The humanized platelet glycoprotein VI Fab inhibitor EMA601 protects from arterial thrombosis and ischaemic stroke in mice.

BACKGROUND AND AIMS: Glycoprotein VI (GPVI) is a platelet collagen/fibrin(ogen) receptor and an emerging pharmacological target for the treatment of thrombotic and thrombo-inflammatory diseases, notably ischaemic stroke. A first anti-human GPVI (hGPVI) antibody Fab-fragment (ACT017/glenzocimab, KD: 4.1 nM) recently passed a clinical phase 1b/2a study in patients with acute ischaemic stroke and was found to be well tolerated, safe, and potentially beneficial. In this study, a novel humanized anti-GPVI antibody Fab-fragment (EMA601; KD: 0.195 nM) was developed that inhibits hGPVI function with very high potency in vitro and in vivo. METHODS: Fab-fragments of the mouse anti-hGPVI IgG Emf6.1 were tested for functional GPVI inhibition in human platelets and in hGPVI expressing (hGP6tg/tg) mouse platelets. The in vivo effect of Emf6.1Fab was assessed in a tail bleeding assay, an arterial thrombosis model and the transient middle cerebral artery occlusion (tMCAO) model of ischaemic stroke. Using complementary-determining region grafting, a humanized version of Emf6.1Fab (EMA601) was generated. Emf6.1Fab/EMA601 interaction with hGPVI was mapped in array format and kinetics and quantified by bio-layer interferometry. RESULTS: Emf6.1Fab (KD: 0.427 nM) blocked GPVI function in human and hGP6tg/tg mouse platelets in multiple assays in vitro at concentrations ≥5 µg/mL. Emf6.1Fab (4 mg/kg)-treated hGP6tg/tg mice showed potent hGPVI inhibition ex vivo and were profoundly protected from arterial thrombosis as well as from cerebral infarct growth after tMCAO, whereas tail-bleeding times remained unaffected. Emf6.1Fab binds to a so far undescribed membrane proximal epitope in GPVI. The humanized variant EMA601 displayed further increased affinity for hGPVI (KD: 0.195 nM) and fully inhibited the receptor at 0.5 µg/mL, corresponding to a >50-fold potency compared with ACT017. CONCLUSIONS: EMA601 is a conceptually novel and promising anti-platelet agent to efficiently prevent or treat arterial thrombosis and thrombo-inflammatory pathologies in humans at risk.

MAGT1 Deficiency Dysregulates Platelet Cation Homeostasis and Accelerates Arterial Thrombosis and Ischemic Stroke in Mice.

BACKGROUND: MAGT1 (magnesium transporter 1) is a subunit of the oligosaccharide protein complex with thiol-disulfide oxidoreductase activity, supporting the process of N-glycosylation. MAGT1 deficiency was detected in human patients with X-linked immunodeficiency with magnesium defect syndrome and congenital disorders of glycosylation, resulting in decreased cation responses in lymphocytes, thereby inhibiting the immune response against viral infections. Curative hematopoietic stem cell transplantation of patients with X-linked immunodeficiency with magnesium defect causes fatal bleeding and thrombotic complications. METHODS: We studied the role of MAGT1 deficiency in platelet function in relation to arterial thrombosis and hemostasis using several in vitro experimental settings and in vivo models of arterial thrombosis and transient middle cerebral artery occlusion model of ischemic stroke. RESULTS: MAGT1-deficient mice (Magt1-/y) displayed accelerated occlusive arterial thrombus formation in vivo, a shortened bleeding time, and profound brain damage upon focal cerebral ischemia. These defects resulted in increased calcium influx and enhanced second wave mediator release, which further reinforced platelet reactivity and aggregation responses. Supplementation of MgCl2 or pharmacological blockade of TRPC6 (transient receptor potential cation channel, subfamily C, member 6) channel, but not inhibition of store-operated calcium entry, normalized the aggregation responses of Magt1-/y platelets to the control level. GP (glycoprotein) VI activation of Magt1-/y platelets resulted in hyperphosphorylation of Syk (spleen tyrosine kinase), LAT (linker for activation of T cells), and PLC (phospholipase C) γ2, whereas the inhibitory loop regulated by PKC (protein kinase C) was impaired. A hyperaggregation response to the GPVI agonist was confirmed in human platelets isolated from a MAGT1-deficient (X-linked immunodeficiency with magnesium defect) patient. Haploinsufficiency of TRPC6 in Magt1-/y mice could normalize GPVI signaling, platelet aggregation, and thrombus formation in vivo. CONCLUSIONS: These results suggest that MAGT1 and TRPC6 are functionally linked. Therefore, deficiency or impaired functionality of MAGT1 could be a potential risk factor for arterial thrombosis and stroke.

Beyond change scores: Employing an improved statistical approach to analyze the impact of entry fitness on physical performance during British Army basic training in men and women.

The aim was to use a robust statistical approach to examine whether physical fitness at entry influences performance changes between men and women undertaking British Army basic training (BT). Performance of 2 km run, seated medicine ball throw (MBT) and isometric mid-thigh pull (MTP) were assessed at entry and completion of Standard Entry (SE), Junior Entry-Short (JE-Short), and Junior Entry-Long (JE-Long) training for 2350 (272 women) recruits. Performance change was analyzed with entry performance as a covariate (ANCOVA), with an additional interaction term allowing different slopes for courses and genders (p < 0.05). Overall, BT courses saw average improvements in 2 km run performance (SE: -6.8% [-0.62 min], JE-Short: -4.6% [-0.43 min], JE-Long: -7.7% [-0.70 min]; all p < 0.001) and MBT (1.0-8.8% [0.04-0.34 m]; all p < 0.05) and MTP (4.5-26.9% [6.5-28.8 kg]; all p < 0.001). Regression models indicate an expected form of "regression to the mean" whereby test performance change was negatively associated with entry fitness in each course (those with low baseline fitness exhibit larger training improvements; all interaction effects: p < 0.001, η p 2 $$ {\eta}_{\mathrm{p}}^2 $$ > 0.006), particularly for women. However, when matched for entry fitness, men displayed considerable improvements in all tests, relative to women. Training courses were effective in developing recruit physical fitness, whereby the level of improvement is, in large part, dependent on entry fitness. Factors including age, physical maturity, course length, and physical training, could also contribute to the variability in training response between genders and should be considered when analyzing and/or developing physical fitness in these cohorts for future success of military job-task performance.

Relieved or disappointed? Children's understanding of how others feel at the cessation of events.

People's emotional states are influenced not just by events occurring in the present but also by how events have unfolded in the past and how they are likely to unfold in the future. To what extent do young children understand the ways in which past events can affect current emotions even if they are no longer ongoing? In the current study, we explored children's ability to understand how others feel at the cessation of events-as events change from being present to being past. We asked 97 4- to 6-year-olds (40.2% female) and 35 adults (54.3% female) to judge how characters felt once particular types of events had ended relative to how they felt during these events. We found that from age 4, children judged (as adults do) that the character would feel positive at the cessation of negative events-what we refer to as temporal relief. This understanding of relief occurs earlier than has been shown in previous research. However, children were less likely than adults to judge others as feeling sad at the cessation of positive events-what we refer to as temporal disappointment. Overall, our findings suggest that children not only understand that the cessation of events can affect others' emotions but also recognize that people feel differently following the cessation of positive, negative, and neutral events.

Progression and Resolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in Golden Syrian Hamsters.

To catalyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, including development of novel interventive and preventive strategies, the progression of disease was characterized in a robust coronavirus disease 2019 (COVID-19) animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at 2, 4, 7, 14, and 28 days after inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal time points, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 after inoculation, corresponding with widespread necrosis and inflammation. At day 7, when the presence of infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 after inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

Association between external training loads and injury incidence during 44 weeks of military training.

Military training is physically arduous and associated with high injury incidence. Unlike in high-performance sport, the interaction between training load and injury has not been extensively researched in military personnel. Sixty-three (43 men, 20 women; age 24 ± 2 years; stature 1.76 ± 0.09 m; body mass 79.1 ± 10.8 kg) British Army Officer Cadets undergoing 44 weeks of training at the Royal Military Academy Sandhurst volunteered to participate. Weekly training load (cumulative 7-day moderate-vigorous physical activity [MVPA], vigorous PA [VPA], and the ratio between MVPA and sedentary-light PA [SLPA; MVPA:SLPA]) was monitored using a wrist-worn accelerometer (GENEActiv, UK). Self-report injury data were collected and combined with musculoskeletal injuries recorded at the Academy medical center. Training loads were divided into quartiles with the lowest load group used as the reference to enable comparisons using odds ratios (OR) and 95% confidence intervals (95% CI). Overall injury incidence was 60% with the most common injury sites being the ankle (22%) and knee (18%). High (load; OR; 95% CI [>2327 mins; 3.44; 1.80-6.56]) weekly cumulative MVPA exposure significantly increased odds of injury. Similarly, likelihood of injury significantly increased when exposed to low-moderate (0.42-0.47; 2.45 [1.19-5.04]), high-moderate (0.48-0.51; 2.48 [1.21-5.10]), and high MVPA:SLPA loads (>0.51; 3.60 [1.80-7.21]). High MVPA and high-moderate MVPA:SLPA increased odds of injury by ~2.0 to 3.5 fold, suggesting that the ratio of workload to recovery is important for mitigating injury occurrence.

Is rumination associated with psychological distress after a cancer diagnosis? A systematic review.

Objective: The aim of this work was to review evidence on the association between psychological rumination and distress in those diagnosed with cancer. Methods: Six databases were searched for studies exploring rumination alongside overall assessments of psychological distress, depression, anxiety, or stress. Results: Sixteen studies were identified. Rumination was associated with distress cross-sectionally and longitudinally. However, once baseline depression was controlled for, the association was no longer seen. The emotional valence of ruminative thoughts and the style in which they were processed, rather than their topic, was associated with distress. Brooding and intrusive rumination were associated with increased distress, deliberate rumination had no association, and reflection/instrumentality had mixed findings. Conclusions: This review highlights that it is not necessarily the topic of content, but the style and valence of rumination that is important when considering its association with distress. The style of rumination should be the target of clinical intervention, including brooding and intrusion.

Monoglyceride Lipase Deficiency Is Associated with Altered Thrombogenesis in Mice.

Monoglyceride lipase (MGL) hydrolyzes monoacylglycerols (MG) to glycerol and one fatty acid. Among the various MG species, MGL also degrades 2-arachidonoylglycerol, the most abundant endocannabinoid and potent activator of the cannabinoid receptors 1 and 2. We investigated the consequences of MGL deficiency on platelet function using systemic (Mgl-/-) and platelet-specific Mgl-deficient (platMgl-/-) mice. Despite comparable platelet morphology, loss of MGL was associated with decreased platelet aggregation and reduced response to collagen activation. This was reflected by reduced thrombus formation in vitro, accompanied by a longer bleeding time and a higher blood volume loss. Occlusion time after FeCl3-induced injury was markedly reduced in Mgl-/- mice, which is consistent with contraction of large aggregates and fewer small aggregates in vitro. The absence of any functional changes in platelets from platMgl-/- mice is in accordance with lipid degradation products or other molecules in the circulation, rather than platelet-specific effects, being responsible for the observed alterations in Mgl-/- mice. We conclude that genetic deletion of MGL is associated with altered thrombogenesis.

Thymosin ß4 is essential for thrombus formation by controlling the G-actin/F-actin equilibrium in platelets.

Coordinated rearrangements of the actin cytoskeleton are pivotal for platelet biogenesis from megakaryocytes but also orchestrate key functions of peripheral platelets in hemostasis and thrombosis, such as granule release, the formation of filopodia and lamellipodia, or clot retraction. Along with profilin (Pfn) 1, thymosin ß4 (encoded by Tmsb4x) is one of the two main G-actin-sequestering proteins within cells of higher eukaryotes, and its intracellular concentration is particularly high in cells that rapidly respond to external signals by increased motility, such as platelets. Here, we analyzed constitutive Tmsb4x knockout (KO) mice to investigate the functional role of the protein in platelet production and function. Thymosin ß4 deficiency resulted in a macrothrombocytopenia with only mildly increased platelet volume and an unaltered platelet life span. Megakaryocyte numbers in the bone marrow and spleen were unaltered, however, Tmsb4x KO megakaryocytes showed defective proplatelet formation in vitro and in vivo. Thymosin ß4-deficient platelets displayed markedly decreased G-actin levels and concomitantly increased F-actin levels resulting in accelerated spreading on fibrinogen and clot retraction. Moreover, Tmsb4x KO platelets showed activation defects and an impaired immunoreceptor tyrosine-based activation motif (ITAM) signaling downstream of the activating collagen receptor glycoprotein VI. These defects translated into impaired aggregate formation under flow, protection from occlusive arterial thrombus formation in vivo and increased tail bleeding times. In summary, these findings point to a critical role of thymosin ß4 for actin dynamics during platelet biogenesis, platelet activation downstream of glycoprotein VI and thrombus stability.

Children's understanding of counterfactual and temporal relief in others.

Developmentalists have investigated relief as a counterfactually mediated emotion, but not relief experienced when negative events end-so-called temporal relief. This study represents the first body of work to investigate the development of children's understanding of temporal relief and compare it with their understanding of counterfactual relief. Across four experiments (407 children aged 4-11 years and 60 adults; 52% female), we examined children's ability to attribute counterfactual and temporal relief to others. In Experiment 1, 7- to 10-year-olds typically judged that two characters would feel equally happy despite avoiding or enduring an event that was unpleasant for one character. Using forced-choice procedures, Experiments 2 to 4 showed that a fledgling ability to attribute relief to others emerges at 5 to 6 years of age and that the tendency to make these attributions increases with age. The experiments in this study provide the first positive evidence in the literature as to when children can begin to attribute both counterfactual and temporal instances of relief to others. Overall, there was little evidence for separate developmental trajectories for understanding counterfactual and temporal relief, although in Experiment 4 there was an indication that, under scaffolded contexts, some children find it easier to attribute counterfactual relief rather than temporal relief to others.