Response to COVID-19 vaccination in patients on cancer therapy: Analysis in a SARS-CoV-2-naïve population.

BACKGROUND: Cancer patients have increased morbidity and mortality from COVID-19, but may respond poorly to vaccination. The Evaluation of COVID-19 Vaccination Efficacy and Rare Events in Solid Tumors (EVEREST) study, comparing seropositivity between cancer patients and healthy controls in a low SARS-CoV-2 community-transmission setting, allows determination of vaccine response with minimal interference from infection. METHODS: Solid tumor patients from The Canberra Hospital, Canberra, Australia, and healthy controls who received COVID-19 vaccination between March 2021 and January 2022 were included. Blood samples were collected at baseline, pre-second vaccine dose and at 1, 3 (primary endpoint), and 6 months post-second dose. SARS-CoV-2 anti-spike-RBD (S-RBD) and anti-nucleocapsid IgG antibodies were measured. RESULTS: Ninety-six solid tumor patients and 20 healthy controls were enrolled, with median age 62 years, and 60% were female. Participants received either AZD1222 (65%) or BNT162b2 (35%) COVID-19 vaccines. Seropositivity 3 months post vaccination was 87% (76/87) in patients and 100% (20/20) in controls (p = .12). Seropositivity was observed in 84% of patients on chemotherapy, 80% on immunotherapy, and 96% on targeted therapy (differences not satistically significant). Seropositivity in cancer patients increased from 40% (6/15) after first dose, to 95% (35/37) 1 month after second dose, then dropped to 87% (76/87) 3 months after second dose. CONCLUSION: Most patients and all controls became seropositive after two vaccine doses. Antibody concentrations and seropositivity showed a decrease between 1 and 3 months post vaccination, highlighting need for booster vaccinations. SARS-CoV-2 infection amplifies S-RBD antibody responses; however, cannot be adequately identified using nucleocapsid serology. This underlines the value of our COVID-naïve population in studying vaccine immunogenicity.

Platelets mediate the clearance of senescent red blood cells by forming prophagocytic platelet-cell complexes.

ABSTRACT: In humans, ∼0.1% to 0.3% of circulating red blood cells (RBCs) are present as platelet-RBC (P-RBC) complexes, and it is 1% to 2% in mice. Excessive P-RBC complexes are found in diseases that compromise RBC health (eg, sickle cell disease and malaria) and contribute to pathogenesis. However, the physiological role of P-RBC complexes in healthy blood is unknown. As a result of damage accumulated over their lifetime, RBCs nearing senescence exhibit physiological and molecular changes akin to those in platelet-binding RBCs in sickle cell disease and malaria. Therefore, we hypothesized that RBCs nearing senescence are targets for platelet binding and P-RBC formation. Confirming this hypothesis, pulse-chase labeling studies in mice revealed an approximately tenfold increase in P-RBC complexes in the most chronologically aged RBC population compared with younger cells. When reintroduced into mice, these complexes were selectively cleared from the bloodstream (in preference to platelet-free RBC) through the reticuloendothelial system and erythrophagocytes in the spleen. As a corollary, patients without a spleen had higher levels of complexes in their bloodstream. When the platelet supply was artificially reduced in mice, fewer RBC complexes were formed, fewer erythrophagocytes were generated, and more senescent RBCs remained in circulation. Similar imbalances in complex levels and senescent RBC burden were observed in humans with immune thrombocytopenia (ITP). These findings indicate that platelets are important for binding and clearing senescent RBCs, and disruptions in platelet count or complex formation and clearance may negatively affect RBC homeostasis and may contribute to the known risk of thrombosis in ITP and after splenectomy.

An implementation strategy package (video education, HIV self-testing, and co-location) improves PrEP implementation for pregnant women in antenatal care clinics in western Kenya.

Background: Pre-exposure prophylaxis (PrEP) is recommended by the World Health Organization and the Kenyan Ministry of Health for HIV prevention in pregnancy and postpartum for women at risk for HIV. Integration of PrEP into antenatal care is promising, but delivery gaps exist in the face of healthcare provider shortages in resource-limited settings. Methods: Between May and November 2021, we conducted a difference-in-differences study (3 months pre-intervention data collection and 3 months post-intervention data collection) analyzing four intervention facilities, where the strategies were implemented, and four comparison facilities, where no strategies were implemented. We tested a combination of three implementation strategies-video-based PrEP information in the waiting bay, HIV self-testing, and dispensing of PrEP in the antenatal care rooms-to improve PrEP delivery. We compared absolute changes in the proportion of antenatal attendees screened for PrEP (PrEP penetration), the proportion receiving all PrEP-specific steps in a visit (HIV testing, risk screening, and PrEP counseling) (PrEP fidelity), and client PrEP knowledge, client satisfaction, and waiting time and service time (a priori outcomes); post hoc, we compared the proportion offered PrEP (PrEP offer) and completing HIV testing. We measured provider perceptions of the acceptability and appropriateness of the implementation strategies. Results: We observed significant improvements in PrEP penetration, PrEP offer, satisfaction, and knowledge (p < 0.05) and improvements in fidelity that trended towards significance (p = 0.057). PrEP penetration increased 5 percentage points (p = 0.008), PrEP fidelity increased 8 percentage points (p = 0.057), and PrEP offer increased 4 percentage points (p = 0.003) in intervention vs. comparison facilities. Client PrEP knowledge increased by 1.7 out of 6 total points (p < 0.001) and client satisfaction increased by 0.7 out of 24 total points (p = 0.003) in intervention vs. comparison facilities. We observed no changes in service time (0.09-min decrease; p = 0.435) and a small increase in waiting time (0.33-min increase; p = 0.005). HIV testing among those eligible did not change (1.5 percentage point decrease, p = 0.800). Providers felt the implementation strategies were acceptable and appropriate (median acceptability: 20/20; median appropriateness: 19.5/20). However, absolute levels of each step of the PrEP cascade remained suboptimal. Conclusions: An implementation strategy package with video information, HIV self-testing, and co-location of medication dispensing enhanced PrEP delivery across several implementation outcomes and client satisfaction, while not substantially increasing wait time or decreasing provider-client contact time. Clinical trial registration: ClinicalTrials.gov , identifier, NCT04712994.

Tissue inhibitors of metalloproteinases (TIMPs) modulate platelet ADAM10 activity.

Platelet-specific collagen receptor glycoprotein (GP)VI is stable on the surface of circulating platelets but undergoes ectodomain cleavage on activated platelets. Activation-dependent GPVI metalloproteolysis is primarily mediated by A Disintegrin And Metalloproteinase (ADAM) 10. Regulation of platelet ADAMs activity is not well-defined however Tissue Inhibitors of Metalloproteinases (TIMPs) may play a role. As levels of TIMPs on platelets and the control of ADAMs-mediated shedding by TIMPs has not been evaluated, we quantified the levels of TIMPs on the surface of resting and activated platelets from healthy donors by flow cytometry and multiplex ELISA. Variable levels of all TIMPs could be detected on platelets. Plasma contained significant quantities of TIMP1 and TIMP2, but only trace amounts of TIMP3 and TIMP4. Recombinant TIMP3 strongly ablated resting and activated platelet ADAM10 activity, when monitored using a quenched fluorogenic peptide substrate with ADAM10 specificity. Whilst ADAM10-specific inhibitor GI254023X or ethylenediamine tetraacetic acid (EDTA) could modulate ligand-initiated shedding of GPVI, only recombinant TIMP2 achieved a modest (~20%) inhibition. We conclude that some platelet TIMPs are able to modulate platelet ADAM10 activity but none strongly regulate ligand-dependent shedding of GPVI. Our findings provide new insights into the regulation of platelet receptor sheddase activity.

What do we know? Platelet receptor GPVI initiates platelet adhesion and aggregation and is proteolytically cleaved from the activated platelet surfaceThe metalloproteinases responsible belong to the ADAMs family of enzymes which are inhibited by TIMPsWhat did we discover? Plasma contains significant amounts of TIMP1 and TIMP2Circulating platelets bear significant amounts of TIMPs 1, 2, and 3Recombinant TIMP3 strongly inhibits resting and activated platelet ADAM10 activityExogenous addition of TIMP2 mildly blocked ligand-initiated shedding of GPVIWhat is the impact? TIMPs may modulate ADAM10 activity under resting conditions and stabilize GPVI levels in response to platelet activationAnti-GPVI agents are being evaluated as anti-thrombotic agents, however, acute loss of GPVI in trauma or settings of thrombocytopenia is linked with clinical bleedingUnderstanding how GPVI levels are regulated is important as agents that modulate GPVI function are emerging as important therapeutics for clinical applications in Thrombosis and Hemostasis fields.

Selecting implementation strategies to improve implementation of integrated PrEP for pregnant and postpartum populations in Kenya: a sequential explanatory mixed methods analysis.

BACKGROUND: There is a higher risk for HIV acquisition during pregnancy and postpartum. Pre-exposure prophylaxis (PrEP) is recommended during this period for those at high risk of infection; integrated delivery in maternal and child health (MCH) clinics is feasible and acceptable but requires implementation optimization. METHODS: The PrEP in Pregnancy, Accelerating Reach and Efficiency study (PrEPARE; NCT04712994) engaged stakeholders to prioritize determinants of PrEP delivery (using Likert scores) and prioritize PrEP delivery implementation strategies. Using a sequential explanatory mixed methods design, we conducted quantitative surveys with healthcare workers at 55 facilities in Western Kenya and a stakeholder workshop (including nurses, pharmacists, counselors, and county and national policymakers), yielding visual plots of stakeholders' perceived feasibility and effectiveness of the strategies. A stepwise elimination process was used to identify seven strategies for empirical testing. Facilitator debriefing reports from the workshop were used to qualitatively assess the decision-making process. RESULTS: Among 146 healthcare workers, the strongest reported barriers to PrEP delivery were insufficient providers and inadequate training, insufficient space, and high volume of patients. Sixteen strategies were assessed, 14 of which were included in the final analysis. Among rankings from 182 healthcare workers and 44 PrEP policymakers and implementers, seven strategies were eliminated based on low post-workshop ranking scores (bottom 50th percentile) or being perceived as low feasibility or low effectiveness for at least 50% of the workshop groups. The top seven strategies included delivering PrEP within MCH clinics instead of pharmacies, fast-tracking PrEP clients to reduce waiting time, delivering PrEP-related health talks in waiting bays, task shifting PrEP counseling, task shifting PrEP risk assessments, training different providers to deliver PrEP, and retraining providers on PrEP delivery. All top seven ranked strategies were grouped into bundles for subsequent testing. Facilitator debriefing reports generally aligned with rankings but noted how stakeholders' decision-making changed when considering the impact of strategies on facility staff and non-PrEP clients. CONCLUSIONS: The most impactful barriers to integrated PrEP delivery in MCH clinics were insufficient staffing and space. Implementation strategies prioritized through multiple methods of stakeholder input focused on co-location of services and increasing clinic efficiency. Future testing of these stakeholder-prioritized strategy bundles will be conducted to assess the effectiveness and implementation outcomes.

Integrating PrEP in maternal and child health clinics in Kenya: analysis of a service availability and readiness assessment (SARA) survey.

Background: Risk of HIV acquisition is high during pregnancy and postpartum, and pre-exposure prophylaxis (PrEP) is recommended for peripartum populations. Integrating PrEP into maternal and child health (MCH) clinics is feasible and acceptable. Understanding clinics' service availability and readiness is essential for effective scale up. Methods: The PrEP in Pregnancy, Accelerating Reach and Efficiency study (PrEPARE; NCT04712994) engaged PrEP-experienced facilities previously linked to a programmatic or research study in Western Kenya to document available services and commodities via a modified service availability and readiness assessment (SARA) survey with 20 PrEP tracer items covering: staffing/guidelines, services/equipment, and medicines/commodities. Facilities' prior study engagement occurred between 2017 and 2019; SARA survey data was collected between April 2020 and June 2021. Descriptive statistics were stratified by prior study engagement. ANOVA tests assessed associations between facility characteristics and gaps. Fisher's tests assessed differences in commodity availability and stockouts. Results: Of the 55 facilities surveyed, 60% had received PrEP training in the last two years, 95% offered PrEP integrated into MCH, and 64% and 78% had both auditory and visual privacy in PrEP and HIV testing service (HTS) delivery spaces, respectively. Supervision frequency was heterogeneous, but 82% had received a supervision visit within 3 months. Availability of commodities was variable and the most commonly unavailable commodities were PrEP in MCH (71% available) and risk assessment screening tool (RAST) and PrEP cards (60% and 75% available, respectively). The number of service and commodity gaps per facility ranged from zero to eight (median: 3; IQR: 2, 5). The most frequent gaps were: PrEP training and risk assessment cards (40% each), lack of privacy in PrEP (36%) and HIV testing services (31%) spaces, PrEP pills in MCH (29%), and PrEP cards (25%). There were no differences in mean number of gaps by county, previous study engagement, or public vs. private status. Level 4 facilities had fewer gaps (mean 2.2) than level 2, 3, and 5 facilities (mean 5.7, 4.5, and 5.3 respectively; p < 0.001). Conclusions: PrEP service availability and readiness was generally high across MCH facilities. However, there is a need for increased frequency of provider training and supportive supervision focused on fidelity. To address key commodity stockouts such as PrEP pills, implementation of electronic logistics management information systems may be needed. Targeting these gaps is essential to effectively scale up integrated PrEP delivery, especially among facilities with limited infrastructure.

Glycoprotein VI is Critical for the Detection and Progression of Abdominal Aortic Aneurysms.

A common feature in patients with abdominal aortic aneurysms (AAA) is the formation of a nonocclusive intraluminal thrombus (ILT) in regions of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation impacts the pathogenesis of AAA. Using RNA-sequencing, we identify that the platelet-associated transcripts are significantly enriched in the ILT compared to the adjacent aneurysm wall and healthy control aortas. We found that the platelet specific receptor glycoprotein VI (GPVI) is among the top enriched genes in AAA ILT and is increased on the platelet surface of AAA patients. Examination of a specific indicator of platelet activity, soluble GPVI (sGPVI), in two independent AAA patient cohorts is highly predictive of a AAA diagnosis and associates more strongly with aneurysm growth rate when compared to D-dimer in humans. Finally, intervention with the anti-GPVI antibody (J) in mice with established aneurysms blunted the progression of AAA in two independent mouse models. In conclusion, we show that levels of sGPVI in humans can predict a diagnosis of AAA and AAA growth rate, which may be critical in the identification of high-risk patients. We also identify GPVI as a novel platelet-specific AAA therapeutic target, with minimal risk of adverse bleeding complications, where none currently exist. KEY POINTS: Soluble glycoprotein VI, which is a platelet-derived blood biomarker, predicts a diagnosis of AAA, with high sensitivity and specificity in distinguishing patients with fast from slow-growing AAA.Blockade of glycoprotein VI in mice with established aneurysms reduces AAA progression and mortality, indicating therapeutic potential.

Comparison of methods to engage diverse stakeholder populations in prioritizing PrEP implementation strategies for testing in resource-limited settings: a cross-sectional study.

BACKGROUND: There is a lack of consensus about how to prioritize potential implementation strategies for HIV pre-exposure prophylaxis (PrEP) delivery. We compared several prioritization methods for their agreement and pragmatism in practice in a resource-limited setting. METHODS: We engaged diverse stakeholders with clinical PrEP delivery and PrEP decision-making experience across 55 facilities in Kenya to prioritize 16 PrEP delivery strategies. We compared four strategy prioritization methods: (1) "past experience surveys" with experienced practitioners reflecting on implementation experience (N = 182); (2 and 3) "pre- and post-small-group ranking" surveys before and after group discussion (N = 44 and 40); (4) "go-zone" quadrant plots of perceived effectiveness vs feasibility. Kendall's correlation analysis was used to compare strategy prioritization using the four methods. Additionally, participants were requested to group strategies into three bundles with up to four strategies/bundle by phone and online survey. RESULTS: The strategy ranking correlation was strongest between the pre- and post-small-group rankings (Tau: 0.648; p < 0.001). There was moderate correlation between go-zone plots and post-small-group rankings (Tau: 0.363; p = 0.079) and between past-experience surveys and post-small-group rankings (Tau: 0.385; p = 0.062). For strategy bundling, participants primarily chose bundles of strategies in the order in which they were listed, reflecting option ordering bias. Neither the phone nor online approach was effective in selecting strategy bundles. Participants agreed that the strategy ranking activities conducted during the workshop were useful in prioritizing a final set of strategies. CONCLUSIONS: Both experienced and inexperienced stakeholder participants' strategy rankings tended to prioritize strategies perceived as feasible. Small group discussions focused on feasibility and effectiveness revealed moderately different priorities than individual rankings. The strategy bundling approach, though less time- and resource-intensive, was not effective. Future research should further compare the relative effectiveness and pragmatism of methodologies to prioritize implementation strategies.

A review of evidence-based management of infectious ocular surface disease in shelter-housed domestic cats.

Infectious ocular surface disease (IOSD) is a common problem in shelter-housed domestic cats and has a widespread negative impact on animal welfare. While the common etiological agents are well-described, addressing IOSD in large groups of animals presents a management challenge to the clinician and logistical challenges to shelter employees. Treatments, diagnostics, and prevention strategies that are effective in privately owned or experimental animals may be impractical or ineffective in the shelter environment. This review article focuses on the relative prevalence of etiological agents in feline IOSD, practical diagnostic testing protocols, prevention strategies, and treatment of IOSD in shelter-housed cats. Discrepancies between experimental laboratory-based studies and clinical trials assessing therapeutics for treatment of feline herpes virus are highlighted. Further high-quality clinical trials are necessary to determine optimal preventative and therapeutic protocols for IOSD in shelter-housed cats.

Healthcare Workers' Perceptions of On-Site Childcare.

GOAL: Measures taken by healthcare organizations to address COVID-19 highlighted the long-standing lack of childcare infrastructure required to support healthcare workers. This study, designed to provide evidence to support operations at an academic medical center, looked at the influence that in-house and emergency childcare could have on the retention, recruitment, and productivity of healthcare workers. This study also outlined the implications that childcare, or its lack, has for healthcare organizations during and after the COVID-19 pandemic. METHODS: We conducted a 35-question electronic employee survey (under institutional review board approval) during pandemic-induced public school closures, which included both quantitative and qualitative (write-in) questions. PRINCIPAL FINDINGS: The survey results showed that weekday on-site childcare was very or extremely important to more than half of survey respondents, the majority of whom were staff members (28%) or physicians (25%), followed by administrators (15%), researchers (12%), others (10%), nurses (5%), educators (2%), and residents (1%). Sixty percent of respondents reported that emergency on-site childcare was extremely important (34%) or very important (26%). Almost half (49%) reported that emergency childcare needs have disrupted their work in the past year, including canceling of clinics or surgical cases. Analysis of qualitative comments via a strategy based on coding and categorization showed that, when asked how childcare influences their work choices, employees responded that childcare availability has limited the hours or times they could work, that lack of childcare has prevented career growth, that they left a previous job or will leave their current job because of childcare needs, or that they stayed at a previous job or have remained in their current job longer because of the availability of childcare. PRACTICAL APPLICATIONS: Although data from this mixed-methods study support findings in the literature that there is a need for in-house and emergency childcare, the data suggest that current employees at this academic medical center do not currently expect it, likely because such childcare is not generally available at most academic institutions. With increased rates of burnout and healthcare workers leaving the field since COVID-19, offering in-house and emergency childcare provides hospital systems with new opportunities to retain and recruit physicians, nurses, and staff, as well as to improve their well-being and productivity.

Cross-sectional Associations of Multiracial Identity with Self-Reported Asthma and Poor Health Among American Indian and Alaska Native Adults.

INTRODUCTION: American Indian and Alaska Native (AI/AN) multiracial subgroups are underrecognized in health outcomes research. METHODS: We performed a cross-sectional analysis of Behavioral Risk Factor Surveillance System surveys (2013-2019), including adults who self-identified as AI/AN only (single race AI/AN, n = 60,413) or as AI/AN and at least one other race (multiracial AI/AN, (n = 6056)). We used log binomial regression to estimate the survey-weighted prevalence ratios (PR) and 95% confidence intervals (CI) of lifetime asthma, current asthma, and poor self-reported health among multiracial AI/AN adults compared to single race AI/AN adults, adjusting for age, obesity, and smoking status. We then examined whether associations differed by sex and by Latinx identity. RESULTS: Lifetime asthma, current asthma, and poor health were reported by 25%, 18%, and 30% of multiracial AI/AN adults and 18%, 12%, and 28% single race AI/AN adults. Multiracial AI/AN was associated with a higher prevalence of lifetime (PR 1.30, 95% CI 1.18-1.43) and current asthma (PR 1.36, 95% CI 1.21-1.54), but not poor health. Associations did not differ by sex. The association of multiracial identity with current asthma was stronger among AI/AN adults who identified as Latinx (PR 1.77, 95% CI 1.08-2.94) than non-Latinx AI/AN (PR 1.18, 95% CI 1.04-1.33), p-value for interaction 0.03. CONCLUSIONS: Multiracial AI/AN adults experience a higher prevalence of lifetime and current asthma compared to single race AI/AN adults. The association between multiracial identity and current asthma is stronger among AI/AN Latinx individuals. The mechanisms for these findings remain under-explored and merit further study.

Pan-Canadian consensus recommendations for proton beam therapy access in Canada.

PURPOSE: Proton Beam Therapy (PBT)is a treatment option for select cancer patients. It is currently not available in Canada. Assessment and referral processes for out-of-country treatment for eligible patients vary by jurisdiction, leading to variability in access to this treatment for Canadian cancer patients. The purpose of this initiative was to develop a framework document to inform consistent and equitable PBT access for appropriate patients through the creation of pan-Canadian PBT access consensus recommendations. MATERIALS AND METHODS: A modified Delphiprocess was used to develop pan-Canadian recommendations with input from 22 PBT clinical and administrative experts across all provinces, external peer-review by provincial cancer and system partners, and feedback from a targeted community consultation. This was conducted by electronic survey and live discussion. Consensus threshold was set at 70% agreement. RESULTS: Fourconsensus rounds resulted in a final set of 27 recommendations divided into three categories: patient eligibility (n = 9); program level (n = 10); and system level (n = 8). Patient eligibility included: anatomic site (n = 4), patient characteristics (n = 3), clinical efficacy (n = 2). Program level included: regulatory and staff requirements (n = 5), equipment and technologies (n = 4), quality assurance (n = 1). System level included: referral process (n = 5), costing, budget impact and quality adjusted life years (n = 2), eligible patient estimates (n = 1). Recommendations were released nationally in June 2021 and distributed to all 43 cancer programs in Canada. CONCLUSION: A pan-Canadian consensus-building approach was successful in creating an evidence-based, peer-reviewed suite of recommendations thatsupportapplication of consistent clinical criteria to inform treatment options, facility set-up and access to high quality proton therapy.

Cryoprecipitate as an alternative to platelet transfusion in thrombocytopenia.

Platelet transfusions are not always available for bleeding in severe thrombocytopenia, as storage outside of major centers is limited by their short shelf-life. Data are lacking to support alternative available blood products; however, additional fibrinogen has been shown to enhance clot formation in vitro. To test the hypothesis that cryoprecipitate supplementation could improve clot formation in severe thrombocytopenia, eight hematological malignancy patients with platelet counts under 10 × 109/L each had 10 units of apheresis cryoprecipitate transfused prior to planned prophylactic platelet transfusions. The primary endpoint of thromboelastometry amplitude at 20 min increased by a mean of 5.1 mm (p < 0.01) following cryoprecipitate transfusion despite persisting thrombocytopenia. Thromboelastometry clotting times reduced by a mean of 7.8 s (p < 0.05) and alpha angle increased by a mean of 10.6° (p < 0.01). These results are consistent with cryoprecipitate enhancing the strength of the fibrin/platelet meshwork within the forming thrombus. While platelet transfusion remains the standard of care, where platelet supplies are limited, these data provide a rationale for the use of cryoprecipitate to obtain hemostasis in bleeding thrombocytopenic patients.

A novel flow cytometry procoagulant assay for diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti-platelet factor 4 (PF4) antibodies that activate platelets via the low-affinity immunoglobulin G receptor FcγRIIa to drive thrombosis and thrombocytopenia. Functional assays are important for VITT diagnosis, as not all detectable anti-PF4 antibodies are pathogenic, and immunoassays have varying sensitivity. Combination of ligand binding of G protein-coupled receptors (protease-activated receptor-1) and immunoreceptor tyrosine-based activation motif-linked receptors (FcγRIIa) synergistically induce procoagulant platelet formation, which supports thrombin generation. Here, we describe a flow cytometry-based procoagulant platelet assay using cell death marker GSAO and P-selectin to diagnose VITT by exposing donor whole blood to patient plasma in the presence of a protease-activated receptor-1 agonist. Consecutive patients triaged for confirmatory functional VITT testing after screening using PF4/heparin ELISA were evaluated. In a development cohort of 47 patients with suspected VITT, plasma from ELISA-positive patients (n = 23), but not healthy donors (n = 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n = 24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified according to clinicopathologic adjudication, procoagulant flow cytometry identified 93% of VITT cases, including ELISA-negative and serotonin release assay-negative patients. The in vitro effect of intravenous immunoglobulin (IVIg) and fondaparinux trended with the clinical response seen in patients. Induction of FcγRIIa-dependent procoagulant response by patient plasma, suppressible by heparin and IVIg, is highly indicative of VITT, resulting in a sensitive and specific assay that has been adopted as part of a national diagnostic algorithm to identify vaccinated patients with platelet-activating antibodies.

PURA-Related Developmental and Epileptic Encephalopathy: Phenotypic and Genotypic Spectrum.

BACKGROUND AND OBJECTIVES: Purine-rich element-binding protein A (PURA) gene encodes Pur-α, a conserved protein essential for normal postnatal brain development. Recently, a PURA syndrome characterized by intellectual disability, hypotonia, epilepsy, and dysmorphic features was suggested. The aim of this study was to define and expand the phenotypic spectrum of PURA syndrome by collecting data, including EEG, from a large cohort of affected patients. METHODS: Data on unpublished and published cases were collected through the PURA Syndrome Foundation and the literature. Data on clinical, genetic, neuroimaging, and neurophysiologic features were obtained. RESULTS: A cohort of 142 patients was included. Characteristics of the PURA syndrome included neonatal hypotonia, feeding difficulties, and respiratory distress. Sixty percent of the patients developed epilepsy with myoclonic, generalized tonic-clonic, focal seizures, and/or epileptic spasms. EEG showed generalized, multifocal, or focal epileptic abnormalities. Lennox-Gastaut was the most common epilepsy syndrome. Drug refractoriness was common: 33.3% achieved seizure freedom. We found 97 pathogenic variants in PURA without any clear genotype-phenotype associations. DISCUSSION: The PURA syndrome presents with a developmental and epileptic encephalopathy with characteristics recognizable from neonatal age, which should prompt genetic screening. Sixty percent have drug-resistant epilepsy with focal or generalized seizures. We collected more than 90 pathogenic variants without observing overt genotype-phenotype associations.

Phosphoproteomic Analysis of Platelets in Severe Obesity Uncovers Platelet Reactivity and Signaling Pathways Alterations.

OBJECTIVE: Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. Approach and Results: We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- and gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients. CONCLUSIONS: Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.

A Dual-Antigen Enzyme-Linked Immunosorbent Assay Allows the Assessment of Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Seroprevalence in a Low-Transmission Setting.

Estimates of seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have been hampered by inadequate assay sensitivity and specificity. Using an enzyme-linked immunosorbent assay-based approach that combines data about immunoglobulin G responses to both the nucleocapsid and spike receptor binding domain antigens, we show that excellent sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (95% Confidence Interval, 0-1.15%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.

Salt and water balance after sweat loss: A study of Bikram yoga.

Bikram yoga is practiced in a room heated to 105°F with 40% humidity for 90 min. During the class a large volume of water and electrolytes are lost in the sweat, specifically, sodium is lost, the main cation of the extracellular fluid. There is little known about the volume of sweat and the amount of sodium lost in sweat during Bikram yoga or the optimum quantity of fluid required to replace these losses. The participants who took part in this small feasibility study were five females with a mean age of 47.4 ± 4.7 years and 2.6 ± 1.6 years of experience at Bikram yoga. The total body weight, water consumed, serum sodium concentration, serum osmolality, and serum aldosterone levels were all measured before and after a Bikram yoga practice. Sweat sodium chloride concentration and osmolality were measured at the end of the practice. The mean estimated sweat loss was 1.54 ± 0.65 L, while the amount of water consumed during Bikram yoga was 0.38 ± 0.22 L. Even though only 25% of the sweat loss was replenished with water intake during the Bikram yoga class, we did not observe a change in serum sodium levels or serum osmolality. The sweat contained 82 ± 16 mmol/L of sodium chloride for an estimated total of 6.8 ± 2.1 g of sodium chloride lost in the sweat. The serum aldosterone increased 3.5-fold from before to after Bikram yoga. There was a decrease in the extracellular body fluid compartment of 9.7%. Sweat loss in Bikram yoga predominately produced a volume depletion rather than the dehydration of body fluids. The sweating-stimulated rise in serum aldosterone levels will lead to increased sodium reabsorption from the kidney tubules and restore the extracellular fluid volume over the next 24 hr.

The molecular basis of immune-based platelet disorders.

Platelets have a predominant role in haemostasis, the maintenance of blood volume and emerging roles as innate immune cells, in wound healing and in inflammatory responses. Platelets express receptors that are important for platelet adhesion, aggregation, participation in inflammatory responses, and for triggering degranulation and enhancing thrombin generation. They carry a cargo of granules bearing enzymes, adhesion molecules, growth factors and cytokines, and have the ability to generate reactive oxygen species. The platelet is at the frontline of a host of cellular responses to invading pathogens, injury, and infection. Perhaps because of this intrinsic responsibility of a platelet to rapidly respond to thrombotic, pathological and immunological factors as part of their infantry role; platelets are susceptible to targeted attack by the adaptive immune system. Such attacks are often transitory but result in aberrant platelet activation as well as significant loss of platelet numbers and platelet function, paradoxically leading to elevated risks of both thrombosis and bleeding. Here, we discuss the main molecular events underlying immune-based platelet disorders with specific focus on events occurring at the platelet surface leading to activation and clearance.