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Supplementary material and methods. Supplementary table 1: cell lines and growth conditions. Supplementary table 2: Notch ligands and control protein for plate coating. Supplementary table 3: qPCR primers. Supplementary table 4: flow cytometry antibodies. Supplementary table 5: ICC/IHC antibodies. Supplementary table 6: titration of Notch1-JAG1 binding inhibition by JAG1 antibodies. Supplementary table 7: amino acid sequence of JAG1 antibodies. Supplementary table 8: blood analysis on antibody-treated tumor-bearing rats.

1: JAG1 antibody characterization. 2: antibody inhibition of JAG1 mediated Notch signalling in tumor cell lines. 3: antibody inhibition of JAG1 signalling in vascular cells. 4: antibody inhibition of JAG1 signalling in HUVEC-HUVSMC co-culture. 5: effect of anti-Notch pathway treatment on MDA-MB231 2D growth and J1-65D titration and effect on 3D growth and gene expression. 6: J1-65D treatment of established OVCAR3 xenograft. 7: JAG1 antibodies cross-reactivity with rat Jag1. 8: anti-JAG1 treatment effect in a rat brain metastasis model.

The role of Notch signaling and its ligand JAGGED1 (JAG1) in tumor biology has been firmly established, making them appealing therapeutic targets for cancer treatment. Here, we report the development and characterization of human/rat-specific JAG1-neutralizing mAbs. Epitope mapping identified their binding to the Notch receptor interaction site within the JAG1 Delta/Serrate/Lag2 domain, where E228D substitution prevented effective binding to the murine Jag1 ortholog. These antibodies were able to specifically inhibit JAG1-Notch binding in vitro, downregulate Notch signaling in cancer cells, and block the heterotypic JAG1-mediated Notch signaling between endothelial and vascular smooth muscle cells. Functionally, in vitro treatment impaired three-dimensional growth of breast cancer cell spheroids, in association with a reduction in cancer stem cell number. In vivo testing showed variable effects on human xenograft growth when only tumor-expressed JAG1 was targeted (mouse models) but a more robust effect when stromal-expressed Jag1 was also targeted (rat MDA-MB-231 xenograft model). Importantly, treatment of established triple receptor-negative breast cancer brain metastasis in rats showed a significant reduction in neoplastic growth. MRI imaging demonstrated that this was associated with a substantial improvement in blood–brain barrier function and tumor perfusion. Lastly, JAG1-targeting antibody treatment did not cause any detectable toxicity, further supporting its clinical potential for cancer therapy.

Biological recording in Britain has increased in accessibility and popularity due to recent technological advances. However, remote locations may still be under-recorded, particularly for aquatic plants and taxonomically challenging groups. We describe a set of notable botanical discoveries made in 2021 at Corrour in the Scottish Highlands (v.c.97 Westerness), including Baldellia repens, Illecebrum verticillatum (new to Scotland) and six British altitudinal records. At the time of writing, there are now four Nationally Rare vascular plant taxa recorded on the estate and 29 Nationally Scarce taxa. These findings demonstrate the value of collaboration between land managers, ecologists, BSBI staff and the local community. Both B. repens and I. verticillatum are well established at Corrour in considerable abundance and with clear evidence of regeneration. B. repens also occurs in the Tay catchment and may have arrived at Corrour via vegetative dispersal by waterfowl. The origin of I. verticillatum is more ambiguous but suggested mechanisms of dispersal include forestry, the railway or hydroelectricity workings. Despite a likely element of accidental human-mediated spread, I. verticillatum should be considered an intriguing addition to the flora of Westerness. Climate change could facilitate further establishment of this taxa in northern parts of Britain, and it is likely that other new records of both B. repens and I. verticillatum await.

Summary Background Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key mediator of signs and symptoms in preclinical models of osteoarthritis. We explored the efficacy, safety, and pharmacokinetics of an anti-GM-CSF antibody, otilimab, in patients with hand osteoarthritis. Methods This double-blind, randomised, placebo-controlled phase 2a study was done in 16 centres in the Netherlands, Germany, Poland, the UK, and the USA. Patients aged 18 years or older with inflammatory hand osteoarthritis, who had received at least one course of unsuccessful non-steroidal anti-inflammatory drugs, with two or more swollen and tender interphalangeal joints (on the same hand), signs of inflammation or synovitis identified with MRI in the affected hand, and a self-reported 24 h average hand pain intensity over the past 7 days of 5 or more on a 0–10 numerical rating scale were eligible for inclusion. Patients were randomly assigned (1:1) via interactive response technology (blocked randomisation; block size of four) to receive either subcutaneous otilimab 180 mg or placebo, administered weekly from week 0 to week 4, then every other week until week 10. Patients, investigators, and trial staff were masked to treatment; at least one administrator at each site was unmasked to prepare and administer treatment. The primary endpoint was change from baseline in 24 h average hand pain numeric rating scale averaged over 7 days before the visit at week 6. Secondary endpoints were: change from baseline in 24 h average and worst hand pain intensity at each visit; proportion of patients showing 30% and 50% reductions in 24 h average and worst hand pain intensity at each visit; change from baseline in Australian and Canadian Hand Osteoarthritis Index (AUSCAN) 3·1 numeric rating scale questionnaire components at each visit; change in number of swollen and tender hand joints at each visit; change from baseline in Patient and Physician Global Assessment of disease activity; serum concentration of otilimab; and safety parameters. Efficacy endpoints were assessed in the intention-to-treat population. The safety population included all patients who received at least one dose of study treatment. The study is registered with ClinicalTrials.gov , NCT02683785 . Findings Between March 17, 2016, and Nov 29, 2017, 44 patients were randomly assigned (22 in the placebo group and 22 in the otilimab group). At week 6, difference in change from baseline in 24 h average hand pain numeric rating scale between the otilimab and placebo groups was −0·36 (95% CI −1·31 to 0·58; p=0·44); at week 12, the difference was −0·89 (–2·06 to 0·28; p=0·13). Patients receiving otilimab showed greater improvement in AUSCAN components versus placebo at each visit. The change from baseline in the 24 h worst hand pain numeric rating scale in the otilimab group at week 6 showed a difference over placebo of −0·33 (95% CI −1·28 to 0·63; p=0·49); at week 12, this difference was −1·01 (95% CI −2·22 to 0·20; p=0·098). The proportion of patients achieving 30% or higher or 50% or higher reduction from baseline in the 24 h average and worst hand pain numeric rating scale scores was consistently greater (although non-significant) with otilimab versus placebo. Similarly, patients receiving otilimab showed greater improvement in AUSCAN pain, functional impairment, and stiffness scores versus placebo at each visit. No differences were observed between otilimab and placebo in the change from baseline in the number of swollen and tender joints across the study. The Patient Global Assessment was consistently lower than placebo at all visits; the Physician Global Assessment showed reductions across the study period, but the changes were similar in both treatment groups. Median otilimab serum concentrations increased during weekly dosing from 1730 ng/mL at week 1 to a maximum of 3670 ng/mL at week 4, but declined after transitioning to dosing every other week (weeks 6–10). In total, 84 adverse events were reported by 24 patients: 54 adverse events in 13 (59%) patients in the otilimab group and 30 adverse events in 11 (50%) patients in the placebo group. The most common adverse events were cough (reported in 4 [9%] patients; 2 in each group), and nasopharyngitis (in 3 [7%] patients; 1 in the placebo group and 2 in the otilimab group). Three serious adverse events occurred in this study (all in the otilimab group) and were deemed not related to the study medication. There were no deaths during the study. Interpretation There was no significant difference between otilimab and placebo in the primary endpoint, although we noted a non-significant trend towards a reduction in pain and functional impairment with otilimab, which supports a potential role for GM-CSF in hand osteoarthritis-associated pain. There were no unexpected safety findings in this study, with no treatment-related serious adverse events reported. Funding GlaxoSmithKline.

In an age where caring for others may seem like a relic of a bygone era, we all must be reminded of the importance of active caring in the classroom and beyond. This article explores the work of Nel Noddings, an educational scholar at the forefront of caring research, and her four dimensions for bringing active caring into the classroom. Modeling, dialogue, practice, and confirmation provide teachers with concrete starting points for reflecting on the ways in which they cultivate caring in their music classrooms and how they develop caring relationships with students that reflect the ways they need and want to be cared for. Music educators often work with the same students over the course of many years—it is to our advantage to use this time to teach caring and teach with care.

BackgroundTraumatic injury is a leading cause of death worldwide. There is a crucial need to develop therapies that improve critically injured patient outcomes. Current trauma research models are ethically and financially challenging, with poor translation. However, traumatic injury and haemorrhagic shock can be modelled using ex-vivo normothermic perfusion (EVNP), a methodology adapted from transplantation. The aim of this study was to develop a 24hr EVNP duel porcine limb and kidney model.MethodEight porcine forelimbs, bilateral kidneys and blood were retrieved via standard protocols. Following ResultsPerfusion was maintained for 24hrs in all limbs, with blood flows of 345.03mls/min (±54.78 SD) and MAP of 77.57mmHg (±3.82 SD). Three kidneys achieved 24hr perfusion, with flows of 214.53mls/min (±41.6 SD) and MAP of 80.58mmHg (±0.51 SD). Biochemical analysis showed a statistically significant potassium elevation at 24hrs compared to baseline, p=0.0078. A further three kidneys were disconnected from the circuit at 7, 11 and 12hrs, and two kidneys showed decline in flow >15 hrs due to declining haemodynamics. Compared to baseline, evidence of cell death was observed in 24hr muscle samples. In the end-point kidney samples, tubular degeneration, protein loss and necrosis extended along the nephron.ConclusionsLimb EVNP can be successfully achieved for 24hrs, but further protocol improvements are required to sustain renal perfusion for 24hrs alongside adjustments to reduce the ischaemic insult and cell death.

The chemical characteristics of rapeseed meal (RSM) produced from two cultivars of UK-grown rapeseed, by both supercritical carbon dioxide extraction (ScCO2) and cold-pressed hexane extraction (CpH...

Closed chest compressions (CCC) are recommended for medical cardiac arrest, but there is little evidence to support their inclusion for traumatic cardiac arrest (TCA). This laboratory study evaluated CCC following haemorrhage-induced TCA and whether resuscitation with blood improved survival compared to saline. The study was conducted with the authority of UK Animals (Scientific Procedures) Act 1986 (received institutional ethical approval and a Home Office Licence) using 39 terminally anesthetised, instrumented, juvenile Large White pigs. Following baseline measurements, animals underwent captive bolt injury to the right thigh and controlled haemorrhage (30% blood volume). Sixty minutes later there was a further haemorrhage to a MAP of 20 mmHg. The randomised resuscitation protocol was initiated within 5 min: CCC (Group 1); IV whole blood (Group 2); IV 0.9% saline (Group 3); IV whole blood + CCC (Group 4); and IV saline + CCC (Group 5). Fluid was administered as 3 × 10 ml/kg boluses using the Belmont® Rapid Infuser. The LUCAS™ II Chest Compression System delivered CCC. Primary Outcome was attainment of return of spontaneous circulation (ROSC MAP ≥ 50 mmHg) at Study End (fifteen minutes post-resuscitation) and secondary outcomes included haemodynamics. Mortality (MAP≤10 mmHg) was significantly higher in Group 1 compared to Groups 2 and 3 (P 0.0001). Resuscitation with whole blood was significantly better than saline (P = 0.0069), no animals in Group 3 attained ROSC. The addition of chest compressions to fluid resuscitation resulted in a significantly worse outcome with saline resuscitation (P = 0.0023) but not with whole blood (P = 0.4411). Cardiovascular variables at the end of the Resuscitation Phase and Study End were significantly worse for Group 5 compared to Group 3. Some significant differences were present at the end of the Resuscitation Phase for Group 4 versus Group 2 but these differences were no longer present by Study End. CCC were associated with increased mortality and compromised haemodynamics compared to intravenous fluid resuscitation. Whole blood resuscitation was better than saline.

A study was conducted to 1) investigate the influence of rapeseed cultivar on the range of variation in the chemical composition and feed value of rapeseed meals (RSM) for broilers and 2) to compare between single-cultivar RSM and a batch of UK-sourced proprietary rapeseed meal. Chemical composition was determined for a total of 10 single-cultivar RSM produced via prepressed solvent-extraction and a proprietary RSM. To investigate RSM digestibility a total of 360 birds were housed, six birds per pen, and between day 13 to 21 of age they received a diet containing per kg 250 g RSM and 750 g basal feed based on wheat and soybean meal. Titaniumdioxide (7 g/kg) was added as an inert marker. For the determination nitrogen-corrected metabolisable energy (AMEn) total excreta and feed intake were quantified over the last 4 d of the experiment. On the final day of the experiment ileal digesta were collected for determination of preceacal crude protein digestibility (pcCPD). With the exception of neutral detergent fibre (NDF), glucosinolates and soluble non-starch polysaccharides the coefficients of variation (CV%) for all major components were similar across all RSM (0.5

Chlorine is a toxic industrial chemical produced in vast quantities globally, being used in a range of applications such as water purification, sanitation and industrial processes. Its use and transport cannot be restricted; exposure may occur following accidental or deliberate releases. The OPCW recently verified the use of chlorine gas against civilians in both Syria and Iraq. Chlorine inhalation produces damage to the lungs, which may result in the development of an acute lung injury, respiratory failure and death. Treatment remains an intractable problem. Our objective was to develop a clinically relevant pre-clinical model of a moderate to severe lung injury in the pig. This would enable future assessment of therapeutic drugs or interventions to be implemented in the pre-hospital phase after exposure. Due to the irritant nature of chlorine, a number of strategies for exposing terminally anesthetized pigs needed to be investigated. A number of challenges (inconsistent acute changes in respiratory parameters; early deaths), resulted in a moderate to severe lung injury not being achieved. However, most pigs developed a mild lung injury by 12 h. Further investigation is required to optimize the model and enable the assessment of therapeutic candidates. In this paper we describe the exposure strategies used and discuss the challenges encountered in establishing a model of chlorine-induced lung injury. A key aim is to assist researchers navigating the challenges of producing a clinically relevant model of higher dose chlorine exposure where animal welfare is protected by use of terminal anesthesia.

Background Parachute airdrop offers a rapid transfusion supply option for humanitarian aid and military support. However, its impact on longer-term RBC survival is undocumented. This study aimed to determine post-drop quality of RBCs in concentrates (RCC), and both RBCs and plasma in whole blood (WB) during subsequent storage. Study design and methods Twenty-two units of leucodepleted RCC in saline, adenine, glucose, mannitol (SAGM) and 22 units of nonclinical issue WB were randomly allocated for air transportation, parachute drop, and subsequent storage (parachute), or simply storage under identical conventional conditions (4 ± 2°C) (control). All blood products were 6-8 days post-donation. Parachute units were packed into Credo Cubes, (Series 4, 16 L) inside a PeliCase (Peli 0350) and rigged as parachute delivery packs. Packs underwent a 4-h tactical flight (C130 aircraft), then parachuted from 250 to 400 ft before ground recovery. The units were sampled aseptically before and after airdrop at weekly intervals. A range of assays quantified the RBC storage lesion and coagulation parameters. Results Blood units were maintained at 2-6°C and recovered intact after recorded ground impacts of 341-1038 m s-2 . All units showed a classical RBC storage lesion and increased RBC microparticles during 42 days of storage. Fibrinogen and clotting factors decreased in WB during storage. Nevertheless, no significant difference was observed between Control and Parachute groups. Air transportation and parachute delivery onto land did not adversely affect, or shorten, the shelf life of fresh RBCs or WB. Discussion Appropriately packaged aerial delivery by parachute can be successfully used for blood supply.

Purpose: The study was to identify the impact of physical activity (PA) and body composition on insulin resistance in older adults and investigate racial/ethnic difference in these relationships. Method: This secondary data analysis project used 717 participants who were 55 years and older without diabetes from the 2017-2018 National Health and Nutrition Examination Survey. Major variables included homeostatic model assessment of insulin resistance (HOMA-IR), body mass index (BMI), waist circumference (WC), and recreational PA. The covariates included were demographics, nutrition intake, and lifestyle (smoking and alcohol usage). Hierarchical multiple regressions were conducted to analyze the data. Results: Body composition and recreational PA had interaction and main effects on HOMA-IR in the overall sample. Level of HOMA-IR decreased with increasing levels of recreational PA in individuals with lower levels of BMI, but in individuals with higher levels of BMI, HOMA-IR increased. Level of HOMA-IR increased with increasing levels of recreational PA across the range of WCs, but the rate of increase was slightly more pronounced at higher level of WCs. These results varied across racial/ethnic groups. Conclusion: Overall, both recreational PA and body composition are important factors that affect level of HOMA-IR, but body composition seems to be the dominant factor. PA is only beneficial for older adults with low BMI. Interventions for reducing insulin resistance should be tailored to specific racial/ethnic groups.

It is widely recognised that a diagnosis of a long-term physical health condition (LTC) is likely to have a significant impact on a person’s mental health. This is highlighted in the Five Year Forward View for Mental Health (NHS England, 2016) where significant numbers of patients projected to be seen through the expansion of Improving Access to Psychological Therapies (IAPT) services are to come from within the LTC community. IAPT services offer evidence-based therapeutic interventions for common mental health issues – anxiety disorders and depression. The South East Staffordshire IAPT services have developed an integrated pathway as a Wave 2 site for the delivery of cognitive behavioural therapy (CBT) adaptations for LTC. The main themes outlined in this paper focus on the innovations and service developments of IAPT-LTC including: the importance of engagement between mental health and medical healthcare professionals, identifying the key professionals in medical healthcare to enhance engagement, extended training for clinicians with in-house continued professional development, as an extension to the National IAPT-approved top-up training for LTC, and developments in clinical supervision structures and practice, along with future developments in the field of IAPT-LTC. These themes have direct relevance to CBT practitioners working within the LTC community in IAPT services. The four contrasting case studies demonstrate how the application of CBT can successfully be adapted to condition related beliefs and behaviours, despite the complexity of the medical condition. Findings show how integrated services and engaging with medical healthcare professionals had profound benefits for the patients, IAPT therapists and medical healthcare professionals. Key learning aims (1) The good practice points in the development of the IAPT-LTC pathway within South East Staffordshire IAPT services. (2) The successful design and implementation of the IAPT-LTC pathway within South East Staffordshire IAPT services. (3) The key considerations of the interaction for patients between their physical and mental health symptoms. (4) The application of CBT adaptations for people with complex LTCs can be effective in improving psychological wellbeing and physical condition management.

Background: Acute trauma coagulopathy (ATC) after military trauma has not been comprehensively studied. ATC is defined as a prolonged prothrombin time ratio (PTr) or reduced clot amplitude (A5) in viscoelastic testing. Compared to civilian trauma, military trauma has more injuries from explosions and gunshot wounds (GSWs), potentially leading to a different pathophysiology for traumatic coagulopathy. This study aimed to characterize military ATC on admission to a military hospital in Afghanistan and to explore any differences due to the mechanism of injury. Methods: Severely injured military casualties were enrolled in the study. Blood samples were taken on admission and after routine testing, waste plasma was prepared, frozen, and transported to the United Kingdom for in-depth hemostatic analysis. Results: Seventy-seven percent of casualties had ATC defined by a PTr greater than 1.2 and 19% when defined by rotational thromboelastometry (ROTEM) A5 less than 36 mm. Coagulation factor depletion correlated with degree of shock, particularly factor V (p < 0.01), factor X (p < 0.01), and fibrinogen levels (p < 0.01). Thrombin generation was well preserved. Fibrinolytic biomarkers were raised correlating with the degree of shock (p < 0.01), and 8% of casualties had hyperfibrinolysis on ROTEM analysis. Plasmin-antiplasmin complexes (p < 0.01) and d-dimer levels (p = 0.01) were higher and clot firmness lower (p = 0.02) in those injured by explosion compared to GSW's. Conclusions: ATC was present and correlated with shock, similar to civilian trauma. Thrombin generation remained adequate. Fibrinogen and factor V levels were disproportionately low but still sufficient to allow clot formation. Fibrinolysis is a key feature, probably due to a tissue plasminogen activator surge at the time of injury. Blast injuries are associated with a greater activation of fibrinolysis than GSWs.

Knowledge on haemostatic changes in humans infected with Ebola virus is limited due to safety concerns and access to patient samples. Ethical approval was obtained to collect plasma samples from patients in Sierra Leone infected with Ebola virus over time and samples were analysed for clotting time, fibrinogen, and D-dimer levels. Plasma from healthy volunteers was also collected by two methods to determine effect of centrifugation on test results as blood collected in Sierra Leone was not centrifuged. Collecting plasma without centrifugation only affected D-dimer values. Patients with Ebola virus disease had higher PT and APTT and D-dimer values than healthy humans with plasma collected in the same manner. Fibrinogen levels in patients with Ebola virus disease were normal or lower than values measured in healthy people. Clotting times and D-dimer levels were elevated during infection with Ebola virus but return to normal over time in patients that survived and therefore could be considered prognostic. Informative data can be obtained from plasma collected without centrifugation which could improve patient monitoring in hazardous environments.

Background There are significant barriers to accessible, quality mental health care for young adolescents affected by adversity. In an attempt to overcome this, the World Health Organization (WHO) has developed the Early Adolescent Skills for Emotions (EASE) psychological intervention for young adolescents with internalizing problems. EASE is group-based (seven sessions for adolescents, three sessions for their caregivers) and can be delivered by non-specialist providers. This paper outlines the study protocols for two trials of EASE in the Middle-East - one in Lebanon and one in Jordan. Methods We will conduct two, single-blind, two-arm, individually randomised group treatment trials in Lebanon and Jordan, with at least 445 young adolescents per trial. Adolescents will be screened eligible for the trial if they demonstrate levels of psychological distress indicative of internalizing problems requiring treatment. Participants will be randomly assigned to receive the EASE programme, or enhanced usual care (one home-visit psychoeducation session). The primary outcome is reduction in overall child-reported psychological distress over time, with 3 months post-treatment as the primary end-point. Secondary child-reported outcomes include post-traumatic stress symptoms, depression symptoms, daily functioning, and wellbeing. Secondary caregiver-reported outcomes include parenting style, overall child distress, and caregiver psychological distress. Coping strategy use will be explored as a mediator of treatment effects, and relevant moderators of treatment effects will be explored. Discussion These trials will provide the first assessments of the effectiveness of the EASE intervention for use in the middle-east, with important implications for the use of low-cost, non-specialist interventions for this age range. Trial registration: Lebanon: ISRCTN, ISRCTN75375136; registered 11th March 2019, https://doi.org/10.1186/ISRCTN75375136. Jordan: Australia New Zealand Clinical Trials Registry, ACTRN12619000341123; registered 5th March 2019, https://www.anzctr.org.au/

UNLABELLED We report a new microcirculatory assessment device, the Braedius Cytocam, an Incident Dark Field (IDF) video microscope, and compare it with a precursor device utilising side stream dark field (SDF) imaging. METHODS Time matched measurements were made with both devices from the sublingual microcirculation of pigs subjected to traumatic injury and hemorrhagic shock at baseline and during a shock phase. Images were analysed for vessel density, microcirculatory flow and image quality. RESULTS There were no differences in density or flow data recorded from the two devices at baseline [TVD IDF 14.2 ± 2.4/TVD SDF 13.2 ± 2.0, p 0.17] [MFI IDF 3 (2.8-3.0)/MFI SDF 3 (2.9-3.0), p 0.36] or during the shock state [TVD IDF 11.64 ± 3.3/TVD SDF 11.4 ± 4.0 p = 0.98] [MFI IDF 1.9 (0.6-2.7)/MFI SDF 1.7 (0.3-2.6) p 0.55]. Bland and Altman analysis showed no evidence of significant bias. Vessel contrast was significantly better with the IDF device for both capillaries [17.1 ± 3.9 (IDF) v 3.4 ± 3.6 (SDF), p = 0.0006] and venules [36.1 ± 11.4 (IDF) v 26.4 ± 7.1 (SDF) p 0.014] CONCLUSION The Braedius Cytocam showed comparable vessel detection to a precursor device during both baseline and low flow (shock) states.

Intermodal preferential looking (IMPL) is widely used in experimental studies of infant development, especially language development. Control measures vary, and it is not clear how these affect findings. We examined effects of parental awareness of stimuli. Infants (17-19 months) looked at paired pictures, one name-known and one name-unknown, each assigned target status in 50 % of trials. Infants looked longer at a name-known than a name-unknown target, regardless of parents' awareness. When parents were aware, looking to a name-unknown target increased over a paired name-known non-target. There is evidence that infants' looking at pictures in this paradigm is not due to direct matching of targets to novel names, but is influenced by additional cues present, in a way that could alter the conclusions of studies of infant word learning and other aspects of infant learning. Implications of these findings are discussed, emphasising replicability and theoretical conclusions drawn from studies using this method.

Background Circulating cell‐free DNA (cfDNA) is not found in healthy subjects, but is readily detected after thermal injury and may contribute to the risk of multiple organ failure. The hypothesis was that a postburn reduction in DNase protein/enzyme activity could contribute to the increase in cfDNA following thermal injury. Methods Patients with severe burns covering at least 15 per cent of total body surface area were recruited to a prospective cohort study within 24 h of injury. Blood samples were collected from the day of injury for 12 months. Results Analysis of blood samples from 64 patients revealed a significant reduction in DNase activity on days 1–28 after injury, compared with healthy controls. DNase protein levels were not affected, suggesting the presence of an enzyme inhibitor. Further analysis revealed that actin (an inhibitor of DNase) was present in serum samples from patients but not those from controls, and concentrations of the actin scavenging proteins gelsolin and vitamin D‐binding protein were significantly reduced after burn injury. In a pilot study of ten military patients with polytrauma, administration of blood products resulted in an increase in DNase activity and gelsolin levels. Conclusion The results of this study suggest a novel biological mechanism for the accumulation of cfDNA following thermal injury by which high levels of actin released by damaged tissue cause a reduction in DNase activity. Restoration of the actin scavenging system could therefore restore DNase activity, and reduce the risk of cfDNA‐induced host tissue damage and thrombosis., Circulating cell‐free DNA (cfDNA) is not found in healthy subjects but is readily detected after thermal injury and may contribute to the risk of multiple organ failure. The authors hypothesized that a postburn reduction in DNase protein/enzyme activity could contribute to the increase in cfDNA after thermal injury. The results of this study suggest a novel biological mechanism for the accumulation of cfDNA following thermal injury whereby high levels of actin released by damaged tissue cause a reduction in DNase activity. Restoration of the actin scavenging system could therefore restore DNase activity, and reduce the risk of cfDNA‐induced host tissue damage and thrombosis. NET, neutrophil extracellular trap. DNAse activity reduced following thermal injury, likely driven in part by raised circulating actin

Severe injury and hemorrhagic shock (HS) result in multiple changes to hematopoietic differentiation, which contribute to the development of immunosuppression and multiple organ failure (MOF). Understanding the changes that take place during the acute injury phase may help predict which patients will develop MOF and provide potential targets for therapy. Obtaining bone marrow from humans during the acute injury phase is difficult so published data are largely derived from peripheral blood samples, which infer bone marrow changes that reflect the sustained inflammatory response. This preliminary and opportunistic study investigated leucopoietic changes in rat bone marrow 6 h following traumatic injury and HS. Terminally anesthetized male Porton Wistar rats were allocated randomly to receive a sham operation (cannulation with no injury) or femoral fracture and HS. Bone marrow cells were flushed from rat femurs and immunophenotypically stained with specific antibody panels for lymphoid (CD45R, CD127, CD90, and IgM) or myeloid (CD11b, CD45, and RP‐1) lineages. Subsequently, cell populations were fluorescence‐activated cell sorted for morphological assessment. Stage‐specific cell populations were identified using a limited number of antibodies, and leucopoietic changes were determined 6 h following trauma and HS. Myeloid subpopulations could be identified by varying levels CD11b expression, CD45, and RP‐1. Trauma and HS resulted in a significant reduction in total CD11b + myeloid cells including both immature (RP‐1(−)) and mature (RP‐1+) granulocytes. Multiple B‐cell lymphoid subsets were identified. The total percentage of CD90+ subsets remained unchanged following trauma and HS, but there was a reduction in the numbers of maturing CD90(−) cells suggesting movement into the periphery. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.