Analysis of blood coagulation in mice: pre-analytical conditions and evaluation of a home-made assay for thrombin-antithrombin complexes

BACKGROUND: The use of mouse models for the study of thrombotic disorders has gained increasing importance. Methods for measurement of coagulation activation in mice are, however, scarce. The primary aim of this study was to develop a specific mouse thrombin-antithrombin (TAT) ELISA for measurement of coagulation activation and to compare it with two commercially available assays for human TAT complexes. In addition, we aimed to improve methods for mouse plasma anticoagulation and preparation. METHODS AND RESULTS: First, for the measurement of TAT-complexes in plasma a mouse specific TAT-ELISA was developed using rabbit polyclonal antibodies raised against mouse thrombin and rat antithrombin, respectively. This ELISA detected an increase in TAT levels in a mouse model of endotoxemia. Two commercial human TAT ELISAs appeared to be less specific for mouse thrombin-rat antithrombin complexes. Second, to prevent clotting of mouse blood sodium citrate was either mixed with blood during collection in a syringe or was injected intravenously immediately prior to blood collection. Intravenous sodium citrate completely inhibited blood coagulation resulting in plasma with consistently low TAT levels. Sodium citrate mixed with blood during collection resulted in increased TAT levels in 4 out of 16 plasma samples. Third, heparinase was added to plasma samples after in vivo injection of different heparin doses to test its neutralizing effect. Heparinase neutralized up to a 20 U of heparin/mouse and resulted in accurate APTT and factor VIII determinations. CONCLUSION: These procedures and reagents for plasma preparation and coagulation testing will improve studies on thrombotic disorders in mice

The Great Debate at 'Immunotherapy Bridge', Naples, December 5, 2019

As part of the 2019 Immunotherapy Bridge congress (December 4–5, Naples, Italy), the Great Debate session featured counterpoint views from leading experts on six topical issues in immunotherapy today. These were the use of chimeric antigen receptor T cell therapy in solid tumors, whether the Immunoscore should be more widely used in clinical practice, whether antibody-dependent cellular cytotoxicity is important in the mode of action of anticytotoxic T-lymphocyte-associated protein 4 antibodies, whether the brain is immunologically unique or just another organ, the role of microbiome versus nutrition in affecting responses to immunotherapy, and whether chemotherapy is immunostimulatory or immunosuppressive. Discussion of these important topics are summarized in this report

Genomics-assisted breeding in four major pulse crops of developing countries: present status and prospects

The global population is continuously increasing and is expected to reach nine billion by 2050. This huge population pressure will lead to severe shortage of food, natural resources and arable land. Such an alarming situation is most likely to arise in developing countries due to increase in the proportion of people suffering from protein and micronutrient malnutrition. Pulses being a primary and affordable source of proteins and minerals play a key role in alleviating the protein calorie malnutrition, micronutrient deficiencies and other undernourishment-related issues. Additionally, pulses are a vital source of livelihood generation for millions of resource-poor farmers practising agriculture in the semi-arid and sub-tropical regions. Limited success achieved through conventional breeding so far in most of the pulse crops will not be enough to feed the ever increasing population. In this context, genomics-assisted breeding (GAB) holds promise in enhancing the genetic gains. Though pulses have long been considered as orphan crops, recent advances in the area of pulse genomics are noteworthy, e.g. discovery of genome-wide genetic markers, high-throughput genotyping and sequencing platforms, high-density genetic linkage/QTL maps and, more importantly, the availability of whole-genome sequence. With genome sequence in hand, there is a great scope to apply genome-wide methods for trait mapping using association studies and to choose desirable genotypes via genomic selection. It is anticipated that GAB will speed up the progress of genetic improvement of pulses, leading to the rapid development of cultivars with higher yield, enhanced stress tolerance and wider adaptability

Immunosuppressive effects of radiation on human dendritic cells: reduced IL-12 production on activation and impairment of naïve T-cell priming

Dendritic cells (DC) are professional antigen-presenting cells (APC) of the immune system, uniquely able to prime naïve T-cell responses. They are the focus of a range of novel strategies for the immunotherapy of cancer, a proportion of which include treating DC with ionising radiation to high dose. The effects of radiation on DC have not, however, been fully characterised. We therefore cultured human myeloid DC from CD14+ precursors, and studied the effects of ionising radiation on their phenotype and function. Dendritic cells were remarkably resistant against radiation-induced apoptosis, showed limited changes in surface phenotype, and mostly maintained their endocytic, phagocytic and migratory capacity. However, irradiated DC were less effective in a mixed lymphocyte reaction, and on maturation produced significantly less IL-12 than unirradiated controls, while IL-10 secretion was maintained. Furthermore, peptide-pulsed irradiated mature DC were less effective at naïve T-cell priming, stimulating fewer effector cells with lower cytotoxicity against antigen-specific targets. Hence irradiation of DC in vitro, and potentially in vivo, has a significant impact on their function, and may shift the balance between T-cell activation and tolerisation in DC-mediated immune responses

NEDD9 Is a Positive Regulator of Epithelial-Mesenchymal Transition and Promotes Invasion in Aggressive Breast Cancer

Epithelial to mesenchymal transition (EMT) plays an important role in many biological processes. The latest studies revealed that aggressive breast cancer, especially the triple-negative breast cancer (TNBC) subtype was frequently associated with apparent EMT, but the mechanisms are still unclear. NEDD9/HEF1/Cas-L is a member of the Cas protein family and was identified as a metastasis marker in multiple cancer types. In this study, we wished to discern the role of NEDD9 in breast cancer progression and to investigate the molecular mechanism by which NEDD9 regulates EMT and promotes invasion in triple-negative breast cancer. We showed that expression of NEDD9 was frequently upregulated in TNBC cell lines, and in aggressive breast tumors, especially in TNBC subtype. Knockdown of endogenous NEDD9 reduced the migration, invasion and proliferation of TNBC cells. Moreover, ectopic overexpression of NEDD9 in mammary epithelial cells led to a string of events including the trigger of EMT, activation of ERK signaling, increase of several EMT-inducing transcription factors and promotion of their interactions with the E-cadherin promoter. Data presented in this report contribute to the understanding of the mechanisms by which NEDD9 promotes EMT, and provide useful clues to the evaluation of the potential of NEDD9 as a responsive molecular target for TNBC chemotherapy

Evidence-based guidelines for use of probiotics in preterm neonates

<p>Abstract</p> <p>Background</p> <p>Current evidence indicates that probiotic supplementation significantly reduces all-cause mortality and definite necrotising enterocolitis without significant adverse effects in preterm neonates. As the debate about the pros and cons of routine probiotic supplementation continues, many institutions are satisfied with the current evidence and wish to use probiotics routinely. Because of the lack of detail on many practical aspects of probiotic supplementation, clinician-friendly guidelines are urgently needed to optimise use of probiotics in preterm neonates.</p> <p>Aim</p> <p>To develop evidence-based guidelines for probiotic supplementation in preterm neonates.</p> <p>Methods</p> <p>To develop core guidelines on use of probiotics, including strain selection, dose and duration of supplementation, we primarily used the data from our recent updated systematic review of randomised controlled trials. For equally important issues including strain identification, monitoring for adverse effects, product format, storage and transport, and regulatory hurdles, a comprehensive literature search, covering the period 1966-2010 without restriction on the study design, was conducted, using the databases PubMed and EMBASE, and the proceedings of scientific conferences; these data were used in our updated systematic review.</p> <p>Results</p> <p>In this review, we present guidelines, including level of evidence, for the practical aspects (for example, strain selection, dose, duration, clinical and laboratory surveillance) of probiotic supplementation, and for dealing with non-clinical but important issues (for example, regulatory requirements, product format). Evidence was inadequate in some areas, and these should be a target for further research.</p> <p>Conclusion</p> <p>We hope that these evidence-based guidelines will help to optimise the use of probiotics in preterm neonates. Continued research is essential to provide answers to the current gaps in knowledge about probiotics.</p

Limited diversity associated with duplicated class II MHC-DRB genes in the red squirrel population in the United Kingdom compared with continental Europe

The red squirrel (Sciurus vulgaris) population in the United Kingdom has declined over the last century and is now on the UK endangered species list. This is the result of competition from the eastern grey squirrel (S. carolinensis) which was introduced in the 19th century. However, recent evidence suggests that the rate of population decline is enhanced by squirrelpox disease, caused by a viral infection carried asymptomatically by grey squirrels but to which red squirrels are highly susceptible. Population genetic diversity provides some resilience to rapidly evolving or exotic pathogens. There is currently no data on genetic diversity of extant UK squirrel populations with respect to genes involved in disease resistance. Diversity is highest at loci involved in the immune response including genes clustered within the major histocompatibility complex (MHC). Using the class II DRB locus as a marker for diversity across the MHC region we genotyped 110 red squirrels from locations in the UK and continentalEurope. Twenty four Scvu-DRB alleles at two functional loci; Scvu-DRB1 and Scvu- DRB2, were identified. High levels of diversity were identified at both loci in the continental populations. In contrast, no diversity was observed at the Scvu-DRB2 locus in the mainland UK population while a high level of homozygosity was observed at the Scvu-DRB1 locus. The red squirrel population in the UK appears to lack the extensive MHC diversity associated with continental populations, a feature which may have contributed to their rapid decline

Stochastic Resonance Modulates Neural Synchronization within and between Cortical Sources

Neural synchronization is a mechanism whereby functionally specific brain regions establish transient networks for perception, cognition, and action. Direct addition of weak noise (fast random fluctuations) to various neural systems enhances synchronization through the mechanism of stochastic resonance (SR). Moreover, SR also occurs in human perception, cognition, and action. Perception, cognition, and action are closely correlated with, and may depend upon, synchronized oscillations within specialized brain networks. We tested the hypothesis that SR-mediated neural synchronization occurs within and between functionally relevant brain areas and thus could be responsible for behavioral SR. We measured the 40-Hz transient response of the human auditory cortex to brief pure tones. This response arises when the ongoing, random-phase, 40-Hz activity of a group of tuned neurons in the auditory cortex becomes synchronized in response to the onset of an above-threshold sound at its “preferred” frequency. We presented a stream of near-threshold standard sounds in various levels of added broadband noise and measured subjects' 40-Hz response to the standards in a deviant-detection paradigm using high-density EEG. We used independent component analysis and dipole fitting to locate neural sources of the 40-Hz response in bilateral auditory cortex, left posterior cingulate cortex and left superior frontal gyrus. We found that added noise enhanced the 40-Hz response in all these areas. Moreover, added noise also increased the synchronization between these regions in alpha and gamma frequency bands both during and after the 40-Hz response. Our results demonstrate neural SR in several functionally specific brain regions, including areas not traditionally thought to contribute to the auditory 40-Hz transient response. In addition, we demonstrated SR in the synchronization between these brain regions. Thus, both intra- and inter-regional synchronization of neural activity are facilitated by the addition of moderate amounts of random noise. Because the noise levels in the brain fluctuate with arousal system activity, particularly across sleep-wake cycles, optimal neural noise levels, and thus SR, could be involved in optimizing the formation of task-relevant brain networks at several scales under normal conditions

P2 receptors are involved in the mediation of motivation-related behavior

The importance of purinergic signaling in the intact mesolimbic–mesocortical circuit of the brain of freely moving rats is reviewed. In the rat, an endogenous ADP/ATPergic tone reinforces the release of dopamine from the axon terminals in the nucleus accumbens as well as from the somatodendritic region of these neurons in the ventral tegmental area, as well as the release of glutamate, probably via P2Y1 receptor stimulation. Similar mechanisms may regulate the release of glutamate in both areas of the brain. Dopamine and glutamate determine in concert the activity of the accumbal GABAergic, medium-size spiny neurons thought to act as an interface between the limbic cortex and the extrapyramidal motor system. These neurons project to the pallidal and mesencephalic areas, thereby mediating the behavioral reaction of the animal in response to a motivation-related stimulus. There is evidence that extracellular ADP/ATP promotes goal-directed behavior, e.g., intention and feeding, via dopamine, probably via P2Y1 receptor stimulation. Accumbal P2 receptor-mediated glutamatergic mechanisms seem to counteract the dopaminergic effects on behavior. Furthermore, adaptive changes of motivation-related behavior, e.g., by chronic succession of starvation and feeding or by repeated amphetamine administration, are accompanied by changes in the expression of the P2Y1 receptor, thought to modulate the sensitivity of the animal to respond to certain stimuli