Significance The myeloproliferative neoplasms (MPNs) are a group of hematological malignancies characterized by increased numbers of myeloid blood cells, such as platelets, erythrocytes, and neutrophils. The main causes of illness and death in patients with MPNs are arterial and venous clotting and also, conversely, bleeding complications. However, the causes of these conditions are poorly understood. In this paper, we use a mouse model of MPNs to determine the cell types responsible for abnormal clotting in MPNs. We demonstrate that endothelial cells, the cell type that lines all blood vessels, have a significant role to play in MPN bleeding complications, potentially identifying a new cellular target for MPN therapies.

OBJECTIVES: Four peer-reviewed publications have reported results from randomized controlled trials of convalescent plasma for coronavirus disease 2019 infection; none were conducted in the United States nor used standard plasma as a comparator. To determine if administration of convalescent plasma to patients with coronavirus disease 2019 increases antibodies to severe acute respiratory syndrome coronavirus 2 and improves outcome. DESIGN: Double-blind randomized controlled trial. SETTING: Hospital in New York. PATIENTS: Patients with polymerase chain reaction documented coronavirus disease 2019 infection. INTERVENTIONS: Patients were randomized (4:1) to receive 2 U of convalescent plasma versus standard plasma. Antibodies to severe acute respiratory syndrome coronavirus 2 were measured in plasma units and in trial recipients. MEASUREMENTS AND MAIN RESULTS: Enrollment was terminated after emergency use authorization was granted for convalescent plasma. Seventy-four patients were randomized. At baseline, mean (sd) Acute Physiology and Chronic Health Evaluation II score (23.4 [5.6] and 22.5 [6.6]), percent of patients intubated (19% and 20%), and median (interquartile range) days from symptom onset to randomization of 9 (6–18) and 9 (6–15), were similar in the convalescent plasma versus standard plasma arms, respectively. Convalescent plasma had high neutralizing activity (median [interquartile range] titer 1:526 [1:359–1:786]) and its administration increased antibodies to severe acute respiratory syndrome coronavirus 2 by 14.4%, whereas standard plasma administration led to an 8.6% decrease (p = 0.005). No difference was observed for ventilator-free days through 28 days (primary study endpoint): median (interquartile range) of 28 (2–28) versus 28 (0–28; p = 0.86) for the convalescent plasma and standard plasma groups, respectively. A greater than or equal to 2 point improvement in the World Health Organization scale was achieved by 20% of subjects in both arms (p = 0.99). All-cause mortality through 90 days was numerically lower in the convalescent plasma versus standard plasma groups (27% vs 33%; p = 0.63) but did not achieve statistical significance. A key prespecified subgroup analysis of time to death in patients who were intubated at baseline was statistically significant; however, sample size numbers were small. CONCLUSIONS: Administration of convalescent plasma to hospitalized patients with coronavirus disease 2019 infection increased antibodies to severe acute respiratory syndrome coronavirus disease 2 but was not associated with improved outcome.

Key Points JAK2R564Q is the first germline JAK2 mutation found to contribute to a familial MPN that involves a residue other than V617. The kinase activity of JAK2R564Q and JAK2V617F are the same, but only V617F is able to escape regulation by SOCS3 and p27.

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JAK2V617F is the primary pathogenic mutation in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), a heterogeneous group of haematological malignancies characterised by increases in one or more myeloid lineages. The most common cause of morbidity and mortality in patients with MPNs are thrombohemorrhagic incidents, although the events leading to these clotting abnormalities remain unclear. It is now known that a subset of MPN patients express JAK2V617F in endothelial cells (ECs) which has been linked with a poorer prognosis and an increase in the incidence of thrombosis. Furthermore, using transgenic mouse models we have recently shown that expression of JAK2V617F in ECs plays a critical role in aberrant thrombosis in vivo (PNAS, 2014. 111(6): p. 2295-300). Here, we describe the transcriptomic and phenotypic profiles of JAK2V617F-positive ECs. Lentivirally transduced human umbilical vein ECs (HUVECs) showed a sustained increase in levels of activated JAK2, STAT3 and STAT1 in JAK2V617F-expressing cells compared to WT JAK2. In addition, JAK2V617F also caused a significant increase in expression of total STAT1 protein. Subsequent RNAseq analysis revealed an upregulation of several mRNAs related to inflammation and permeability in JAK2V617F-expressing cells. Expression of interferon-related genes, including serine protease inhibitor B2 (SERPINB2) and early growth response protein 1 (EGR1), and chemokine ligand 2 (CCL2) were also increased in JAK2V617F cells. SERPINB2 and EGR1 have roles in initiating cellular senescence and CCL2 was previously reported to induce permeability. These data, combined with increased expression of total STAT1, suggest that JAK2V617F expression in ECs induces an inflammatory response in this cell type. Moreover, in vitro permeability assays show increased permeability in HUVECs transduced with JAK2V617Fcompared to WT JAK2, which was significantly amplified following stimulation with thrombin. We then determined whether any functional differences existed between patient-derived normal and JAK2V617F-expressing ECs. For this we obtained blood from JAK2V617F-positive polycythaemia vera patients undergoing therapeutic phlebotomy and isolated blood outgrowth endothelial cells (BOECs) by repeated culture of PBMCs. Allelic burden of JAK2V617F, determined by quantitative allele-specific amplification (QuASA) was shown to vary between patient samples, (0% to 59.8% (n=9)) and increased expression of JAK2V617F appeared to correlate with decreased proliferation and significant changes in cellular morphology. In order to quantify these differences, ptychography, a novel label-free imaging technology was used to generate quantitative phase images of BOECs in time-lapse, to measure phenotypic changes in cell morphology, motility and proliferation. These results confirmed that JAK2V617F allelic burden was inversely related to BOEC proliferation. Cells which expressed only WT JAK2 or low levels of JAK2V617F exhibited exponential growth. However, an increase in JAK2V617F resulted in a decrease in BOEC proliferation. Indeed, cells which expressed high JAK2V617F levels (30-60%) failed to proliferate over the 48 hour imaging period. Furthermore, JAK2V617F-high BOECs were also shown to be larger in size, multi-nucleated and less motile compared to JAK2V617F-low. The increase in cell size and decreases in cell motility and proliferation suggest that increased JAK2V617F expression induces senescence in BOECs. These data describe features of ECs which are commonly associated with dysfunctional endothelium: sustained inflammation/permeability, limited cell growth and senescence. The critical role of ECs in maintaining hemostasis and the observed features of EC dysfunction in cells which express JAK2V617F, implicate mutation burden in ECs as a contributing factor towards the thrombotic abnormalities observed in patients with MPNs. Disclosures No relevant conflicts of interest to declare.

Objective: Severe traumatic brain injury (sTBI) often results in disorders of consciousness. Patients emerging from coma frequently exhibit aberrant behaviors such as agitation. These non-purposeful combative behaviors can interfere with medical care. Interestingly, agitation is associated with arousal and is often among the first signs of neurological recovery. A better understanding of these behaviors may shed light on the mechanisms driving the return of consciousness in sTBI patients. This study aims to investigate the association between posttraumatic agitation and the recovery of consciousness.Methods: A retrospective chart review was conducted in 530 adult patients (29.1% female) admitted to Stony Brook University Hospital between January 2011 and December 2019 with a diagnosis of sTBI and Glasgow Coma Scale (GCS) ≤8. Agitation was defined as a Richmond Agitation Sedation Scale (RASS) > +1, or any documentation of equivalently combative and violent behaviors in daily clinical notes. The ability to follow verbal commands was used to define the recovery of consciousness and was assessed daily.Results: Of 530 total sTBI patients, 308 (58.1%) survived. Agitation was present in 169 of all patients and 162 (52.6%) of surviving patients. A total of 273 patients followed commands, and 159 of them developed agitation. Forty patients developed agitation on hospital arrival whereas 119 developed agitation later during their hospital course. Presence of in-hospital agitation positively correlated with command-following (r = 0.315, p < 0.001). The time to develop agitation and time to follow commands showed positive correlation (r = 0.485, p < 0.001). These two events occurred within 3 days in 54 (44.6%) patients, within 7 days in 81 (67.8%) patients, and within 14 days in 96 (80.2%) patients. In 71 (59.7%) patients, agitation developed before command-following; in 36 (30.2%) patients, agitation developed after command-following; in 12 (10.1%) patients, agitation developed on the same day as command-following.Conclusion: Posttraumatic agitation in comatose patients following sTBI is temporally associated with the recovery of consciousness. This behavior indicates the potential for recovery of higher neurological functioning. Further studies are required to identify neural correlates of posttraumatic agitation and recovery of consciousness after sTBI.

Background Convalescent plasma is undergoing randomized trials as a potential therapeutic option for COVID‐19 infection. Little empirical evidence exists regarding the determination of donor eligibility and experiences with donor selection. Study Design and Methods This prospective study was conducted at a tertiary care hospital in New York to select plasma donors for a randomized, double‐blind, controlled convalescent plasma trial. Clearance for donation required successful completion of an online questionnaire and an in‐person screening visit, which included (a) completion of a Donor Health Questionnaire (DHQ), (b) Immunoglobulin G (IgG) antibody testing using an immunochromatographic anti‐ severe acute respiratory coronavirus 2 (SARS‐CoV‐2) test, (c) Polymerase chain reaction (PCR) testing if <28 days from symptom resolution, and (d) routine blood bank testing. Results After receiving 3093 online questionnaires, 521 individuals presented for in‐person screening visits, with 40.1% (n = 209) fully qualifying. Subjects (n = 312) failed to progress due to the following reasons: disqualifying answer from DHQ (n = 30, 9.6%), insufficient antibodies (n = 198, 63.5%), persistent positive PCR tests (n = 14, 4.5%), and blood donation testing labs (n = 70, 22.4%). Importantly, 24.6% and 11.1% of potential donors who reported having PCR‐diagnosed infection had low or undetectable SARS‐CoV‐2 antibody levels, respectively. Surprisingly, 62.9% (56/89) of subjects had positive PCR tests 14–27 days after symptom resolution, with 13 individuals continuing to be PCR positive after 27 days. Conclusion It is feasible for a single site to fully qualify a large number of convalescent plasma donors in a short period of time. Among otherwise qualified convalescent plasma donors, we found high rates of low or undetectable antibody levels and many individuals with persistently positive PCR tests.

Recovery of consciousness after traumatic brain injury (TBI) is heterogeneous and difficult to predict. Structures such as the thalamus and prefrontal cortex are thought to be important in facilitating consciousness. We sought to investigate whether the integrity of thalamo-prefrontal circuits, assessed via diffusion tensor imaging (DTI), was associated with the return of goal-directed behavior after severe TBI. We classified a cohort of severe TBI patients (N = 25, 20 males) into Early and Late/Never outcome groups based on their ability to follow commands within 30 days post-injury. We assessed connectivity between whole thalamus, and mediodorsal thalamus (MD), to prefrontal cortex (PFC) subregions including dorsolateral PFC (dlPFC), medial PFC (mPFC), anterior cingulate (ACC), and orbitofrontal (OFC) cortices. We found that the integrity of thalamic projections to PFC subregions (L OFC, L and R ACC, and R mPFC) was significantly associated with Early command-following. This association persisted when the analysis was restricted to prefrontal-mediodorsal (MD) thalamus connectivity. In contrast, dlPFC connectivity to thalamus was not significantly associated with command-following. Using the integrity of thalamo-prefrontal connections, we created a linear regression model that demonstrated 72% accuracy in predicting command-following after a leave-one-out analysis. Together, these data support a role for thalamo-prefrontal connectivity in the return of goal-directed behavior following TBI.