Enhancing sepsis biomarker development: key considerations from public and private perspectives.

Implementation of biomarkers in sepsis and septic shock in emergency situations, remains highly challenging. This viewpoint arose from a public-private 3-day workshop aiming to facilitate the transition of sepsis biomarkers into clinical practice. The authors consist of international academic researchers and clinician-scientists and industry experts who gathered (i) to identify current obstacles impeding biomarker research in sepsis, (ii) to outline the important milestones of the critical path of biomarker development and (iii) to discuss novel avenues in biomarker discovery and implementation. To define more appropriately the potential place of biomarkers in sepsis, a better understanding of sepsis pathophysiology is mandatory, in particular the sepsis patient's trajectory from the early inflammatory onset to the late persisting immunosuppression phase. This time-varying host response urges to develop time-resolved test to characterize persistence of immunological dysfunctions. Furthermore, age-related difference has to be considered between adult and paediatric septic patients. In this context, numerous barriers to biomarker adoption in practice, such as lack of consensus about diagnostic performances, the absence of strict recommendations for sepsis biomarker development, cost and resources implications, methodological validation challenges or limited awareness and education have been identified. Biomarker-guided interventions for sepsis to identify patients that would benefit more from therapy, such as sTREM-1-guided Nangibotide treatment or Adrenomedullin-guided Enibarcimab treatment, appear promising but require further evaluation. Artificial intelligence also has great potential in the sepsis biomarker discovery field through capability to analyse high volume complex data and identify complex multiparametric patient endotypes or trajectories. To conclude, biomarker development in sepsis requires (i) a comprehensive and multidisciplinary approach employing the most advanced analytical tools, (ii) the creation of a platform that collaboratively merges scientific and commercial needs and (iii) the support of an expedited regulatory approval process

Relationship between ventilator-associated pneumonia and mortality in COVID-19 patients: a planned ancillary analysis of the coVAPid cohort

Background Patients with SARS-CoV-2 infection are at higher risk for ventilator-associated pneumonia (VAP). No study has evaluated the relationship between VAP and mortality in this population, or compared this relationship between SARS-CoV-2 patients and other populations. The main objective of our study was to determine the relationship between VAP and mortality in SARS-CoV-2 patients. Methods Planned ancillary analysis of a multicenter retrospective European cohort. VAP was diagnosed using clinical, radiological and quantitative microbiological criteria. Univariable and multivariable marginal Cox's regression models, with cause-specific hazard for duration of mechanical ventilation and ICU stay, were used to compare outcomes between study groups. Extubation, and ICU discharge alive were considered as events of interest, and mortality as competing event. Findings Of 1576 included patients, 568 were SARS-CoV-2 pneumonia, 482 influenza pneumonia, and 526 no evidence of viral infection at ICU admission. VAP was associated with significantly higher risk for 28-day mortality in SARS-CoV-2 group (adjusted HR 1.65 (95% CI 1.11-2.46), p = 0.013), but not in influenza (1.74 (0.99-3.06), p = 0.052), or no viral infection groups (1.13 (0.68-1.86), p = 0.63). VAP was associated with significantly longer duration of mechanical ventilation in the SARS-CoV-2 group, but not in the influenza or no viral infection groups. VAP was associated with significantly longer duration of ICU stay in the 3 study groups. No significant difference was found in heterogeneity of outcomes related to VAP between the 3 groups, suggesting that the impact of VAP on mortality was not different between study groups. Interpretation VAP was associated with significantly increased 28-day mortality rate in SARS-CoV-2 patients. However, SARS-CoV-2 pneumonia, as compared to influenza pneumonia or no viral infection, did not significantly modify the relationship between VAP and 28-day mortality

a retrospective multicenter study

Funding This study was supported in part by a grant from the French government through the « Programme Investissement d’Avenir» (I-SITE ULNE) managed by the Agence Nationale de la Recherche (coVAPid project). Prof. Ignacio Martin-Loeches has been supported by SFI (Science Foundation Ireland), Grant number 20/COV/0038. The funders of the study had no role in the study design, data collection, analysis or interpretation, writing of the report or deci sion to submit for publication.BACKGROUND: Ventilator-associated pneumonia (VAP) is common in patients with severe SARS-CoV-2 pneumonia. The aim of this ancillary analysis of the coVAPid multicenter observational retrospective study is to assess the relationship between adjuvant corticosteroid use and the incidence of VAP. METHODS: Planned ancillary analysis of a multicenter retrospective European cohort in 36 ICUs. Adult patients receiving invasive mechanical ventilation for more than 48 h for SARS-CoV-2 pneumonia were consecutively included between February and May 2020. VAP diagnosis required strict definition with clinical, radiological and quantitative microbiological confirmation. We assessed the association of VAP with corticosteroid treatment using univariate and multivariate cause-specific Cox's proportional hazard models with adjustment on pre-specified confounders. RESULTS: Among the 545 included patients, 191 (35%) received corticosteroids. The proportional hazard assumption for the effect of corticosteroids on the incidence of VAP could not be accepted, indicating that this effect varied during ICU stay. We found a non-significant lower risk of VAP for corticosteroid-treated patients during the first days in the ICU and an increased risk for longer ICU stay. By modeling the effect of corticosteroids with time-dependent coefficients, the association between corticosteroids and the incidence of VAP was not significant (overall effect p = 0.082), with time-dependent hazard ratios (95% confidence interval) of 0.47 (0.17-1.31) at day 2, 0.95 (0.63-1.42) at day 7, 1.48 (1.01-2.16) at day 14 and 1.94 (1.09-3.46) at day 21. CONCLUSIONS: No significant association was found between adjuvant corticosteroid treatment and the incidence of VAP, although a time-varying effect of corticosteroids was identified along the 28-day follow-up.publishersversionpublishe

a planned ancillary analysis of the coVAPid cohort

Funding: This study was supported in part by a grant from the French government through the «Programme Investissement d’Avenir» (I-SITE ULNE) managed by the Agence Nationale de la Recherche (coVAPid project). The funders of the study had no role in the study design, data collection, analysis, or interpreta tion, writing of the report, or decision to submit for publication.BACKGROUND: Patients with SARS-CoV-2 infection are at higher risk for ventilator-associated pneumonia (VAP). No study has evaluated the relationship between VAP and mortality in this population, or compared this relationship between SARS-CoV-2 patients and other populations. The main objective of our study was to determine the relationship between VAP and mortality in SARS-CoV-2 patients. METHODS: Planned ancillary analysis of a multicenter retrospective European cohort. VAP was diagnosed using clinical, radiological and quantitative microbiological criteria. Univariable and multivariable marginal Cox's regression models, with cause-specific hazard for duration of mechanical ventilation and ICU stay, were used to compare outcomes between study groups. Extubation, and ICU discharge alive were considered as events of interest, and mortality as competing event. FINDINGS: Of 1576 included patients, 568 were SARS-CoV-2 pneumonia, 482 influenza pneumonia, and 526 no evidence of viral infection at ICU admission. VAP was associated with significantly higher risk for 28-day mortality in SARS-CoV-2 (adjusted HR 1.70 (95% CI 1.16-2.47), p = 0.006), and influenza groups (1.75 (1.03-3.02), p = 0.045), but not in the no viral infection group (1.07 (0.64-1.78), p = 0.79). VAP was associated with significantly longer duration of mechanical ventilation in the SARS-CoV-2 group, but not in the influenza or no viral infection groups. VAP was associated with significantly longer duration of ICU stay in the 3 study groups. No significant difference was found in heterogeneity of outcomes related to VAP between the 3 groups, suggesting that the impact of VAP on mortality was not different between study groups. INTERPRETATION: VAP was associated with significantly increased 28-day mortality rate in SARS-CoV-2 patients. However, SARS-CoV-2 pneumonia, as compared to influenza pneumonia or no viral infection, did not significantly modify the relationship between VAP and 28-day mortality. CLINICAL TRIAL REGISTRATION: The study was registered at ClinicalTrials.gov, number NCT04359693.publishersversionpublishe

Hétérogénéité des réponses immunes post-septiques : approche translationnelle

Sepsis following microbial infection is a complex clinical entity, characterized by the simultaneous occurrence of an initial dysregulated inflammatory response and protracted immune suppression associating quantitative and functional abnormalities of immune cells. Most of the immune dysfunction induced by sepsis has been associated with an increased susceptibility to nosocomial infections, first and foremost ventilator-acquired pneumonia (VAP). However, there appears to be variability in the susceptibility to these ICU-acquired infections. First, we identified in patients admitted in the intensive care unit for septic shock an increased risk of VAP after a first pulmonary infection when compared to other sites of infection. Furthermore, in the pandemic context, we have identified that COVID-19 was independently associated with an increased risk of nosocomial pneumonia, thus suggesting that SARS-CoV-2 infection induces particular lung immune. Using two murine models, we then addressed the impact of primary pulmonary and non-pulmonary infectious insults on lung immunity. Mice were first subjected either to polymicrobial peritonitis induced by caecal ligation and puncture (CLP), or to bacterial pneumonia induced by intra-tracheal (i.t.) instillation of Escherichia coli. Respective control mice were subjected to sham surgery or intra-tracheal instillation of phosphate-buffered saline. Seven days later, mice that survived the primary insult were subjected to i.t. instillation of Pseudomonas aeruginosa (PAO1 strain). We assessed survival and pulmonary bacterial clearance of after P. aeruginosa pneumonia, as well as quantitative and functional changes in lung immune cells. When compared to sham-operated mice, post-CLP animals exhibited increased susceptibility to secondary P. aeruginosa pneumonia as demonstrated by defective lung bacterial clearance and increased mortality rate (50% vs. 0%, p< 0.05). In contrast, all post-pneumonia mice survived and even exhibited improved bacterial clearance as compared to their control counterparts. When addressing whole-lung immune cell distribution prior to second hit (day 7), amounts of alveolar macrophages (AM) were decreased in post-CLP mice while increased in post-pneumonia mice. In contrast to post-CLP, AM from post-pneumonia mice express high MHC-II, a defence-ready transcriptomic signature and promotes antigen-specific CD4 T cells proliferation. Additionally, we observed a TLR2-dependent increase in regulatory T cells (Tregs) proportion among CD4 T cells in the lung of post-CLP mice at day 7 when compared to controls and post-pneumonia mice. CD25-mediated depletion of Tregs prior to P. aeruginosa pneumonia restored survival and bacterial clearance in post-CLP mice. Tregs depletion was associated with restoration of numbers and functions of AMin post-CLP mice. Given the difficulties in accessing tissue macrophages in clinical practice, we evaluated the functions of circulating monocytes using an innovative experimental technique based on the encapsulation of cells in lipid droplets in a microfluidic system. We evaluated in real time the individual secretion of TNFα by monocytes from septic patients (n = 7) and healthy volunteers (n = 10) exposed to stimulation by LPS. By comparing the results with those obtained by flow cytometry (membrane expressions of HLA-DR and intracellular expressions of TNFα), we were able to demonstrate the existence of a significant temporal variability and a great heterogeneity of the monocytic responses in septic patients.Le sepsis consécutif à l'infection microbienne est une entité clinique complexe, caractérisée par la survenue simultanée d'une dysrégulation de la réaction inflammatoire initiale et d'une immunodépression associant des anomalies quantitatives et fonctionnelles des cellules de l'immunité innée et de l'immunité adaptative. La plupart des dysfonctions immunitaires induites par le sepsis ont été associées à une susceptibilité accrue aux infections nosocomiales, au premier rang desquelles les pneumonies acquises sous ventilation mécanique (PAVM). Toutefois, il semble exister une variabilité dans la susceptibilité à ces infections acquises en réanimation. Dans un premier temps, nous avons identifié chez les patients pris en charge pour un choc septique un risque accru de PAVM dans les suites d'une première agression pulmonaire en comparaison des autres sites infectieux. Par ailleurs, dans le contexte pandémique, nous avons identifié que la COVID-19 était indépendamment associée à un risque accru de pneumonie nosocomiale, suggérant ainsi que l'infection à SARS-CoV-2 induit des altérations particulières de l'immunité pulmonaire. A l'aide de deux modèles murins d'infection pulmonaire (pneumonie à E. coli) et extra-pulmonaire (ligature ponction caecale, LPC) et de leurs contrôles respectifs (laparotomie simple et instillation de PBS), tous suivis d'une seconde pneumonie à Pseudomonas aeruginosa après 7 jours, nous avons pu mettre en évidence une différence de susceptibilité et de clairance bactérienne dans les suites d'une pneumonie secondaire à P.aeruginosa en fonction du site infectieux initial, ainsi que des modifications quantitatives et fonctionnelles des cellules immunitaires pulmonaires. En comparaison des souris ayant subi une laparotomie simple, les souris post-LPC ont une susceptibilité accrue à une pneumonie secondaire à P. aeruginosa avec une diminution de la clairance bactérienne et une mortalité plus importante (50% versus 0%, p<0.05). A l'inverse, l'ensemble des souris ayant eu une pneumonie à E. coli a survécu à cette seconde pneumonie, avec une augmentation de la clairance bactérienne. L'évaluation de la composition cellulaire pulmonaire au 7ème jour (avant la seconde pneumonie) a montré une diminution du nombre de macrophages alvéolaires (MA) chez les souris post-LPC et une augmentation chez les souris post-pneumonie. A l'inverse des MA post-LPC, les MA de souris post-pneumonie expriment fortement le CMH de classe II, ont une signature transcriptomique orientée vers les défenses antibactériennes et stimulent la prolifération antigène-spécifique de lymphocytes T CD4. De plus, nous avons identifié une augmentation de la proportion des lymphocytes T CD4 régulateurs (Tregs) dans les poumons de souris post-LPC en comparaison des autres conditions, cette augmentation étant dépendante de la voie de signalisation du TLR2. La déplétion des Tregs préalablement à la survenue de la seconde pneumonie à P. aeruginosa par des anticorps bloquants dirigés contre le CD25 restaure la survie et la clairance bactérienne chez les souris post-LPC. Leur déplétion est aussi associée à une restauration du nombre et des fonctions des MA post-LPC. Compte-tenu des difficultés d'accès aux macrophages tissulaire en pratique clinique, nous avons évalué les fonctions des monocytes circulants à l'aide d'une technique expérimentale innovante basée sur l'encapsulation de cellules dans des gouttelettes lipidiques dans un système de microfluidique. Nous avons évalué en temps réel la sécrétion individuelle de TNFα par des monocytes de patients septiques (n=7) et de volontaires sains (n=10) exposés à une stimulation par LPS. En confrontant les résultats à ceux obtenus par cytométrie de flux (expressions membranaire de HLA-DR et intracellulaire de TNFα), nous avons pu mettre en évidence l'existence d'une variabilité temporelle importante et d'une grande hétérogénéité des réponses monocytaires chez les patients septiques

Heterogeneity of post-septic immune responses : a translational approach

Le sepsis consécutif à l'infection microbienne est une entité clinique complexe, caractérisée par la survenue simultanée d'une dysrégulation de la réaction inflammatoire initiale et d'une immunodépression associant des anomalies quantitatives et fonctionnelles des cellules de l'immunité innée et de l'immunité adaptative. La plupart des dysfonctions immunitaires induites par le sepsis ont été associées à une susceptibilité accrue aux infections nosocomiales, au premier rang desquelles les pneumonies acquises sous ventilation mécanique (PAVM). Toutefois, il semble exister une variabilité dans la susceptibilité à ces infections acquises en réanimation. Dans un premier temps, nous avons identifié chez les patients pris en charge pour un choc septique un risque accru de PAVM dans les suites d'une première agression pulmonaire en comparaison des autres sites infectieux. Par ailleurs, dans le contexte pandémique, nous avons identifié que la COVID-19 était indépendamment associée à un risque accru de pneumonie nosocomiale, suggérant ainsi que l'infection à SARS-CoV-2 induit des altérations particulières de l'immunité pulmonaire. A l'aide de deux modèles murins d'infection pulmonaire (pneumonie à E. coli) et extra-pulmonaire (ligature ponction caecale, LPC) et de leurs contrôles respectifs (laparotomie simple et instillation de PBS), tous suivis d'une seconde pneumonie à Pseudomonas aeruginosa après 7 jours, nous avons pu mettre en évidence une différence de susceptibilité et de clairance bactérienne dans les suites d'une pneumonie secondaire à P.aeruginosa en fonction du site infectieux initial, ainsi que des modifications quantitatives et fonctionnelles des cellules immunitaires pulmonaires. En comparaison des souris ayant subi une laparotomie simple, les souris post-LPC ont une susceptibilité accrue à une pneumonie secondaire à P. aeruginosa avec une diminution de la clairance bactérienne et une mortalité plus importante (50% versus 0%, p<0.05). A l'inverse, l'ensemble des souris ayant eu une pneumonie à E. coli a survécu à cette seconde pneumonie, avec une augmentation de la clairance bactérienne. L'évaluation de la composition cellulaire pulmonaire au 7ème jour (avant la seconde pneumonie) a montré une diminution du nombre de macrophages alvéolaires (MA) chez les souris post-LPC et une augmentation chez les souris post-pneumonie. A l'inverse des MA post-LPC, les MA de souris post-pneumonie expriment fortement le CMH de classe II, ont une signature transcriptomique orientée vers les défenses antibactériennes et stimulent la prolifération antigène-spécifique de lymphocytes T CD4. De plus, nous avons identifié une augmentation de la proportion des lymphocytes T CD4 régulateurs (Tregs) dans les poumons de souris post-LPC en comparaison des autres conditions, cette augmentation étant dépendante de la voie de signalisation du TLR2. La déplétion des Tregs préalablement à la survenue de la seconde pneumonie à P. aeruginosa par des anticorps bloquants dirigés contre le CD25 restaure la survie et la clairance bactérienne chez les souris post-LPC. Leur déplétion est aussi associée à une restauration du nombre et des fonctions des MA post-LPC. Compte-tenu des difficultés d'accès aux macrophages tissulaire en pratique clinique, nous avons évalué les fonctions des monocytes circulants à l'aide d'une technique expérimentale innovante basée sur l'encapsulation de cellules dans des gouttelettes lipidiques dans un système de microfluidique. Nous avons évalué en temps réel la sécrétion individuelle de TNFα par des monocytes de patients septiques (n=7) et de volontaires sains (n=10) exposés à une stimulation par LPS. En confrontant les résultats à ceux obtenus par cytométrie de flux (expressions membranaire de HLA-DR et intracellulaire de TNFα), nous avons pu mettre en évidence l'existence d'une variabilité temporelle importante et d'une grande hétérogénéité des réponses monocytaires chez les patients septiques.Sepsis following microbial infection is a complex clinical entity, characterized by the simultaneous occurrence of an initial dysregulated inflammatory response and protracted immune suppression associating quantitative and functional abnormalities of immune cells. Most of the immune dysfunction induced by sepsis has been associated with an increased susceptibility to nosocomial infections, first and foremost ventilator-acquired pneumonia (VAP). However, there appears to be variability in the susceptibility to these ICU-acquired infections. First, we identified in patients admitted in the intensive care unit for septic shock an increased risk of VAP after a first pulmonary infection when compared to other sites of infection. Furthermore, in the pandemic context, we have identified that COVID-19 was independently associated with an increased risk of nosocomial pneumonia, thus suggesting that SARS-CoV-2 infection induces particular lung immune. Using two murine models, we then addressed the impact of primary pulmonary and non-pulmonary infectious insults on lung immunity. Mice were first subjected either to polymicrobial peritonitis induced by caecal ligation and puncture (CLP), or to bacterial pneumonia induced by intra-tracheal (i.t.) instillation of Escherichia coli. Respective control mice were subjected to sham surgery or intra-tracheal instillation of phosphate-buffered saline. Seven days later, mice that survived the primary insult were subjected to i.t. instillation of Pseudomonas aeruginosa (PAO1 strain). We assessed survival and pulmonary bacterial clearance of after P. aeruginosa pneumonia, as well as quantitative and functional changes in lung immune cells. When compared to sham-operated mice, post-CLP animals exhibited increased susceptibility to secondary P. aeruginosa pneumonia as demonstrated by defective lung bacterial clearance and increased mortality rate (50% vs. 0%, p< 0.05). In contrast, all post-pneumonia mice survived and even exhibited improved bacterial clearance as compared to their control counterparts. When addressing whole-lung immune cell distribution prior to second hit (day 7), amounts of alveolar macrophages (AM) were decreased in post-CLP mice while increased in post-pneumonia mice. In contrast to post-CLP, AM from post-pneumonia mice express high MHC-II, a defence-ready transcriptomic signature and promotes antigen-specific CD4 T cells proliferation. Additionally, we observed a TLR2-dependent increase in regulatory T cells (Tregs) proportion among CD4 T cells in the lung of post-CLP mice at day 7 when compared to controls and post-pneumonia mice. CD25-mediated depletion of Tregs prior to P. aeruginosa pneumonia restored survival and bacterial clearance in post-CLP mice. Tregs depletion was associated with restoration of numbers and functions of AMin post-CLP mice. Given the difficulties in accessing tissue macrophages in clinical practice, we evaluated the functions of circulating monocytes using an innovative experimental technique based on the encapsulation of cells in lipid droplets in a microfluidic system. We evaluated in real time the individual secretion of TNFα by monocytes from septic patients (n = 7) and healthy volunteers (n = 10) exposed to stimulation by LPS. By comparing the results with those obtained by flow cytometry (membrane expressions of HLA-DR and intracellular expressions of TNFα), we were able to demonstrate the existence of a significant temporal variability and a great heterogeneity of the monocytic responses in septic patients

Pulmonary infections prime the development of subsequent ICU-acquired pneumonia in septic shock

Abstract Purpose To investigate the determinants and the prognosis of intensive care unit (ICU)-acquired pneumonia in patients with septic shock. Methods This single-center retrospective study was conducted in a medical ICU in a tertiary care center from January 2008 to December 2016. All consecutive patients diagnosed for septic shock within the first 48 h of ICU admission were included. Patients were classified in three groups: no ICU-acquired infections (no ICU-AI), ICU-acquired pneumonia and non-pulmonary ICU-AI. The determinants of ICU-acquired pneumonia and death were investigated by multivariate competitive risk analysis. Results A total of 1021 patients were admitted for septic shock, and 797 patients were alive in the ICU after 48 h of management. The incidence of a first episode of ICU-AI was 31%, distributed into pulmonary (17%) and non-pulmonary ICU-AI (14%). Patients with septic shock caused by pneumonia were at increased risk of further pulmonary ICU-AI with a cumulated incidence of 34.4%. A pulmonary source of the initial septic shock was an independent risk factor for subsequent ICU-acquired pneumonia (cause-specific hazard 2.33, 95% confidence interval [1.55–3.52], p < 0.001). ICU-AI were not associated with a higher risk of ICU mortality after adjustment in a multivariate-adjusted cause-specific proportional hazard model. Conclusion Septic shock of pulmonary origin may represent a risk factor for subsequent ICU-acquired pneumonia without affecting mortality

Impaired biological response to aspirin in therapeutic hypothermia comatose patients resuscitated from out-of-hospital cardiac arrest

International audienc

The balance of thrombosis and hemorrhage in STEMI patients with or without associated cardiac arrest: An observational study

International audienceBackground: Data is scarce on hemorrhagic and thrombotic complications in patients with ST-elevation myocardial infarction (STEMI) associated with out-of-hospital cardiac arrest (OHCA).Methods: This is a monocentric, retrospective study conducted from January 2012 to December 2017 in a tertiary university hospital, which serves as a cardiac arrest center for a large urban area. Over the study period, all consecutive patients who were treated with stent implantation for STEMI with or without OHCA were included. Baseline characteristics, treatments, hemorrhagic and thrombotic events were compared between STEMI patients with and without OHCA. Univariate and multivariate analysis were performed in order to identify predictors of 30-day mortality, occurrence of major bleeding (MB), and early stent thrombosis (ST).Results: A total of 549 patients treated for STEMI without OHCA and 146 patients for STEMI with OHCA were included. The incidence of definite ST and MB after coronary angioplasty was significantly higher in patients with OHCA (2.6% vs. 7.5%, p = 0.004 and 3.3% vs. 19.2%, p < 0.001, respectively). Independent predictors of MB in OHCA patients were anticoagulation therapy (HR = 3.11, 95%CI [1.22-7.98], p = 0.02) and the use of glycoprotein IIb/IIIa inhibitors (HR = 4.16, 95%CI [1.61-10.79], p = 0.003). Independent predictors of mortality in OHCA patients were age (HR = 1.05, 95%CI [1.02-1.09], p = 0.004) and ST (HR = 5.62, 95%CI [1.61-19.65], p = 0.007, with a protective effect of new anti-P2Y12 treatments (HR = 0.20, 95%CI [0.08-0.46], p < 0.001).Conclusion: Patients treated for STEMI associated with OHCA are at higher-risk of ST and MB than those who did not experience cardiac arrest. In this subset of patients, prospective studies are needed to better evaluate the balance of thrombosis and hemorrhage