all enrolled infants, respectively. During the study, 31 infants (18%) had no recorded hemorrhage, 123 (73%) developed minor hemorrhage, and 15 (9%) developed major hemorrhage. Thirteen (87%) of 15 episodes of major hemorrhage occurred in neonates with gestational ages of <28 weeks. Platelet transfusions (n = 415) were administered to 116 infants (69%); for 338 (81%) transfusions, the main recorded reason was low platelet count. Transfusions increased the platelet count from a median of 27 x 10(9) platelets per L (IQR: 19-36 x 10(9) platelets per L) to 79 x 10(9) platelets per L (IQR: 47.5-127 x 10(9) platelets per L). CONCLUSIONS: Although one third of neonates enrolled in this study developed thrombocytopenia of <20 x 10(9) platelets per L, 91% did not develop major hemorrhage. Most platelet transfusions were given to neonates with thrombocytopenia with no bleeding or minor bleeding only.

A novel triple fusion reporter system for use in gene trap mutagenesis.

Gene trapping is an insertional mutagenesis strategy that allows for simultaneous gene identification and mutation in embryonic stem (ES) cells. Gene trap vectors both disrupt coding sequence and report on the genes' endogenous expression. The most popular gene trap reporter to date combines beta-galactosidase expression with neomycin resistance in a fusion protein known as beta-geo. Here we describe a refinement to this reporter that also incorporates real time fluorescent readouts. We have constructed a series of gene trap vectors incorporating a novel tripartite fusion protein consisting of EGFP, beta-galactosidase, and the neomycin or hygromycin resistance activities. Our results indicate that these triple fusions can function efficiently as reporters of endogenous trapped gene expression and subcellular localization. We show that these fusion proteins constitute versatile gene trap reporters whose activity can be detected in real time by fluorescence and in fixed tissue with a sensitive enzymatic activity.

Hex acts with beta-catenin to regulate anteroposterior patterning via a Groucho-related co-repressor and Nodal.

In Xenopus, the establishment of the anteroposterior axis involves two key signalling pathways, canonical Wnt and Nodal-related TGFbeta. There are also a number of transcription factors that feedback upon these pathways. The homeodomain protein Hex, an early marker of anterior positional information, acts as a transcriptional repressor, suppressing induction and propagation of the Spemman organiser while specifying anterior identity. We show that Hex promotes anterior identity by amplifying the activity of canonical Wnt signalling. Hex exerts this activity by inhibiting the expression of Tle4, a member of the Groucho family of transcriptional co-repressors that we identified as a Hex target in embryonic stem (ES) cells and Xenopus embryos. This Hex-mediated enhancement of Wnt signalling results in the upregulation of the Nieuwkoop centre genes Siamois and Xnr3, and the subsequent increased expression of the anterior endodermal marker Cerberus and other mesendodermal genes downstream of Wnt signalling. We also identified Nodal as a Hex target in ES cells. We demonstrate that in Xenopus, the Nodal-related genes Xnr1 and Xnr2, but not Xnr5 and Xnr6, are regulated directly by Hex. The identification of Nodal-related genes as Hex targets explains the ability of Hex to suppress induction and propagation of the organiser. Together, these results support a model in which Hex acts early in development to reinforce a Wnt-mediated, Nieuwkoop-like signal to induce anterior endoderm, and later in this tissue to block further propagation of Nodal-related signals. The ability of Hex to regulate the same targets in both Xenopus and mouse implies this model is conserved.