Mapping systemic lupus erythematosus heterogeneity at the single-cell level.

Patients with systemic lupus erythematosus (SLE) display a complex blood transcriptome whose cellular origin is poorly resolved. Using single-cell RNA sequencing, we profiled ~276,000 peripheral blood mononuclear cells from 33 children with SLE with different degrees of disease activity and 11 matched controls. Increased expression of interferon-stimulated genes (ISGs) distinguished cells from children with SLE from healthy control cells. The high ISG expression signature (ISGhi) derived from a small number of transcriptionally defined subpopulations within major cell types, including monocytes, CD4+ and CD8+ T cells, natural killer cells, conventional and plasmacytoid dendritic cells, B cells and especially plasma cells. Expansion of unique subpopulations enriched in ISGs and/or in monogenic lupus-associated genes classified patients with the highest disease activity. Profiling of ~82,000 single peripheral blood mononuclear cells from adults with SLE confirmed the expansion of similar subpopulations in patients with the highest disease activity. This study lays the groundwork for resolving the origin of the SLE transcriptional signatures and the disease heterogeneity towards precision medicine applications.

IL-1 is a critical regulator of group 2 innate lymphoid cell function and plasticity.

Group 2 innate lymphoid cells (ILC2 cells) are important for type 2 immune responses and are activated by the epithelial cytokines interleukin 33 (IL-33), IL-25 and thymic stromal lymphopoietin (TSLP). Here we demonstrated that IL-1ß was a critical activator of ILC2 cells, inducing proliferation and cytokine production and regulating the expression of epithelial cytokine receptors. IL-1ß also governed ILC2 plasticity by inducing low expression of the transcription factor T-bet and the cytokine receptor chain IL-12Rß2, which enabled the conversion of these cells into an ILC1 phenotype in response to IL-12. This transition was marked by an atypical chromatin landscape characterized by the simultaneous transcriptional accessibility of the locus encoding interferon-γ (IFN-γ) and the loci encoding IL-5 and IL-13. Finally, IL-1ß potentiated ILC2 activation and plasticity in vivo, and IL-12 acted as the switch that determined an ILC2-versus-ILC1 response. Thus, we have identified a previously unknown role for IL-1ß in facilitating ILC2 maturation and plasticity.

Group 1 Innate Lymphoid Cell Lineage Identity Is Determined by a cis-Regulatory Element Marked by a Long Non-coding RNA.

Commitment to the innate lymphoid cell (ILC) lineage is determined by Id2, a transcriptional regulator that antagonizes T and B cell-specific gene expression programs. Yet how Id2 expression is regulated in each ILC subset remains poorly understood. We identified a cis-regulatory element demarcated by a long non-coding RNA (lncRNA) that controls the function and lineage identity of group 1 ILCs, while being dispensable for early ILC development and homeostasis of ILC2s and ILC3s. The locus encoding this lncRNA, which we termed Rroid, directly interacted with the promoter of its neighboring gene, Id2, in group 1 ILCs. Moreover, the Rroid locus, but not the lncRNA itself, controlled the identity and function of ILC1s by promoting chromatin accessibility and deposition of STAT5 at the promoter of Id2 in response to interleukin (IL)-15. Thus, non-coding elements responsive to extracellular cues unique to each ILC subset represent a key regulatory layer for controlling the identity and function of ILCs.

One-Year Outcomes in Patients With Acute Stroke Requiring Mechanical Ventilation.

BACKGROUND: Long-term outcomes of patients with severe stroke remain poorly documented. We aimed to characterize one-year outcomes of patients with stroke requiring mechanical ventilation in the intensive care unit (ICU). METHODS: We conducted a prospective multicenter cohort study in 33 ICUs in France (2017-2019) on patients with consecutive strokes requiring mechanical ventilation for at least 24 hours. Outcomes were collected via telephone interviews by an independent research assistant. The primary end point was poor functional outcome, defined by a modified Rankin Scale score of 4 to 6 at 1 year. Multivariable mixed models investigated variables associated with the primary end point. Secondary end points included quality of life, activities of daily living, and anxiety and depression in 1-year survivors. RESULTS: Among the 364 patients included, 244 patients (66.5% [95% CI, 61.7%-71.3%]) had a poor functional outcome, including 190 deaths (52.2%). After adjustment for non-neurological organ failure, age ≥70 years (odds ratio [OR], 2.38 [95% CI, 1.26-4.49]), Charlson comorbidity index ≥2 (OR, 2.01 [95% CI, 1.16-3.49]), a score on the Glasgow Coma Scale <8 at ICU admission (OR, 3.43 [95% CI, 1.98-5.96]), stroke subtype (intracerebral hemorrhage: OR, 2.44 [95% CI, 1.29-4.63] versus ischemic stroke: OR, 2.06 [95% CI, 1.06-4.00] versus subarachnoid hemorrhage: reference) remained independently associated with poor functional outcome. In contrast, a time between stroke diagnosis and initiation of mechanical ventilation >1 day was protective (OR, 0.56 [95% CI, 0.33-0.94]). A sensitivity analysis conducted after exclusion of patients with early decisions of withholding/withdrawal of care yielded similar results. We observed persistent physical and psychological problems at 1 year in >50% of survivors. CONCLUSIONS: In patients with severe stroke requiring mechanical ventilation, several ICU admission variables may inform caregivers, patients, and their families on post-ICU trajectories and functional outcomes. The burden of persistent sequelae at 1 year reinforces the need for a personalized, multi-disciplinary, prolonged follow-up of these patients after ICU discharge. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03335995.

Combination of Mycological Criteria: a Better Surrogate to Identify COVID-19-Associated Pulmonary Aspergillosis Patients and Evaluate Prognosis?

Diagnosis of coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) remains unclear especially in nonimmunocompromised patients. The aim of this study was to evaluate seven mycological criteria and their combination in a large homogenous cohort of patients. All successive patients (n = 176) hospitalized for COVID-19 requiring mechanical ventilation and who clinically worsened despite appropriate standard of care were included over a 1-year period. Direct examination, culture, Aspergillus quantitative PCR (Af-qPCR), and galactomannan testing were performed on all respiratory samples (n = 350). Serum galactomannan, ß-d-glucan, and plasma Af-qPCR were also assessed. The criteria were analyzed alone or in combination in relation to mortality rate. Mortality was significantly different in patients with 0, ≤2, and ≥3 positive criteria (log rank test, P = 0.04) with death rate of 43.1, 58.1, and 76.4%, respectively. Direct examination, plasma qPCR, and serum galactomannan were associated with a 100% mortality rate. Bronchoalveolar lavage (BAL) galactomannan and positive respiratory sample culture were often found as isolated markers (28.1 and 34.1%) and poorly repeatable when a second sample was obtained. Aspergillus DNA was detected in 13.1% of samples (46 of 350) with significantly lower quantitative cycle (Cq) when associated with at least one other criterion (30.2 versus 35.8) (P < 0.001). A combination of markers and/or blood biomarkers and/or direct respiratory sample examination seems more likely to identify patients with CAPA. Af-qPCR may help identifying false-positive results of BAL galactomannan testing and culture on respiratory samples while quantifying fungal burden accurately.

Accuracy of cumulative volumes of fluid challenge to assess fluid responsiveness in critically ill patients with acute circulatory failure: a pharmacodynamic approach.

BACKGROUND: The relationship between the dose (volume of fluid) and the effect (increase of stroke volume [SV]) has been poorly described. We hypothesised that the analysis of the dynamic response of SV during fluid challenge (FC) helps to determine the optimal volume of FC, along with its diagnostic accuracy parameters for fluid responsiveness. METHODS: A prospective observational study was conducted in critically ill patients with circulatory failure. Patients monitored with oesophageal Doppler and assigned to an FC of 500 ml of crystalloid were included. The areas under the curve (AUC) and 95% confidence intervals (CI95) of the receiver operating characteristic curves for cumulative volumes from 50 to 450 ml were determined for fluid responsiveness (SV increase ≥15% from baseline) along with other parameters of diagnostic accuracy. In the pharmacodynamic analysis, dose-effect and dose-response models were constructed, with determination of median and 90% effective dose (ED50 and ED90). RESULTS: Forty-five patients were included. The AUC increased with cumulative volumes of FC up to 250 ml (AUC250 0.93 [CI95: 0.85-1.00]), followed by a plateau above 0.95 of AUC. The optimal volume was 250 ml, associated with a specificity of 0.89 [CI95: 0.78-1.00], a sensitivity of 0.92 [CI95: 0.69-1.00], and a threshold of 9.6% increase in SV. The ED50 was 156 [CI95: 136-177] ml and the ED90 was 312 [CI95: 269-352] ml. CONCLUSIONS: A volume of FC of 250 ml with a threshold of 9.6% increase in SV showed the highest accuracy in detecting fluid responsiveness in critically ill patients with shock. CLINICAL TRIAL REGISTRATION: .

The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan.

Neutrophils, eosinophils and 'classical' monocytes collectively account for about 70% of human blood leukocytes and are among the shortest-lived cells in the body. Precise regulation of the lifespan of these myeloid cells is critical to maintain protective immune responses and minimize the deleterious consequences of prolonged inflammation. However, how the lifespan of these cells is strictly controlled remains largely unknown. Here we identify a long non-coding RNA that we termed Morrbid, which tightly controls the survival of neutrophils, eosinophils and classical monocytes in response to pro-survival cytokines in mice. To control the lifespan of these cells, Morrbid regulates the transcription of the neighbouring pro-apoptotic gene, Bcl2l11 (also known as Bim), by promoting the enrichment of the PRC2 complex at the Bcl2l11 promoter to maintain this gene in a poised state. Notably, Morrbid regulates this process in cis, enabling allele-specific control of Bcl2l11 transcription. Thus, in these highly inflammatory cells, changes in Morrbid levels provide a locus-specific regulatory mechanism that allows rapid control of apoptosis in response to extracellular pro-survival signals. As MORRBID is present in humans and dysregulated in individuals with hypereosinophilic syndrome, this long non-coding RNA may represent a potential therapeutic target for inflammatory disorders characterized by aberrant short-lived myeloid cell lifespan.

Evaluation of glomerular filtration rate using iohexol plasma clearance in critically ill patients with augmented renal creatinine clearance: A single-centre retrospective study.

BACKGROUND: Augmented renal creatinine clearance (ARC) (≥130 ml min-1 1.73 m-2) is frequent in intensive care unit (ICU) patients and may impact patient outcome. OBJECTIVES: To compare glomerular filtration rate (GFR) measured with iohexol plasma clearance and creatinine clearance in critically ill patients with augmented renal clearance. DESIGN: Single-centre, retrospective study. SETTING: French University Hospital ICU from November 2016 to May 2019. PATIENTS: Adult patients with augmented renal clearance who had a measurement of iohexol plasma clearance. MAIN OUTCOME MEASURE: Agreement between 6 h creatinine clearance (6 h CrCl) and iohexol plasma clearance (GFRio). RESULTS: Twenty-nine patients were included. The median 6 h creatinine clearance was 195 [interquartile range (IQR) 162 to 251] ml min-1 1.73 m-2 and iohexol clearance was 133 [117 to 153] ml min-1 1.73 m-2. Sixteen patients (55%) had hyperfiltration (clearance >130 ml min-1 1.73 m-2) measured with iohexol clearance. Mean bias between iohexol and creatinine clearance was -80 [limits of agreement (LoA) -216 to 56 ml min-1 1.73 m-2]. For Cockcroft and Gault Modification of Diet in Renal Disease equation (MDRD), Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI) formulae, mean biases were, respectively -27 (LoA -99 to 45), -14 (LoA -86 to 59) and 15 (LoA -33 to 64) ml min-1 1.73 m-2. CONCLUSION: In the present study, we found that in patients with augmented renal creatinine clearance, half of the patients do not have hyperfiltration using iohexol clearance measurements. We observed an important bias between 6 h CrCl and GFRio with large LoA. In critically patients with ARC, 6 h CrCl does not reliably estimate GFR and 6 h CrCl nearly systematically overestimates renal function. Comparison of creatinine-based GFR estimations and GFRio show acceptable bias but wide LoA.

Impact of Early Out-of-Bed Mobilization on Functional Outcome in Patients with Aneurysmal Subarachnoid Hemorrhage: A Retrospective Cohort Study.

BACKGROUND: Effects of early mobilization are not well documented in patients with aneurysmal subarachnoid hemorrhage (aSAH). Only a few studies have investigated it through progressive mobilization protocols and suggested that it is safe and feasible. This study aimed to determine the impact of early out-of-bed mobilization (EOM) on 3-month functional outcome and cerebral vasospasm (CVS) occurrence in patients with aSAH. METHODS: A retrospective review of consecutive patients admitted to the intensive care unit with a diagnosis of aSAH was performed. EOM was defined as out-of-bed (OOB) mobilization performed before or on day 4 after aSAH onset. The primary outcome was 3-month functional independence (i.e., a modified Rankin Scale below 3) and the occurrence of CVS. RESULTS: A total of 179 patients with aSAH met the inclusion criteria. Thirty-one patients constituted the EOM group, and 148 patients were in the delayed out-of-bed mobilization group. Functional independence was more frequent in the EOM group than in the delayed out-of-bed mobilization group (n = 26 [84%] vs. n = 83 [56%], P = 0.004). In a multivariable analysis, EOM was an independent predictor of functional independence (adjusted odds ratio = 3.11; 95% confidence interval, 1.11-10.36; P < 0.05). The delay between bleeding and first OOB mobilization was also identified as an independent risk factor for the occurrence of CVS (adjusted odds ratio = 1.12; 95% confidence interval = 1.06-1.18, P < 0.001). CONCLUSIONS: EOM was independently associated with favorable functional outcome after aSAH. The delay between bleeding and OOB mobilization was an independent risk factor for reduced functional independence and CVS occurrence. Prospective randomized trials are necessary to confirm these results and improve clinical practice.

Relationship between positive end-expiratory pressure levels, central venous pressure, systemic inflammation and acute renal failure in critically ill ventilated COVID-19 patients: a monocenter retrospective study in France.

BACKGROUND: The role of positive pressure ventilation, central venous pressure (CVP) and inflammation on the occurrence of acute kidney injury (AKI) have been poorly described in mechanically ventilated patient secondary to coronavirus disease 2019 (COVID-19). METHODS: This was a monocenter retrospective cohort study of consecutive ventilated COVID-19 patients admitted in a French surgical intensive care unit between March 2020 and July 2020. Worsening renal function (WRF) was defined as development of a new AKI or a persistent AKI during the 5 days after mechanical ventilation initiation. We studied the association between WRF and ventilatory parameters including positive end-expiratory pressure (PEEP), CVP, and leukocytes count. RESULTS: Fifty-seven patients were included, 12 (21%) presented WRF. Daily PEEP, 5 days mean PEEP and daily CVP values were not associated with occurrence of WRF. 5 days mean CVP was higher in the WRF group compared to patients without WRF (median [IQR], 12 mm Hg [11-13] vs. 10 mm Hg [9-12]; P=0.03). Multivariate models with adjustment on leukocytes and Simplified Acute Physiology Score (SAPS) II confirmed the association between CVP value and risk of WRF (odd ratio, 1.97; 95% confidence interval, 1.12-4.33). Leukocytes count was also associated with occurrence of WRF in the WRF group (14 G/L [11-18]) and the no-WRF group (9 G/L [8-11]) (P=0.002). CONCLUSIONS: In mechanically ventilated COVID-19 patients, PEEP levels did not appear to influence occurrence of WRF. High CVP levels and leukocytes count are associated with risk of WRF.

Risk factors associated with COVID-19-associated pulmonary aspergillosis in ICU patients: a French multicentric retrospective cohort.

OBJECTIVES: The main objective of this study was to determine the incidence of invasive pulmonary aspergillosis (IPA) in patients with coronavirus disease 2019 (COVID-19) admitted to the intensive care unit (ICU), and to describe the patient characteristics associated with IPA occurrence and to evaluate its impact on prognosis. METHODS: We conducted a retrospective cohort study including all successive COVID-19 patients, hospitalized in four ICUs, with secondary deterioration and one or more respiratory samples sent to the mycology department. We used a strengthened IPA testing strategy including seven mycological criteria. Patients were classified as probable IPA according to the European Organization for Research and Treatment of Cancer (EORTC)/Mycoses Study Group Education and Research Consortium (MSGERC) classification if immunocompromised, and according to the recent COVID-19-associated IPA classification otherwise. RESULTS: Probable IPA was diagnosed in 21 out of the 366 COVID-19 patients (5.7%) admitted to the ICU and in the 108 patients (19.4%) who underwent respiratory sampling for deterioration. No significant differences were observed between patients with and without IPA regarding age, gender, medical history and severity on admission and during hospitalization. Treatment with azithromycin for ≥3 days was associated with the diagnosis of probable IPA (odds ratio 3.1, 95% confidence interval 1.1-8.5, p = 0.02). A trend was observed with high-dose dexamethasone and the occurrence of IPA. Overall mortality was higher in the IPA patients (15/21, 71.4% versus 32/87, 36.8%, p < 0.01). CONCLUSION: IPA is a relatively frequent complication in severe COVID-19 patients and is responsible for increased mortality. Azithromycin, known to have immunomodulatory properties, may contribute to increase COVID-19 patient's susceptibility to IPA.

Influence of systemic hemodynamics on microcirculation during sepsis.

PURPOSE: During sepsis, improvement of hemodynamic may not be related to improvement of microcirculation. The aim of this study was to investigate influence of systemic circulation on microcirculation in septic ICU patients. METHODS: This is a prospective cohort study of septic ICU patients. Microcirculation was investigated with Near infrared spectrometry (NIRS) measuring tissue oxygen saturation (StO2). StO2 desaturation (desStO2) and resaturation (resStO2) slopes were determined. Analyses were made at baseline and after fluid challenges. RESULTS: Seventy-two patients were included. One hundred and sixty measures were performed at baseline. StO2 was 77.8% [72.4-85.0] and resStO2 was 87.3%/min [57.8-141.7]. Univariate analysis showed an association between resStO2 and diastolic arterial pressure (DAP) (p = .001), and norepinephrine dose (p = .033). In multivariate linear regression, there was an association between resStO2 and DAP (ß = 1.85 (0.64 to 3.08), p = .004). Fluid challenges (n = 60) increased CO, and resStO2 (all p < .001). In multivariate analysis, variation of stroke volume was associated with variation of resStO2 (p = .004) after fluid challenge. There was no association between CVP and resStO2. CONCLUSIONS: DAP was the only independent determinant of resStO2 in septic patients. Fluid challenges may improve microcirculation. CVP did not influence resStO2.

Identification of a T follicular helper cell subset that drives anaphylactic IgE.

Cross-linking of high-affinity immunoglobulin E (IgE) results in the life-threatening allergic reaction anaphylaxis. Yet the cellular mechanisms that induce B cells to produce IgE in response to allergens remain poorly understood. T follicular helper (TFH) cells direct the affinity and isotype of antibodies produced by B cells. Although TFH cell-derived interleukin-4 (IL-4) is necessary for IgE production, it is not sufficient. We report a rare population of IL-13-producing TFH cells present in mice and humans with IgE to allergens, but not when allergen-specific IgE was absent or only low-affinity. These "TFH13" cells have an unusual cytokine profile (IL-13hiIL-4hiIL-5hiIL-21lo) and coexpress the transcription factors BCL6 and GATA3. TFH13 cells are required for production of high- but not low-affinity IgE and subsequent allergen-induced anaphylaxis. Blocking TFH13 cells may represent an alternative therapeutic target to ameliorate anaphylaxis.

Molecular tools for metalloprotease sub-proteome generation.

Molecular systems biology, the highly challenging post-genomic research area has many different facets like transcriptomics, proteomics, metabolomics, interactomics, modelling of cell cycles, etc. Among them, functional proteomics and interactomics represent exciting fields of research with high relevance towards biochemistry, medicinal chemistry, therapy, biotechnology and bioinformatics. The number of different proteins expressed by a cell under a set of certain conditions and the high dynamic range of these proteins together with different activation states require methods for sub-proteome generation on a mechanistic basis to reduce the amount of data. This can be achieved by application of tailor-made molecular tools that are based on inhibitors or, more generally, on protein ligands. Immobilised protein ligands proved to be suitable for the generation of sub-proteomes by affinity chromatography or by fishing using magnetic beads. Metalloproteases share a catalytically active metal ion in the active site. They can for example be addressed by hydroxamate type inhibitors like marimastat which are suitable for targeting active metalloproteases on a mechanistic basis aiming at the generation of an activity- and affinity-based sub-proteome. For such purposes, modified hydroxamate type inhibitors can be attached to a solid surface, e.g., chromatography material, magnetic beads, or a surface plasmon resonance sensor chip. The latter technique is a valuable tool for the optimisation of binding and elution conditions of biomolecules in affinity chromatography or on experiments using magnetic beads. Preliminary results are reported on the application of these probes in fishing experiments using magnetic beads.

Migratory CD11b+ conventional dendritic cells induce T follicular helper cell-dependent antibody responses.

T follicular helper (Tfh) cells are a subset of CD4+ T cells that promote antibody production during vaccination. Conventional dendritic cells (cDCs) efficiently prime Tfh cells; however, conclusions regarding which cDC instructs Tfh cell differentiation have differed between recent studies. We found that these discrepancies might exist because of the unusual sites used for immunization in murine models, which differentially bias which DC subsets access antigen. We used intranasal immunization as a physiologically relevant route of exposure that delivers antigen to all tissue DC subsets. Using a combination of mice in which the function of individual DC subsets is impaired and different antigen formulations, we determined that CD11b+ migratory type 2 cDCs (cDC2s) are necessary and sufficient for Tfh induction. DC-specific deletion of the guanine nucleotide exchange factor DOCK8 resulted in an isolated loss of CD11b+ cDC2, but not CD103+ cDC1, migration to lung-draining lymph nodes. Impaired cDC2 migration or development in DC-specific Dock8 or Irf4 knockout mice, respectively, led to reduced Tfh cell priming, whereas loss of CD103+ cDC1s in Batf3-/- mice did not. Loss of cDC2-dependent Tfh cell priming impaired antibody-mediated protection from live influenza virus challenge. We show that migratory cDC2s uniquely carry antigen into the subanatomic regions of the lymph node where Tfh cell priming occurs-the T-B border. This work identifies the DC subset responsible for Tfh cell-dependent antibody responses, particularly when antigen dose is limiting or is encountered at a mucosal site, which could ultimately inform the formulation and delivery of vaccines.