Background COVID‐19 and other respiratory viral infections can cause cardiovascular complications. SARS‐CoV‐2 virions are found in the blood, and circulating viral RNA levels are associated with death. We hypothesized that viremia can induce thrombotic endotheliopathy that contributes to death and studied this relationship in patients hospitalized for COVID‐19 and enrolled in the ACTIV‐4a (Accelerating COVID‐19 Therapeutic Interventions and Vaccines) randomized trial of antithrombotic therapy. Methods We quantified SARS‐CoV‐2 nucleocapsid RNA and protein in plasma and measured their associations with clinical outcomes and biomarkers of thromboinflammation and endotheliopathy. We used Cox regression and Fine–Gray competing risk models to analyze survival and thrombosis. We conducted causal mediation analysis to explore whether thrombotic endotheliopathy mediates the relationship between viral RNA and death. Results In 93 patients, SARS‐CoV‐2 RNA and N‐antigen were higher in nonsurvivors. Baseline viral RNA levels were associated with increased 90‐day death (hazard ratio [HR], 1.31 [95% CI, 1.17–1.46]) and thrombosis (HR, 1.35 [95% CI, 1.24–1.47]). Viral RNA more effectively predicted survivorship than N‐antigen levels. Soluble thrombomodulin, a biomarker of thrombotic endotheliopathy, positively correlated with viral RNA levels. Mediation analysis revealed that soluble thrombomodulin accounts for 12.2% (95% CI, 0.1%–32.3%; P=0.048) after adjustment for age and sex of the relationship between viral RNA and the 90‐day mortality rate. Conclusions Elevated plasma SARS‐CoV‐2 RNA levels are associated with death in ACTIV‐4a, which is causally mediated in part by soluble thrombomodulin. We propose that lung–blood viral dissemination is a potential mechanism for cardiovascular complications of respiratory viruses. Registration URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04505774.

OBJECTIVES:Soluble ST2 (sST2), a decoy receptor for the alarmin interleukin-33 (IL-33), has been implicated in adverse clinical outcomes in acute respiratory failure (ARF). We evaluated sST2 distribution across diverse cohorts of patients with different etiologies of ARF, compared plasma and lower respiratory tract (LRT) concentrations, and examined associations with individual organ dysfunction, biological subphenotypes, and outcomes. DESIGN:Observational study. SETTING:Multicenter cohorts of ARF patients. PATIENTS:A total of 1432 ARF patients, including 863 non-COVID and 569 COVID-19 cases, from five cohorts. INTERVENTIONS:None. MEASUREMENTS AND MAIN RESULTS:sST2 levels were measured in plasma and LRT specimens (when available) and analyzed for associations with ARF etiology, severity, organ dysfunction, systemic host response, subphenotypes, and 30-day mortality. Plasma sST2 levels were higher in non-COVID ARF patients compared with COVID-19 patients ( p < 0.05) and were markedly elevated compared with LRT levels (> 19-fold), with weak intercompartmental correlation. Elevated plasma sST2 levels were associated with extrapulmonary organ dysfunction and a hyperinflammatory ARF subphenotype but not with respiratory indices, including hypoxemia. Plasma sST2 independently predicted 30-day mortality in pooled cohort data, adjusted for age, sex, and illness severity. In longitudinal measurements, nonsurvivors had persistently elevated plasma sST2 levels in the first 2 weeks of critical illness compared with survivors. CONCLUSIONS:Plasma sST2 levels independently predict outcomes in ARF and are strongly associated with extrapulmonary organ dysfunction. The weak correlation between plasma and LRT sST2 levels suggests a predominantly systemic source. These findings highlight the potential of the IL-33/ST2 axis as a therapeutic target and warrant further investigation into its role in multiple organ dysfunction in ARF.

The X-linked A- variant (rs1050828, Val68Met) in G6PDX accounts for glucose-6-phosphate (G6PD) deficiency in approximately 11% of African American males. This common, hypomorphic variant may impact pulmonary host defense and phagocyte function during pneumonia by altering levels of reactive oxygen species produced by host leukocytes. We used CRISPR-Cas9 technology to generate novel mouse strain with "humanized" G6PD A- variant containing non-synonymous Val68Met single nucleotide polymorphism. Male hemizygous or littermate wild-type (WT) controls were inoculated intratracheally with K. pneumoniae (KP2 serotype, ATCC 43816 strain,103 CFU inoculum). We examined leukocyte recruitment, organ bacterial burden, bone marrow neutrophil and macrophage (BMDM) phagocytic capacity, and hydrogen peroxide (H2O2) production. Unexpectedly, G6PD-deficient mice showed decreased lung bacterial burden (p=0.05) compared to controls 24-h post-infection. Extrapulmonary dissemination and bacteremia were significantly reduced in G6PD-deficient mice 48-h post-infection. Bronchoalveolar lavage fluid (BALF) IL-10 levels were elevated in G6PD-deficient mice (p=0.03) compared to controls at 24-h but were lower at 48-h (p=0.03). G6PD A- BMDMs show mildly decreased in vitro phagocytosis of pHrodo-labeled KP2 (p=0.03). Baseline, but not stimulated, H2O2 production by G6PD A- neutrophils was greater compared to WT neutrophils. G6PD A- variant demonstrate higher basal neutrophil H2O2 production and are protected against acute Klebsiella intrapulmonary infection.

BACKGROUND: Medical student involvement in proce-dures, including pelvic exams under anesthesia (EUAs), is a fundamental part of medical education. While guide-lines exist regarding informed consent for medical stu-dent participation, there is ongoing debate and uncertainty regarding the requirement and modality of obtaining explicit consent for pelvic EUAs. This study aims to explore the perceptions and experiences of medi-cal students who do not favor an explicit informed con -sent process for pelvic EUAs.METHODS: An anonymous online questionnaire was dis-tributed to third-and fourth-year medical students at the University of Pittsburgh School of Medicine who had completed their obstetrics and gynecology core clerk-ship. The questionnaire included both quantitative and qualitative sections. Qualitative analysis was conducted using a mixed inductive and deductive coding approach, with key patterns, categories, and themes identified through content analysis.RESULTS: Among the 201 students included in the analy-sis, 50 students did not endorse an explicit informed consent process for pelvic EUAs. Themes that emerged from their open-ended responses included: (1) the belief that medical student involvement is implicitly included in patient agreements at teaching hospitals; (2) the per-ception that pelvic EUAs are an essential first step in gynecologic surgery; (3) the view that pelvic EUAs are comparable to other medical procedures; (4) concern that explicit consent would limit educational opportunities; and (5) the belief that pelvic EUAs are not harmful or traumatic to patients. DISCUSSION: The findings highlight the justifications provided by medical students who do not support explicit informed consent for pelvic EUAs. While some arguments align with previous ethical analyses, this study provides empirical and qualitative insights into students' perspec-tives. The belief that patients implicitly consent to medical student involvement at teaching hospitals warrants further examination, as patient awareness and understanding may vary. The differentiation between pelvic exams and other EUAs, as well as the perception of minimal harm, should be critically evaluated in the context of trauma-informed care and patient autonomy. Furthermore, the interconnec-tedness of educational and surgical aspects of pelvic EUAs should be clarified in patient-physician communication.CONCLUSION: Understanding the perspectives of medi-cal students who do not favor explicit consent for pelvic EUAs is crucial for developing and implementing con -sent processes. The findings emphasize the need for enhanced patient-physician communication, standard-ized frameworks for learner involvement, and curricular adaptations to address patient perceptions and trauma-informed care. Future research should explore these themes in larger and more diverse cohorts to inform best practices in obtaining informed consent for medical stu-dent participation in pelvic EUAs. ( J Surg Ed 81:64-69. (c) 2023 Association of Program Directors in Surgery. Published by Elsevier Inc. All rights reserved.)

BACKGROUND:Donor genetic variation is associated with red blood cell (RBC) storage integrity and post-transfusion recovery. Our previous large-scale genome-wide association study demonstrated that the African G6PD deficient A- variant (rs1050828, Val68Met) is associated with higher oxidative hemolysis after cold storage. Despite a high prevalence of X-linked G6PD mutation in African American population (>10%), blood donors are not routinely screened for G6PD status and its importance in transfusion medicine is relatively understudied. STUDY DESIGN AND METHODS:To further evaluate the functional effects of the G6PD A- mutation, we created a novel mouse model carrying this genetic variant using CRISPR-Cas9. We hypothesize that this humanized G6PD A- variant is associated with reduced G6PD activity with a consequent effect on RBC hemolytic propensity and post-transfusion recovery. RESULTS:G6PD A- RBCs had reduced G6PD protein with ~5% residual enzymatic activity. Significantly increased in vitro hemolysis induced by oxidative stressors was observed in fresh and stored G6PD A- RBCs, along with a lower GSH:GSSG ratio. However, no differences were observed in storage hemolysis, osmotic fragility, mechanical fragility, reticulocytes, and post-transfusion recovery. Interestingly, a 14% reduction of 24-h survival following irradiation was observed in G6PD A- RBCs compared to WT RBCs. Metabolomic assessment of stored G6PD A- RBCs revealed an impaired pentose phosphate pathway (PPP) with increased glycolytic flux, decreasing cellular antioxidant capacity. DISCUSSION:This novel mouse model of the common G6PD A- variant has impaired antioxidant capacity like humans and low G6PD activity may reduce survival of transfused RBCs when irradiation is performed.

Background Fatty acid oxidation (FAO) defects have been implicated in experimental models of acute lung injury and associated with poor outcomes in critical illness. In this study, we examined acylcarnitine profiles and 3-methylhistidine as markers of FAO defects and skeletal muscle catabolism, respectively, in patients with acute respiratory failure. We determined whether these metabolites were associated with host-response ARDS subphenotypes, inflammatory biomarkers, and clinical outcomes in acute respiratory failure. Methods In a nested case–control cohort study, we performed targeted analysis of serum metabolites of patients intubated for airway protection (airway controls), Class 1 (hypoinflammatory), and Class 2 (hyperinflammatory) ARDS patients (N = 50 per group) during early initiation of mechanical ventilation. Relative amounts were quantified by liquid chromatography high resolution mass spectrometry using isotope-labeled standards and analyzed with plasma biomarkers and clinical data. Results Of the acylcarnitines analyzed, octanoylcarnitine levels were twofold increased in Class 2 ARDS relative to Class 1 ARDS or airway controls ( P = 0.0004 and < 0.0001, respectively) and was positively associated with Class 2 by quantile g-computation analysis ( P = 0.004). In addition, acetylcarnitine and 3-methylhistidine were increased in Class 2 relative to Class 1 and positively correlated with inflammatory biomarkers. In all patients within the study with acute respiratory failure, increased 3-methylhistidine was observed in non-survivors at 30 days ( P = 0.0018), while octanoylcarnitine was increased in patients requiring vasopressor support but not in non-survivors ( P = 0.0001 and P = 0.28, respectively). Conclusions This study demonstrates that increased levels of acetylcarnitine, octanoylcarnitine, and 3-methylhistidine distinguish Class 2 from Class 1 ARDS patients and airway controls. Octanoylcarnitine and 3-methylhistidine were associated with poor outcomes in patients with acute respiratory failure across the cohort independent of etiology or host-response subphenotype. These findings suggest a role for serum metabolites as biomarkers in ARDS and poor outcomes in critically ill patients early in the clinical course.

OBJECTIVE:To obtain an overview of medical student attitudes on the need for explicit consent for pelvic exams under anesthesia performed for educational purposes DESIGN: From February to October 2020, 201 medical students at a single medical school in the United States participated in a cross-sectional survey after completion of the obstetrics and gynecology clerkship. Outcome measures included endorsement of need for explicit informed consent for educational pelvic exams under anesthesia, and knowledge of informed consent processes for such exams. SETTING:University of Pittsburgh School of Medicine PARTICIPANTS: Third- and fourth-year medical students RESULTS: Overall, 75% of medical students endorsed a need for explicit informed consent for educational pelvic exams under anesthesia, which extended to prostate, rectal, and breast exams under anesthesia. Additionally, 45% and 77% of these participants indicated that consent for educational pelvic exams under anesthesia should take the form of a separate signature line on the surgical consent form and/or a verbal form, respectively. Only 40% of students correctly identified institutional policy for obtaining informed consent for educational pelvic exams under anesthesia. Rotation with the oncologic surgical service (p = 0.02) and correct identification of institutional informed consent policies (p = 0.002) were associated with decreased perceptions of the importance of explicit informed consent for educational pelvic exams under anesthesia. CONCLUSIONS:Medical students at the institution studied largely support explicit informed consent for educational pelvic and other sensitive exams under anesthesia, but a knowledge gap on institutional informed consent policy exists. Medical students support increased transparency and bodily autonomy. Due to the agreement of patients and medical students and the ethical rationale for this position, it may be appropriate for physicians and institutions to consider new processes of obtaining explicit informed consent for pelvic exams under anesthesia by medical students.

p38 MAPKs are key regulators of cellular adaptation to various stress stimuli, however, their role in mediating erythrocyte cell death and hemolysis is largely unknown. We hypothesized that activation of erythrocyte p38 MAPK is a common event in the stimulation of hemolysis, and that inhibition of p38 MAPK pathways could mitigate hemolysis in hemoglobinopathies. We exposed human erythrocytes to diamide-induced oxidative stress or to hypoosmotic shock in the presence or absence of p38 MAPK inhibitors (SCIO469, SB203580, CMPD1) and used immunoblotting to determine MAPK activity and to identify possible downstream effectors of p38 MAPK. We also evaluated the impact of p38 MAPK inhibitors on stress-induced hemolysis or hypoxia-induced sickling in erythrocytes from mouse models of sickle cell disease. We found that human erythrocytes express conventional MAPKs (MKK3, p38 MAPK, MAPKAPK2) and identified differential MAPK activation pathways in each stress condition. Specifically, p38 MAPK inhibition in diamide-treated erythrocytes was associated with decreased phosphorylation of Src tyrosine kinases and Band 3 protein. Conversely, hypoosmotic shock induced MAPKAPK2 and RSK2 phosphorylation, which was inhibited by SCIO469 or CMPD1. Relevant to hemoglobinopathies, sickle cell disease was associated with increased erythrocyte MKK3, p38 MAPK, and MAPKAPK2 expression and phosphorylation as compared with erythrocytes from healthy individuals. Furthermore, p38 MAPK inhibition was associated with decreased hemolysis in response to diamide treatments or osmotic shock, and with decreased erythrocyte sickling under experimental hypoxia. These findings provided insights into MAPK-mediated signaling pathways that regulate erythrocyte function and hemolysis in response to extracellular stressors or human diseases.

Background Hydroxychloroquine (HCQ) is widely used in the treatment of malaria, rheumatologic disease such as lupus, and most recently, COVID-19. These uses raise concerns about its safe use in the setting of glucose-6-phosphate dehydrogenase (G6PD) deficiency, especially as 11% of African American men carry the G6PD A- variant. However, limited data exist regarding the safety of HCQ in this population. Study design and methods Recently, we created a novel “humanized” mouse model containing the G6PD deficiency A- variant (Val68Met) using CRISPR technology. We tested the effects of high-dose HCQ administration over 5 days on hemolysis in our novel G6PD A- mice. In addition to standard hematologic parameters including plasma hemoglobin, erythrocyte methemoglobin, and reticulocytes, hepatic and renal function were assessed after HCQ. Results Residual erythrocyte G6PD activity in G6PD A- mice was ~6% compared to wild-type (WT) littermates. Importantly, we found no evidence of clinically significant intravascular hemolysis, methemoglobinemia, or organ damage in response to high-dose HCQ. Conclusions Though the effects of high doses over prolonged periods was not assessed, this study provides early, novel safety data of the use of HCQ in the setting of G6PD deficiency secondary to G6PD A-. In addition to novel safety data for HCQ, to our knowledge, we are the first to present the creation of a “humanized” murine model of G6PD deficiency.

Background: Growing evidence suggest that donor genetic variation is associated with RBCs storage integrity and post-transfusion recovery. In collaboration with the REDS III program, we performed a large-scale genome-wide association study (GWAS) study in ~13,000 healthy human blood donors, which demonstrated that RBCs with an African American G6PD-deficient A- variant (rs1050828, Val68Met) was associated with higher oxidative hemolysis after cold storage in normal volunteers. Despite a high prevalence of X-linked G6PD mutation in African American population (> 10%), blood donors are not routinely checked for G6PD deficiency and its importance in transfusion medicine is relatively understudied. We hypothesize that G6PD V68M SNP is associated with G6PD deficiency and modulates RBCs in vitro hemolytic propensity and in vivo post-transfusion recovery. Methods and Results: CRISPR-Cas9 technology was used to create non-synonymous human G6PD X-linked SNP (Val68Met) in C57B6 mice, and the desired genotypes were isolated by strategic back-crossing and sequential genotyping to ensure passage of SNP. G6PD enzymatic activity in erythrocytes was measured in fresh collected blood using a colorimetric assay kit. The predisposition of fresh and stored RBCs (after 11 days cold storage) to hemolysis was evaluated by subjecting washed mouse RBCs to selected stress assays, including osmotic fragility, mechanical fragility, and oxidative hemolysis using AAPH, diamide or H2O2. Hemolysis was measured by detection of supernatant cell-free hemoglobin using Drabkin's assay. Hematological values were measured using a Hemavet® 950 Hematology Analyzer System. Reticulocyte count was obtained using thiazole orange staining and analyzed by flow cytometry. We found severe disruption of G6PD enzymatic activity in erythrocytes from G6PD V68M SNP mice compared to WT mice (~5% residual activity in hemizygous male and ~60% in heterozygous female mice). Significant increased oxidative hemolysis was observed in both fresh and stored mouse RBCs with G6PD SNP, consistent with the GWAS study in human. G6PD V68M SNP hemizygous male mice had higher mean corpuscular volume (MCV) and lower mean corpuscular hemoglobin concentration (MCHC) compared to WT mice. However, no difference was observed in storage hemolysis, osmotic fragility, mechanical fragility and reticulocyte counts. Transfusion experiments with stored red blood cells from G6PD hemizygote males into GFP positive recipients will evaluate red blood cell recovery and half-life after standard cold storage and transfusion. Conclusions: We successfully generated a novel mouse strain carrying a "humanized" African American G6PD V68M variant which resembles the phenotype of humans with G6PD deficiency and increased oxidative hemolysis. Studies are undertaken to further investigate the effects of G6PD V68M SNP on RBCs structure, functions and in vivo post-transfusion recovery. Disclosures Gladwin: Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning.

OBJECTIVE:Sexual health is important to quality of life; however, the sexual health of gynecologic cancer patients is infrequently and inadequately addressed. We sought to understand patient experiences and preferences for sexual health care to help inform strategies for improvement. METHODS/MATERIALS:An anonymous, cross-sectional survey of outpatient gynecologic cancer patients at a large academic medical center was performed as part of a larger study examining patient and caregiver needs. The survey explored patient-provider discussions about sexuality across 3 domains (experiences, preferences, barriers) and 4 phases of cancer care (diagnosis, treatment, treatment completion, follow-up). Age, relationship status, sexual activity, and cancer type were recorded. RESULTS:Mean age was 63 years. Most patients had ovarian cancer (38%) or endometrial cancer (32%). Thirty-seven percent received treatment within the last month, 55% were in a relationship, and 35% were sexuality active. Thirty-four percent reported sexuality as somewhat or very important, whereas 27% felt that it was somewhat or very important to discuss. Importance of sexuality was associated with age, relationship status, and sexual activity but not cancer type. Fifty-seven percent reported never discussing sexuality. Age was associated with sexuality discussions, whereas relationship status, sexual activity, and cancer type were not. The most common barrier to discussion was patient discomfort. Follow-up was identified as the best time for discussion. Sexuality was most often discussed with a physician or advanced practice provider and usually brought up by the provider. CONCLUSIONS:Demographic predictors of importance of sexuality to the patient are age, relationship status, and sexual activity. Providers primarily use age as a proxy for importance of sexuality; however, relationship status and sexual activity may represent additional ways to screen for patients interested in discussing sexual health. Patient discomfort with discussing sexuality is the primary barrier to sexual health discussions, and awareness of this is key to developing effective approaches to providing sexual health care.