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Abstract Background Delirium, an acute brain dysfunction, is proposed to be highly prevalent in clinical care and shown to significantly increase the risk of mortality and dementia. Objectives To report on the global prevalence of clinically documented delirium and delirium-related clinical practices in wards caring for paediatric and adult patients in healthcare facilities. Design A prospective, cross-sectional, 39-question survey completed on World Delirium Awareness Day, 15 March 2023. Participants Clinicians or researchers with access to clinical data. Main Outcome and Measure The primary outcome was the prevalence of clinically documented delirium at 8:00 a.m. (4 h) and 8:00 p.m. (±4 h). Secondary outcomes included delirium-related care practices and barriers to use. Descriptive statistics were calculated and multilevel modelling was completed. Results 1664 wards submitted surveys from 44 countries, reporting on delirium assessments at 8:00 a.m. (n = 36 048) and 8:00 p.m. (n = 32 867); 61% reported use of validated delirium assessment tools. At 8:00 a.m., 18% (n = 2788/15 458) and at 8:00 p.m., 17.7% (n = 2454/13 860) were delirium positive. Top prevention measures were pain management (86.7%), mobilisation (81.4%) and adequate fluids (80.4%). Frequently reported pharmacologic interventions were benzodiazepines (52.7%) and haloperidol (46.2%). Top barriers included the shortage of staff (54.3%), lack of time to educate staff (48.6%) and missing knowledge about delirium (38%). Conclusion and Relevance In this study, approximately one out of five patients were reported as delirious. The reported high use of benzodiazepines needs further evaluation as it is not aligned with best-practice recommendations. Findings provide a benchmark for future quality improvement projects and research.

BACKGROUND Despite an overall poor prognosis, about 15% of patients with advanced-stage tubo-ovarian high-grade serous carcinoma (HGSC) survive 10 or more years after standard treatment. METHODS We evaluated the tumor microenvironment of this exceptional, understudied group using a large international cohort enriched for long-term survivors (LTS; 10+ years; n = 374) compared with mid-term (MTS; 5–7.99 years; n = 433) and short-term survivors (STS; 2–4.99 years; n = 416). Primary tumor samples were immunostained and scored for intraepithelial and intrastromal densities of 10 immune-cell subsets (including T cells, B cells, plasma cells, myeloid cells, PD-1 + cells, and PD-L1 + cells) and epithelial content. RESULTS Positive associations with LTS compared with STS were seen for 9 of 10 immune-cell subsets. In particular, the combination of intraepithelial CD8 + T cells and intrastromal B cells showed near 5-fold increased odds of LTS compared with STS. All of these associations were stronger in tumors with high epithelial content and/or the C4/Differentiated molecular subtype, despite immune-cell densities generally being higher in tumors with low epithelial content and/or the C2/Immunoreactive molecular subtype. CONCLUSION The tumor microenvironment of HGSC LTS is distinguished by the intersection of T and B cell coinfiltration, high epithelial content, and C4/differentiated molecular subtype, features which may inspire new approaches to immunotherapy. FUNDING Ovarian Cancer Research Program (OCRP) of the Congressionally Directed Medical Research Program (CDMRP), U.S. Department of Defense (DOD); American Cancer Society; BC Cancer Foundation; Canada’s Networks of Centres of Excellence; Canadian Cancer Society; Canadian Institutes of Health Research; Cancer Councils of New South Wales, Victoria, Queensland, South Australia, and Tasmania, Cancer Foundation of Western Australia; Cancer Institute NSW; Cancer Research UK; Deutsche Forschungsgesellschaft; ELAN Funds of the University of Erlangen-Nuremberg; Fred C. and Katherine B. Andersen Foundation; Genome BC; German Cancer Research Center; German Federal Ministry of Education and Research, Programme of Clinical Biomedical Research; Instituto de Salud Carlos III; Mayo Foundation; Minnesota Ovarian Cancer Alliance; Ministerio de Economía y Competitividad; Medical Research Council (MRC); National Center for Advancing Translational Sciences; National Health and Medical Research Council of Australia (NHMRC); Ovarian Cancer Australia; Peter MacCallum Foundation; Sydney West Translational Cancer Research Centre; Terry Fox Research Institute; The Eve Appeal (The Oak Foundation); UK National Institute for Health Research Biomedical Research Centres at the University of Cambridge; University of Pittsburgh School of Medicine; U.S. National Cancer Institute of the National Institutes of Health; VGH & UBC Hospital Foundation; Victorian Cancer Agency.

Abstract Background Transforming growth factor beta (TGFβ) and its receptors (TGFβRs) are widely expressed by most human and murine cells and are implicated in several physiological and pathological conditions. In the context of atherosclerosis, TGFβ has been identified as a pleiotropic molecule involved in both pro- and anti-atherogenic processes, with its effects varying depending on the cell type it signals through. Purpose Given that myeloid cells play a major role in atherogenesis, we sought out to unravel the involvement of myeloid TGFβ signaling in atherosclerosis. Methods Aortic leukocytes from ApoE-/- mice fed a Western diet (WD) for 20 weeks were subjected to CITE-Seq analysis to assess the expression profile of Tgfβ and Tgfβrs. To investigate the effect of myeloid Tgfβ signaling on atherosclerosis, LysMCre+Tgfβr1flox/floxTgfβr2flox/floxmT/mGflox/floxApoE-/- (M-Tgfβr1/2dkoApoE-/-) mice and their LysMCre- littermates (ApoE-/-) were fed a WD for 6 weeks. Leukocyte phenotyping was assessed by flow cytometry (FC) and qPCR. Atherosclerotic plaque size in the aortic root was assessed by immunohistochemistry. Results CITE-Seq revealed that aortic ApoE-/- leukocytes highly expressed Tgfb1, whereas Tgfb2 and Tgfb3 were very lowly expressed. Tgfbr1 was mainly expressed in myeloid cells with macrophages and neutrophils showing the highest expression. Tgfbr2 was primarily expressed in T and B cells, while still showing a high level of expression in macrophages. Importantly, Tgfbr3 was not expressed in myeloid cells. Thus, to study the effect of myeloid Tgfβ signaling on atherosclerosis and to mitigate the potential for aberrant signaling that might arise from deleting only one receptor, we generated ApoE-/- mice with myeloid lineage tracing mT/mG alleles and combined deletion of Tgfbr1 and Tgfbr2 in myeloid cells using the LysMCre model. FC analysis of eGFP+ cells, i.e., cells where LysMCre is active, revealed a ~60-80% Tgfbr1/2 deletion efficiency in bone marrow-derived macrophages (BMDM) and in blood, splenic and BM CD11b+ cells, which was accompanied by decreased Smad2/3 signaling. M-Tgfβr1/2dkoApoE-/- mice showed a ~35% increase in blood and splenic CD11b+ cells and a ~25% increase in splenic macrophages compared to ApoE-/- controls, while BM CD11b+ cells were not affected. Tgfbr1/2-deficient BMDMs had increased Il-6, Il-1b and CD38 expression compared to controls, suggesting that Tgfbr1/2-deficient macrophages have acquired a pro-inflammatory phenotype. Interestingly, M-Tgfβr1/2dkoApoE-/- mice had ~30% fewer blood and splenic T cells compared to controls, which was explained by decreased naïve CD4+ and CD8+ T cells. M-Tgfβr1/2dkoApoE-/- mice had a ~50% increase in atherosclerotic plaque burden compared to ApoE-/- controls. Conclusion Our data suggest that impaired myeloid Tgfβ signaling leads to a pro-atherogenic environment in ApoE-/- mice. These effects may be mediated by an altered crosstalk between T cells and Tgfβr1/2-deficient macrophages.

Abstract Background Atherosclerosis is a chronic inflammatory disease and the primary underlying cause of cardiovascular disease (CVD). Recently, intracellular metabolic pathways have been identified as master switches of immune cell function and thus seem promising targets for therapeutic interventions aimed at lowering inflammation and thereby atherogenesis. Decades of research have demonstrated the importance of the amino acid (AA) L-arginine (Arg) in CVD. Although most research has focused on endothelium-dependent effects of Arg by the nitric oxide synthase (NOS), Arg metabolism - mainly through its metabolizing enzyme arginase 1 (Arg1) - has also been shown to regulate immune cell responses independent of its endothelial functions. Despite these findings, the cell-specific contribution of Arg/Arg1 metabolism on atherogenesis is still largely unknown. Purpose Profiling the Arg metabolism in endothelial and immune cells to identify cell-specific changes during athero-progression. Methods To investigate Arg metabolism in atherogenesis, we fed apolipoprotein E deficient (Apoe-­/­-) mice a Western diet (WD) for 6 and 12 weeks. Chow diet-fed aged-matched Apoe-­/-­ mice were used as littermate controls and C57BL/6 mice as steady-state controls. Systemic and local Arg concentrations were measured by mass-spectrometry. Arg metabolism in organs was studied by qPCR. Arg1 expression in atherosclerotic lesions was examined by immunohistochemistry (IHC). To assess cell-specific expression of Arg-related enzymes, aortic leukocytes from WD-fed Apoe-­/-­ mice were subjected to CITE-seq analysis. The role of T cell Arg1 was determined by nor-NOHA inhibition in Jurkat cells. Results AA measurements showed that systemic Arg was reduced in early and advanced atherogenesis (p<0.02), and splenic Arg was reduced in advanced atherogenesis (p=0.006) compared with steady state. In advanced atherosclerosis, Arg1 was decreased in the liver but increased in the spleen and aorta, whereas Nos2 was increased in the liver and aorta compared with steady state. To identify the cell types driving these local changes, we performed IHC on atherosclerotic lesions. Arg1+ lesional macrophages accumulated in advanced compared to early lesions (p=0.03), whereas Arg1+ endothelial cells tended to decrease (p=0.2). Surprisingly, we also found an accumulation of Arg1+ CD4+ T cells in lesions and a further upward trend with athero-progression. CITE-seq confirmed a constitutive expression of Arg1 in lymphoid clusters. In vitro experiments with Jurkat cells showed that arginase inhibition suppressed T cell proliferation and promoted apoptosis and migration (all p<0.0001). Conclusion Our data indicate that Arg metabolism is strongly regulated during athero-progression and suggest a so far unknown role of T cell-intrinsic Arg/Arg1 metabolism in the disease. Murine atherosclerosis studies are now being performed to decipher the cell-specific contribution of Arg/Arg1 metabolism in atherosclerosis.

Abstract Background Patients with carotid artery disease undergoing carotid endarterectomy (CEA) appear to be at high risk of all-cause mortality during their follow-up. However, the possible predictors of this adverse outcome are not elucidated yet. Purpose We aimed to test cardiovascular (CV) risk factors as possible predictors of all-cause mortality in patients undergoing CEA and to determine whether there is a difference between the mortality rate of our study cohort and the general population. We hypothesize that CEA-treated patients have a higher mortality rate during follow-up after surgery than the general population, which is not related to the presence of CV risk factors. Methods We prospectively enrolled and followed up patients undergoing CEA. Univariate and multivariable Cox regression were performed to test whether common CV risk factors (demographics, laboratory values, comorbidities, and events before CEA) were predictive of all-cause mortality. We then compared the standardized mortality ratio (SMR) of our population and the age-sex-matched Minnesota (MN) general population at 5,10, and 21 years of follow-up after CEA. Results A total of 710 patients were followed for a median of 7.1 [2.9-11.9] years after CEA. Mortality occurred in 198 (27.0%) patients during the follow-up. The median time to event was 11.0 [10.0-12.0] years. Age was the only predictor of all-cause mortality in CEA-treated patients in multivariable models adjusted for known CV risk factors (Model 1: HR 1.03 [95%CI 1.01-1.06]; p=0.004; Model 2: HR 1.02 [95%CI 1.00-1.04]; p=0.023) (Table 1). However, the CEA-treated group had a higher mortality rate than would have been expected in the MN population (log-rank p<0.001) (Fig.1A). Compared to the MN population, at 5 years of follow-up only patients <60 years old had a higher SMR (4.53 [1.95-8.92]; p=0.001), whereas, at 10 years and 21 years of follow-up, our study population had a higher mortality rate at any age (Fig.1B). Conclusions The elevated mortality risk post-CEA in our cohort cannot be solely attributed to chronological aging. The underlying process of atherosclerosis likely contributes to all-cause mortality, and plaque characteristics may offer additional insight into the risk stratification of this patient group.All cause mortality predictors after CEAStudy population vs. MN population SMR

Abstract Background Patients with carotid artery disease undergoing carotid endarterectomy (CEA) have a high risk of experiencing Major Adverse Cardiovascular Events (MACE) after surgery. The occurrence of MACE in CEA-treated patients is not predicted by common cardiovascular (CV) risk factors. Purpose We aimed to identify the association between plasma and plaque inflammatory cytokines and the incidence of MACE in the follow-up after CEA. We hypothesized that MACE is related to a higher level of circulating inflammatory cytokines at the time of surgery. Methods We prospectively enrolled patients with carotid artery disease undergoing CEA. Plasma and carotid plaques were collected during the surgery. Inflammatory cytokines (MCP-1/CCL2, IL-8/CXCL8, IFN-gamma, IL-10, IL-1beta/IL-1F2, IL-6, PDGF-AA, PDGF-AB/BB, TNF-alpha, VEGF) were measured in plasma and tissue homogenates using a customized 10-plex Luminex assay. Baseline demographics and clinical data for all the patients were extracted from electronic health records. MACE after CEA was defined as a composite measure of nonfatal cardiac events such as myocardial infarction with or without revascularization, cerebrovascular events (ischemic stroke, TIA, and amaurosis fugax), and all-cause mortality. Results Of 107 patients who underwent CEA with a median age of 73 (67-78) years, 57 (53.2%) experienced MACE during a median follow-up of 5.2 [2.2-9.3] years: 23 (40.3%) cardiac events, 9 (15.7%) CVA, and 25 (43.8%) all-cause mortality. Plaque cytokines levels did not differ between the groups with and without MACE in the follow-up after CEA. Instead, plasma cytokines were associated with MACE in the follow-up after CEA, as summarized in Figure 1. In the logistic regression analysis, plasma levels of MCP-1/CCL2, IFN-gamma, and TNF-alpha were independent predictors of MACE in follow-up post-CEA in univariate and multivariable models adjusted for CV risk factors such as age, BMI, systolic and diastolic blood pressure, low-density lipoprotein, high-density lipoprotein, and glucose level: MCP1/CCL2 (HR 1.009 [1.002-1.015]; p=0.007), IFN-gamma (HR 1.368 [1.014-1.845]; p=0.040), and TNF-alpha (HR 1.121 [1.022-1.228]; p=0.015) (Table 1). Conclusions Plasma levels of CCL2, IFN-gamma, and TNF-alpha are associated with the high incidence of MACE in the follow-up after CEA, indicating that these are potential biomarkers of MACE in this patient population and potential therapeutic targets in patients' progression of systemic atherosclerosis.Plasma cytokines and MACE after CEACytokines logistic regression analysis

Abstract Background One of the main features of vulnerability in atherosclerotic carotid plaques is intraplaque hemorrhage (IPH), which is related to a higher incidence of cerebrovascular events in patients with carotid artery disease. Additionally, inflammation plays a pivotal role in the progression of atherosclerotic disease. Purpose We aimed to determine plasma and tissue levels of inflammatory cytokines in patients with carotid artery disease hypothesizing that plaques with IPH are characterized by the presence of a more inflamed environment. Methods We retrospectively enrolled 104 consecutive patients undergoing carotid endarterectomy (CEA) with symptomatic (ischemic stroke, TIA, amaurosis fugax) or asymptomatic ipsilateral carotid artery disease. Plasma and carotid plaque specimens were collected at CEA. The tissue samples were stained with H&E to identify IPH and classified as specimens with and without IPH. Frozen homogenates of both plasma and tissue were analyzed using a customized 10-plex Luminex assay to determine the levels of inflammatory cytokines such as MCP-1/CCL2, IL-8/CXCL8, IFN-gamma, IL-10, IL-1beta/IL-1F2, IL-6, PDGF-AA, PDGF-AB/BB, TNF-alpha, and VEGF. Results IPH was identified in 53 patients (51.0 %). Cytokine levels in plasma and tissue from patients with and without IPH are reported in Table 1. In the univariate logistic regression MCP-1/CCL2, IL-8/CXCL8, IFN-gamma, IL-10, IL6, PDGF-AA, and PDGF-AB/BB were associated with IPH (Table 2). In the multivariate logistic regression, IL-6 was an independent predictor of IPH (HR 1.026 [95% CI 1.005-1.047]; p=0.014) after adjusting for cardiovascular risk factors such as age, BMI, sex, hypertension, diabetes mellitus, hyperlipidemia, ipsilateral carotid stenosis %. Conclusions Carotid plaques with IPH have an elevated inflammatory burden, potentially impacting adverse outcomes in this patient cohort. The local inflammatory cytokines in carotid plaques with IPH might serve as biomarkers and therapeutic targets in patients with carotid artery disease.

Inositol 1,4,5-trisphosphate (IP3) receptor type 1 ( ITPR1 ), 2 ( ITPR2 ), and 3 ( ITPR3 ) encode the IP3 receptor (IP3R), a key player in intracellular calcium release. In four unrelated patients, we report that an identical ITPR3 de novo variant—NM_002224.3:c.7570C>T, p.Arg2524Cys—causes, through a dominant-negative effect, a complex multisystemic disorder with immunodeficiency. This leads to defective calcium homeostasis, mitochondrial malfunction, CD4 + lymphopenia, a quasi-absence of naïve CD4 + and CD8 + cells, an increase in memory cells, and a distinct TCR repertoire. The calcium defect was recapitulated in Jurkat knock-in. Site-directed mutagenesis displayed the exquisite sensitivity of Arg 2524 to any amino acid change. Despite the fact that all patients had severe immunodeficiency, they also displayed variable multisystemic involvements, including ectodermal dysplasia, Charcot-Marie-Tooth disease, short stature, and bone marrow failure. In conclusion, unlike previously reported ITPR1-3 deficiencies leading to narrow, mainly neurological phenotypes, a recurrent dominant ITPR3 variant leads to a multisystemic disease, defining a unique role for IP3R3 in the tetrameric IP3R complex.

Abstract Background: Breast cancer has been associated with monogenic, polygenic, and epidemiologic (clinical, reproductive, and lifestyle) risk factors, but studies evaluating the combined effects of these factors have been limited. Methods: We extended previous work in breast cancer risk modeling, incorporating pathogenic variants (PV) in six breast cancer predisposition genes and a 105-SNP polygenic risk score (PRS), to include an epidemiologic risk score (ERS) in a sample of non-Hispanic White women drawn from prospective cohorts and population-based case–control studies, with 23,518 cases and 22,832 controls, from the Cancer Risk Estimates Related to Susceptibility (CARRIERS) Consortium. Results: The model predicts 4.4-fold higher risk of breast cancer for postmenopausal women with no predisposition PV and median PRS, but with the highest versus lowest ERS. Overall, women with CHEK2 PVs had >20% lifetime risk of breast cancer. However, 15.6% of women with CHEK2 PVs and a family history of breast cancer, and 45.1% of women with CHEK2 PVs but without a family history of breast cancer, had low (<20%) predicted lifetime risk and thus were below the threshold for MRI screening. CHEK2 PV carriers at the 10th percentile of the joint distribution of ERS and PRS, without a family history of breast cancer, had a predicted lifetime risk similar to the general population. Conclusions: These results illustrate that an ERS, alone and combined with the PRS, can contribute to clinically relevant risk stratification. Impact: Integrating monogenic, polygenic, and epidemiologic risk factors in breast cancer risk prediction models may inform personalized screening and prevention efforts.