An atlas of cells in the human tonsil.

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

Cardiovascular Risk Age Reflects Arterial Status: Middle-Aged People Showed Equivalent Arterial Stiffness to Older People in the Same Risk Category.

AIM: The concept of risk age may help overcome an excessive weight of age in cardiovascular risk functions. This study aimed to evaluate the equivalence of risk age with arterial stiffness by comparing people with increased risk age and individuals with the same chronological and risk age. In order to materialize this aim, we categorized individuals based on cardiovascular risk and compared groups with increased risk factors (other than age) and groups with normal levels. METHODS: This is a cross-sectional population-level study carried out in Girona province within the context of the REGICOR study (Girona Heart Registry). In this study, individuals aged 35-90 years who had a brachial-ankle pulse wave velocity measurement and with no previous cardiovascular disease or peripheral arterial disease were included. Cardiovascular risk was estimated with the FRESCO (in 35-79 year-olds), SCORE2 (in 35-69 year-olds), and SCORE2-OP (in 70-90 year-olds) functions and categorized to calculate and compare (in each category) the median chronological age in the group with increased risk factors and the reference. Arterial stiffness was assessed with the brachial-ankle pulse wave velocity (baPWV). The analyses were carried out separately by sex. RESULTS: In this study, 2499 individuals were included, with a mean age of 59.7 and 46.9% of men. Men presented worse health condition, including a higher mean cardiovascular disease risk score. Both men and women with increased levels of risk factors showed worse health condition than the respective men and women with optimal levels. In each risk category, the groups with higher risk age than chronological age (increased risk factors) were similar in baPWV values to the groups with the same chronological and risk ages (reference), who were consistently older. CONCLUSIONS: In categories with the same cardiovascular risk, the arterial stiffness of participants with a higher risk factor burden (increased risk age) matched that of older participants with the rest of the risk factors at optimal levels (same chronological and risk age). These results support the guidelines on the utilization of risk age to explain heightened cardiovascular risk, particularly among individuals in middle age.

Tumor heterogeneity and tumor-microglia interactions in primary and recurrent IDH1-mutant gliomas.

The isocitrate dehydrogenase (IDH) gene is recurrently mutated in adult diffuse gliomas. IDH-mutant gliomas are categorized into oligodendrogliomas and astrocytomas, each with unique pathological features. Here, we use single-nucleus RNA and ATAC sequencing to compare the molecular heterogeneity of these glioma subtypes. In addition to astrocyte-like, oligodendrocyte progenitor-like, and cycling tumor subpopulations, a tumor population enriched for ribosomal genes and translation elongation factors is primarily present in oligodendrogliomas. Longitudinal analysis of astrocytomas indicates that the proportion of tumor subpopulations remains stable in recurrent tumors. Analysis of tumor-associated microglia/macrophages (TAMs) reveals significant differences between oligodendrogliomas, with astrocytomas harboring inflammatory TAMs expressing phosphorylated STAT1, as confirmed by immunohistochemistry. Furthermore, inferred receptor-ligand interactions between tumor subpopulations and TAMs may contribute to TAM state diversity. Overall, our study sheds light on distinct tumor populations, TAM heterogeneity, TAM-tumor interactions in IDH-mutant glioma subtypes, and the relative stability of tumor subpopulations in recurrent astrocytomas.

Dietary palmitic acid promotes a prometastatic memory via Schwann cells.

Fatty acid uptake and altered metabolism constitute hallmarks of metastasis1,2, yet evidence of the underlying biology, as well as whether all dietary fatty acids are prometastatic, is lacking. Here we show that dietary palmitic acid (PA), but not oleic acid or linoleic acid, promotes metastasis in oral carcinomas and melanoma in mice. Tumours from mice that were fed a short-term palm-oil-rich diet (PA), or tumour cells that were briefly exposed to PA in vitro, remained highly metastatic even after being serially transplanted (without further exposure to high levels of PA). This PA-induced prometastatic memory requires the fatty acid transporter CD36 and is associated with the stable deposition of histone H3 lysine 4 trimethylation by the methyltransferase Set1A (as part of the COMPASS complex (Set1A/COMPASS)). Bulk, single-cell and positional RNA-sequencing analyses indicate that genes with this prometastatic memory predominantly relate to a neural signature that stimulates intratumoural Schwann cells and innervation, two parameters that are strongly correlated with metastasis but are aetiologically poorly understood3,4. Mechanistically, tumour-associated Schwann cells secrete a specialized proregenerative extracellular matrix, the ablation of which inhibits metastasis initiation. Both the PA-induced memory of this proneural signature and its long-term boost in metastasis require the transcription factor EGR2 and the glial-cell-stimulating peptide galanin. In summary, we provide evidence that a dietary metabolite induces stable transcriptional and chromatin changes that lead to a long-term stimulation of metastasis, and that this is related to a proregenerative state of tumour-activated Schwann cells.

A single-cell tumor immune atlas for precision oncology.

The tumor immune microenvironment is a main contributor to cancer progression and a promising therapeutic target for oncology. However, immune microenvironments vary profoundly between patients, and biomarkers for prognosis and treatment response lack precision. A comprehensive compendium of tumor immune cells is required to pinpoint predictive cellular states and their spatial localization. We generated a single-cell tumor immune atlas, jointly analyzing published data sets of >500,000 cells from 217 patients and 13 cancer types, providing the basis for a patient stratification based on immune cell compositions. Projecting immune cells from external tumors onto the atlas facilitated an automated cell annotation system. To enable in situ mapping of immune populations for digital pathology, we applied SPOTlight, combining single-cell and spatial transcriptomics data and identifying colocalization patterns of immune, stromal, and cancer cells in tumor sections. We expect the tumor immune cell atlas, together with our versatile toolbox for precision oncology, to advance currently applied stratification approaches for prognosis and immunotherapy.

Single human oocyte transcriptome analysis reveals distinct maturation stage-dependent pathways impacted by age.

Female fertility is inversely correlated with maternal age due to a depletion of the oocyte pool and a reduction in oocyte developmental competence. Few studies have addressed the effect of maternal age on the human mature oocyte (MII) transcriptome, which is established during oocyte growth and maturation, however, the pathways involved remain unclear. Here, we characterize and compare the transcriptomes of a large cohort of fully grown germinal vesicle stage (GV) and in vitro matured (IVM-MII) oocytes from women of varying reproductive age. First, we identified two clusters of cells reflecting the oocyte maturation stage (GV and IVM-MII) with 4445 and 324 putative marker genes, respectively. Furthermore, we identified genes for which transcript representation either progressively increased or decreased with age. Our results indicate that the transcriptome is more affected by age in IVM-MII oocytes (1219 genes) than in GV oocytes (596 genes). In particular, we found that transcripts of genes involved in chromosome segregation and RNA splicing significantly increased representation with age, while genes related to mitochondrial activity showed a lower representation. Gene regulatory network analysis facilitated the identification of potential upstream master regulators of the genes involved in those biological functions. Our analysis suggests that advanced maternal age does not globally affect the oocyte transcriptome at GV or IVM-MII stages. Nonetheless, hundreds of genes displayed altered transcript representation, particularly in IVM-MII oocytes, which might contribute to the age-related quality decline in human oocytes.

SPOTlight: seeded NMF regression to deconvolute spatial transcriptomics spots with single-cell transcriptomes.

Spatially resolved gene expression profiles are key to understand tissue organization and function. However, spatial transcriptomics (ST) profiling techniques lack single-cell resolution and require a combination with single-cell RNA sequencing (scRNA-seq) information to deconvolute the spatially indexed datasets. Leveraging the strengths of both data types, we developed SPOTlight, a computational tool that enables the integration of ST with scRNA-seq data to infer the location of cell types and states within a complex tissue. SPOTlight is centered around a seeded non-negative matrix factorization (NMF) regression, initialized using cell-type marker genes and non-negative least squares (NNLS) to subsequently deconvolute ST capture locations (spots). Simulating varying reference quantities and qualities, we confirmed high prediction accuracy also with shallowly sequenced or small-sized scRNA-seq reference datasets. SPOTlight deconvolution of the mouse brain correctly mapped subtle neuronal cell states of the cortical layers and the defined architecture of the hippocampus. In human pancreatic cancer, we successfully segmented patient sections and further fine-mapped normal and neoplastic cell states. Trained on an external single-cell pancreatic tumor references, we further charted the localization of clinical-relevant and tumor-specific immune cell states, an illustrative example of its flexible application spectrum and future potential in digital pathology.

Levels of ankle-brachial index and the risk of diabetes mellitus complications.

OBJECTIVE: We sought to compare the association of categorized ankle-brachial index (ABI) with mortality and complications of diabetes in persons with no symptoms of peripheral arterial disease (PAD) and in primary cardiovascular disease prevention. RESEARCH DESIGN AND METHODS: This is a retrospective cohort study of persons with type 2 diabetes aged 35-85 years, from 2006 to 2011. Data were obtained from the Sistema d'Informació per al Desenvolupament de la Investigació en Atenció Primària (SIDIAPQ). Participants had an ABI measurement that was classified into six categories. For each category of ABI, we assessed the incidence of mortality; macrovascular complications of diabetes: acute myocardial infarction (AMI), ischemic stroke, and a composite of these two; and microvascular complications of this metabolic condition: nephropathy, retinopathy, and neuropathy. We also estimated the HRs for these outcomes by ABI category using Cox proportional hazards models. RESULTS: Data from 34 689 persons with type 2 diabetes were included. The mean age was 66.2; 51.5% were men; and the median follow-up was 6.0 years. The outcome with the highest incidence was nephropathy, with 24.4 cases per 1000 person-years in the reference category of 1.1≤ABI≤1.3. The incidences in this category for mortality and AMI were 15.4 and 4.1, respectively. In the Cox models, low ABI was associated with increased risk and was significant from ABI lower than 0.9; below this level, the risk kept increasing steeply. High ABI (over 1.3) was also associated with significant increased risk for most outcomes. CONCLUSIONS: The studied categories of ABI were associated with different risks of type 2 diabetes complications in persons asymptomatic for PAD, who were in primary cardiovascular prevention. These findings could be useful to optimize preventive interventions according to the ABI category in this population.

Role of Low Ankle-Brachial Index in Cardiovascular and Mortality Risk Compared with Major Risk Conditions.

Cardiovascular prevention is of particular interest in persons with asymptomatic peripheral arterial disease. We aimed to quantify its association with mortality and cardiovascular outcomes, compared to other indicators of high risk. We performed a retrospective cohort study using the Database of the Catalan primary care system (SIDIAPQ), for 2006-2015, including 35-85-year-old patients with an ankle-brachial index (ABI) measurement, classified according to the presence of diabetes, cardiovascular disease, and low ABI (<0.9). We calculated the incidences and hazard ratios (HRs) for all-cause mortality, acute myocardial infarction, and ischemic stroke. During a median follow-up of 5.9 years, we analyzed 58,118 persons. The mean (SD) age was 66.6 (10.7) years and 53.4% were men. Compared to the reference group with no diabetes, no previous cardiovascular disease, and normal ankle-brachial index, the HR for all-cause mortality was 1.42 (1.25-1.63) in the group with low ABI, 1.35 (1.26-1.45) in those with diabetes, 1.50 (1.34-1.69) in those with previous cardiovascular disease, and 1.84 (1.68-2.01) in those with low ABI and diabetes. In conclusion, participants with low ABI showed increased mortality, acute myocardial infarction, and ischemic stroke incidence in all the subgroups. Patients with low ankle-brachial index plus diabetes presented increased mortality, acute myocardial infarction, and ischemic stroke risk, all at rates similar to those with previous cardiovascular disease.

Hypertension and high ankle brachial index: the overlooked combination.

OBJECTIVE: Assessment of asymptomatic organ damage in the management of hypertension includes low (<0.9) ankle brachial index (ABI) values. No recommendations are given for patients with high ABI (≥1.3), despite evidence of an association with increased risk. We aimed to study the association of high ABI with all-cause mortality and cardiovascular outcomes in a hypertensive population. METHODS: In anonymized clinical records from the Catalan Primary Care (SIDIAP) database, we designed a large cohort of hypertensive patients aged 35-85 years at the start date. Participants were excluded if they had previous heart failure, coronary heart disease, stroke, diabetes mellitus, or chronic kidney disease. The study population was categorized according to ABI values. Cox proportional hazards models were used to assess all-cause mortality, heart failure, acute myocardial infarction, and stroke. RESULTS: From 2006 through 2015, SIDIAP records included 44 657 hypertensive patients with an ABI measurement 9126 of whom met inclusion criteria. The median follow-up (first to third quartiles) was 6.0 years (4.7-7.6). High ABI (≥ 1.3) was associated with an increase in mortality risk, hazard ratio, and 95% confidence interval: 1.44 (1.10-1.88), similar to the group with ABI at least 0.9 and less than 1.1, hazard ratio 1.36 (1.12-1.65), and lower than all groups with ABI less than 0.9. High ABI values tended to associate with heart failure, hazard ratio 1.34 (0.95-1.91), but the relation of high ABI with acute myocardial infarction and stroke was nonsignificant, hazard ratios 1.30 (0.72-2.35) and 0.97 (0.65-1.42), respectively. CONCLUSION: Patients with high ABI values and hypertension presented an increased all-cause mortality risk that could be considered when advising such patients.

Statins for primary prevention of cardiovascular events and mortality in old and very old adults with and without type 2 diabetes: retrospective cohort study.

OBJECTIVE: To assess whether statin treatment is associated with a reduction in atherosclerotic cardiovascular disease (CVD) and mortality in old and very old adults with and without diabetes. DESIGN: Retrospective cohort study. SETTING: Database of the Catalan primary care system (SIDIAP), Spain, 2006-15. PARTICIPANTS: 46 864 people aged 75 years or more without clinically recognised atherosclerotic CVD. Participants were stratified by presence of type 2 diabetes mellitus and as statin non-users or new users. MAIN OUTCOME MEASURES: Incidences of atherosclerotic CVD and all cause mortality compared using Cox proportional hazards modelling, adjusted by the propensity score of statin treatment. The relation of age with the effect of statins was assessed using both a categorical approach, stratifying the analysis by old (75-84 years) and very old (≥85 years) age groups, and a continuous analysis, using an additive Cox proportional hazard model. RESULTS: The cohort included 46 864 participants (mean age 77 years; 63% women; median follow-up 5.6 years). In participants without diabetes, the hazard ratios for statin use in 75-84 year olds were 0.94 (95% confidence interval 0.86 to 1.04) for atherosclerotic CVD and 0.98 (0.91 to 1.05) for all cause mortality, and in those aged 85 and older were 0.93 (0.82 to 1.06) and 0.97 (0.90 to 1.05), respectively. In participants with diabetes, the hazard ratio of statin use in 75-84 year olds was 0.76 (0.65 to 0.89) for atherosclerotic CVD and 0.84 (0.75 to 0.94) for all cause mortality, and in those aged 85 and older were 0.82 (0.53 to 1.26) and 1.05 (0.86 to 1.28), respectively. Similarly, effect analysis of age in a continuous scale, using splines, corroborated the lack of beneficial statins effect for atherosclerotic CVD and all cause mortality in participants without diabetes older than 74 years. In participants with diabetes, statins showed a protective effect against atherosclerotic CVD and all cause mortality; this effect was substantially reduced beyond the age of 85 years and disappeared in nonagenarians. CONCLUSIONS: In participants older than 74 years without type 2 diabetes, statin treatment was not associated with a reduction in atherosclerotic CVD or in all cause mortality, even when the incidence of atherosclerotic CVD was statistically significantly higher than the risk thresholds proposed for statin use. In the presence of diabetes, statin use was statistically significantly associated with reductions in the incidence of atherosclerotic CVD and in all cause mortality. This effect decreased after age 85 years and disappeared in nonagenarians.

Association of Classic Cardiovascular Risk Factors and Lifestyles With the Cardio-ankle Vascular Index in a General Mediterranean Population.

INTRODUCTION AND OBJECTIVES: The cardio-ankle vascular index (CAVI) assesses arterial stiffness. We aimed to describe the distribution of CAVI in a Mediterranean population, to determine the proportion of CAVI ≥ 9 by sex and coronary risk level, and to assess the association of CAVI with classic cardiovascular risk factors and lifestyle patterns. METHODS: This cross-sectional study was based on the population of Girona province. The CAVI was measured using the VaSera VS-1500. RESULTS: Of 2613 individuals included in this study, the prevalence of CAVI ≥ 9 was 46.8% in men and 36.0% in women and significantly increased with coronary risk: from 21.1% and 24.8%, respectively to 76.7%, in the low-risk group, and 61.9% in the high-risk group. The CAVI increased with age in both sexes, being higher in men across all age groups. In men, CAVI ≥ 9 was associated with hypertension (OR, 2.70; 95%CI, 1.90-3.87) and diabetes (OR, 2.38; 95%CI, 1.52-3.78), body mass index (BMI) ≤ 25 to < 30 (OR, 0.44; 95%CI, 0.27-0.72) and BMI ≥ 30 (OR, 0.28; 95%CI, 0.14-0.58), and physical activity (OR, 0.66; 95%CI, 0.47-0.92). In women, CAVI ≥ 9 was associated with hypertension (OR, 2.22; 95%CI, 1.59-3.09), hypercholesterolemia (OR, 1.40; 95%CI, 1.01-1.94), and BMI ≥ 30 (OR, 0.38; 95%CI, 0.20-0.71). CONCLUSIONS: The CAVI increases with age and is higher in men than in women. This index is associated with classic risk factors and coronary risk. It could be a good predictive biomarker, but further follow-up studies are required to assess its added value to cardiovascular risk stratification.

Statins and new-onset atrial fibrillation in a cohort of patients with hypertension. Analysis of electronic health records, 2006-2015.

Hypertension is the most prevalent risk factor for new-onset atrial fibrillation (AF). But few studies have addressed the effect of statins on the incidence of this arrhythmia in patients with hypertension. This study aimed to evaluate the effect of statins on new-onset of this arrhythmia in a hypertensive population, accounting for AF risk. Data from the Information System for the Development of Research in Primary Care was used to recruit a retrospective cohort of ≥55-year-old hypertensive individuals with no ischemic vascular disease, in 2006-2007, followed up through 2015. The effect of initiating statin treatment on new-onset atrial fibrillation was assessed with Cox proportional hazards models adjusted by the propensity score of receiving statin treatment, in the overall study population and stratified by AF risk. Of 100 276 included participants, 9814 initiated statin treatment. The AF incidence per 1000 person-years (95% confidence interval) was 12.5 (12.3-12.8). Statin use associated with a significant (9%) reduction in AF incidence. Differences in absolute AF incidence were higher in the highest AF risk subgroup, and the estimated number needed to treat to avoid one case was 720. The relative effect was poor, similar across groups, and non-significant, as was the association of statins with adverse effects. We found a limited protective effect of statins over new-onset AF in this hypertensive population with no ischemic vascular disease. If there is no further indication, hypertensive patients would not benefit from statin use solely for AF primary prevention.

Differences in cardio-ankle vascular index in a general Mediterranean population depending on the presence or absence of metabolic cardiovascular risk factors.

BACKGROUND AND AIMS: The main aim of this study is to describe the differences in the cardio-ankle vascular index (CAVI) in individuals with metabolic cardiovascular risk factors or a previous history of vascular diseases (WCVRF) compared to healthy individuals (free of risk factors and previous history of vascular diseases; FCVRF) in a general Mediterranean population. The secondary aim is to describe the proportion of CAVI≥9 depending on the cardiovascular risk category in both CVRF groups by sex. METHODS: The study is a descriptive analysis of 2613 participants randomly selected in the Girona province (Catalonia, Spain). RESULTS: CAVI mean differences between sexes and age categories in both CVRF groups followed the same pattern, the FCVRF group (men 25.2%; women 14.4%) in turn had a lower prevalence of CAVI≥9 than the WCVRF group (men 57.9%; women 51.8%). The percentage of men and women with CAVI≥9 with low risk was 13.9% and 11.3% in the FCVRF group, and 31.8% and 42.0% in the WCVRF group; with moderate risk, it was 55.8% and 10.0% in the FCVRF group and 60.3% and 49.0% in the WCVRF group. CONCLUSIONS: In both sexes, FCVRF groups had a lower prevalence of CAVI≥9 as well as lower mean CAVI scores, across all 10 year-age categories from 40 to 69 years, than WCVRF groups. Moreover, CAVI≥9 was frequent in individuals with low and moderate coronary risk in the WCVRF group but also in the FCVRF group. These results suggest that CAVI assessment to detect asymptomatic arteriosclerosis could be a useful tool to improve cardiovascular risk stratification.