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1.
J Clin Microbiol ; 60(3): e0216921, 2022 Mar 16.
Article in English | MEDLINE | ID: covidwho-1799236

ABSTRACT

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.


Subject(s)
COVID-19 , Invasive Pulmonary Aspergillosis , Pulmonary Aspergillosis , Bronchoalveolar Lavage Fluid/microbiology , COVID-19/complications , COVID-19/diagnosis , Humans , Invasive Pulmonary Aspergillosis/complications , Mannans/analysis , Prognosis , Sensitivity and Specificity
2.
J Clin Microbiol ; 60(4): e0229821, 2022 04 20.
Article in English | MEDLINE | ID: covidwho-1759280

ABSTRACT

Critically ill patients with coronavirus disease 2019 (COVID-19) may develop COVID-19-associated pulmonary aspergillosis (CAPA), which impacts their chances of survival. Whether positive bronchoalveolar lavage fluid (BALF) mycological tests can be used as a survival proxy remains unknown. We conducted a post hoc analysis of a previous multicenter, multinational observational study with the aim of assessing the differential prognostic impact of BALF mycological tests, namely, positive (optical density index of ≥1.0) BALF galactomannan (GM) and positive BALF Aspergillus culture alone or in combination for critically ill patients with COVID-19. Of the 592 critically ill patients with COVID-19 enrolled in the main study, 218 were included in this post hoc analysis, as they had both test results available. CAPA was diagnosed in 56/218 patients (26%). Most cases were probable CAPA (51/56 [91%]) and fewer were proven CAPA (5/56 [9%]). In the final multivariable model adjusted for between-center heterogeneity, an independent association with 90-day mortality was observed for the combination of positive BALF GM and positive BALF Aspergillus culture in comparison with both tests negative (hazard ratio, 2.53; 95% CI confidence interval [CI], 1.28 to 5.02; P = 0.008). The other independent predictors of 90-day mortality were increasing age and active malignant disease. In conclusion, the combination of positive BALF GM and positive BALF Aspergillus culture was associated with increased 90-day mortality in critically ill patients with COVID-19. Additional study is needed to explore the possible prognostic value of other BALF markers.


Subject(s)
COVID-19 , Invasive Pulmonary Aspergillosis , Pulmonary Aspergillosis , Aspergillus , Bronchoalveolar Lavage Fluid , COVID-19/complications , Critical Illness , Galactose/analogs & derivatives , Humans , Intensive Care Units , Invasive Pulmonary Aspergillosis/complications , Invasive Pulmonary Aspergillosis/diagnosis , Mannans , Mycology , Prognosis , Sensitivity and Specificity
3.
Clin Respir J ; 16(4): 329-334, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1741359

ABSTRACT

INTRODUCTION: Coronavirus disease-2019 (COVID-19) may lead to acute respiratory distress syndrome requiring extracorporeal membrane oxygenation (ECMO). Patterns of inflammatory bronchoalveolar cells in COVID-19 patients treated with ECMO are not well described. OBJECTIVE: We aimed to describe inflammatory cell subpopulations in blood and bronchoalveolar lavages (BALs) obtained in critically ill COVID-19 patients shortly after ECMO implementation. METHODS: BAL was performed in the middle lobe in 12 consecutive ECMO-treated COVID-19 patients. Trained cytologists analyzed peripheral blood and BAL cells using flow cytometry and routine staining, respectively. Data were interpreted in relation to dexamethasone administration and weaning from ECMO and ventilator. RESULTS: High neutrophil proportions (66% to 88% of total cells) were observed in the absence of bacterial superinfection and more frequently in dexamethasone-free patients (83% [82-85] vs. 29% [8-68], P = 0.006), suggesting that viral infection could be responsible of predominantly neutrophilic lung inflammation. Successful weaning from ECMO/ventilator could not be predicted by the peripheral white blood and BAL cell pattern. CONCLUSION: High neutrophil proportions can be observed in critically ill COVID-19 patients despite the lack of microbiological evidence on BAL of bacterial superinfection. Dexamethasone was associated with lower neutrophil proportions in BAL. Our study was probably underpowered to provide BAL cell pattern helpful to predict weaning from ECMO/ventilator.


Subject(s)
COVID-19 , Extracorporeal Membrane Oxygenation , Superinfection , Bronchoalveolar Lavage Fluid , COVID-19/therapy , Critical Illness , Dexamethasone/therapeutic use , Humans , Respiration, Artificial
4.
Sci Rep ; 12(1): 4040, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735284

ABSTRACT

To provide novel data on surfactant levels in adult COVID-19 patients, we collected bronchoalveolar lavage fluid less than 72 h after intubation and used Fourier Transform Infrared Spectroscopy to measure levels of dipalmitoylphosphatidylcholine (DPPC). A total of eleven COVID-19 patients with moderate-to-severe ARDS (CARDS) and 15 healthy controls were included. CARDS patients had lower DPPC levels than healthy controls. Moreover, a principal component analysis was able to separate patient groups into distinguishable subgroups. Our findings indicate markedly impaired pulmonary surfactant levels in COVID-19 patients, justifying further studies and clinical trials of exogenous surfactant.


Subject(s)
Bronchoalveolar Lavage Fluid/chemistry , COVID-19/pathology , Pulmonary Surfactants/analysis , 1,2-Dipalmitoylphosphatidylcholine/analysis , Adult , Aged , COVID-19/virology , Case-Control Studies , Female , Humans , Male , Middle Aged , Principal Component Analysis , Pulmonary Surfactants/metabolism , SARS-CoV-2/isolation & purification , Severity of Illness Index , Spectrophotometry, Infrared/methods
5.
PLoS Pathog ; 18(2): e1010259, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1690683

ABSTRACT

At the end of 2019 Wuhan witnessed an outbreak of "atypical pneumonia" that later developed into a global pandemic. Metagenomic sequencing rapidly revealed the causative agent of this outbreak to be a novel coronavirus denoted SARS-CoV-2. To provide a snapshot of the pathogens in pneumonia-associated respiratory samples from Wuhan prior to the emergence of SARS-CoV-2, we collected bronchoalveolar lavage fluid samples from 408 patients presenting with pneumonia and acute respiratory infections at the Central Hospital of Wuhan between 2016 and 2017. Unbiased total RNA sequencing was performed to reveal their "total infectome", including viruses, bacteria and fungi. We identified 35 pathogen species, comprising 13 RNA viruses, 3 DNA viruses, 16 bacteria and 3 fungi, often at high abundance and including multiple co-infections (13.5%). SARS-CoV-2 was not present. These data depict a stable core infectome comprising common respiratory pathogens such as rhinoviruses and influenza viruses, an atypical respiratory virus (EV-D68), and a single case of a sporadic zoonotic pathogen-Chlamydia psittaci. Samples from patients experiencing respiratory disease on average had higher pathogen abundance than healthy controls. Phylogenetic analyses of individual pathogens revealed multiple origins and global transmission histories, highlighting the connectedness of the Wuhan population. This study provides a comprehensive overview of the pathogens associated with acute respiratory infections and pneumonia, which were more diverse and complex than obtained using targeted PCR or qPCR approaches. These data also suggest that SARS-CoV-2 or closely related viruses were absent from Wuhan in 2016-2017.


Subject(s)
COVID-19/epidemiology , Disease Outbreaks , Pneumonia/epidemiology , Respiratory Tract Infections/epidemiology , SARS-CoV-2/isolation & purification , Acute Disease , Adolescent , Adult , Aged , Aged, 80 and over , Bronchoalveolar Lavage Fluid/microbiology , COVID-19/virology , China/epidemiology , Cohort Studies , Female , Gene Expression Profiling , Humans , Male , Metagenomics , Middle Aged , Phylogeny , Pneumonia/microbiology , Respiratory Tract Infections/microbiology , Young Adult
6.
Mycoses ; 65(4): 411-418, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1685386

ABSTRACT

BACKGROUND: The diagnosis of invasive pulmonary aspergillosis (IPA) in intensive care unit (ICU) patients is challenging, and the role of Aspergillus-PCR in bronchoalveolar lavage (BAL) is unknown. OBJECTIVES: This study evaluated diagnostic accuracy of Aspergillus-PCR in BAL in IPA in three different cohorts: ICU-admitted patients with COVID-19, ICU-admitted patients without COVID-19 and immunocompromised patients. METHODS: All stored available BAL samples collected from three patient groups were tested with Aspergillus-PCR (AsperGenius® ). IPA was diagnosed according to appropriate criteria for each patient group. RESULTS: We included 111 BAL samples from 101 patients: 52 (51%) patients admitted to ICU for COVID-19, 24 (24%) admitted to ICU for other reasons and 25 (25%) immunocompromised. There were 31 cases of IPA (28%). Aspergillus-PCR sensitivity was 64% (95% CI 47-79) and specificity 99% (95% CI 93-100). Aspergillus-PCR sensitivity was 40% (95%CI 19-64) in ICU COVID-19, 67% (95% CI 21-93) in non-COVID-19 ICU patients and 92% (95%CI 67-98) in the immunocompromised. The concordance between positive BAL-GM and BAL-PCR in patients with and without IPA was significantly lower in ICU patients (32%; 43% in COVID-19, 18% in non-COVID-19) than in the immunocompromised (92%), p < .001. CONCLUSIONS: Aspergillus-PCR in BAL improves the diagnostic accuracy of BAL-GM in ICU patients.


Subject(s)
COVID-19 , Invasive Pulmonary Aspergillosis , Aspergillus/genetics , Bronchoalveolar Lavage , Bronchoalveolar Lavage Fluid , COVID-19/diagnosis , Critical Illness , Galactose , Humans , Invasive Pulmonary Aspergillosis/diagnosis , Mannans/analysis , Polymerase Chain Reaction , Sensitivity and Specificity
7.
Sci Rep ; 12(1): 1626, 2022 01 31.
Article in English | MEDLINE | ID: covidwho-1661980

ABSTRACT

The ongoing COVID-19 pandemic is one of the biggest health challenges of recent decades. Among the causes of mortality triggered by SARS-CoV-2 infection, the development of an inflammatory "cytokine storm" (CS) plays a determinant role. Here, we used transcriptomic data from the bronchoalveolar lavage fluid (BALF) of COVID-19 patients undergoing a CS to obtain gene-signatures associated to this pathology. Using these signatures, we interrogated the Connectivity Map (CMap) dataset that contains the effects of over 5000 small molecules on the transcriptome of human cell lines, and looked for molecules which effects on transcription mimic or oppose those of the CS. As expected, molecules that potentiate immune responses such as PKC activators are predicted to worsen the CS. In addition, we identified the negative regulation of female hormones among pathways potentially aggravating the CS, which helps to understand the gender-related differences in COVID-19 mortality. Regarding drugs potentially counteracting the CS, we identified glucocorticoids as a top hit, which validates our approach as this is the primary treatment for this pathology. Interestingly, our analysis also reveals a potential effect of MEK inhibitors in reverting the COVID-19 CS, which is supported by in vitro data that confirms the anti-inflammatory properties of these compounds.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , COVID-19/complications , COVID-19/drug therapy , Computer Simulation , Cytokine Release Syndrome/etiology , Cytokine Release Syndrome/prevention & control , Glucocorticoids/therapeutic use , Pandemics , Protein Kinase Inhibitors/therapeutic use , SARS-CoV-2 , Anti-Inflammatory Agents/pharmacology , Bronchoalveolar Lavage Fluid/virology , COVID-19/blood , COVID-19/epidemiology , Cytokine Release Syndrome/mortality , Cytokines/blood , Female , Gene Expression Profiling/methods , Glucocorticoids/pharmacology , Humans , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Signaling System/drug effects , Male , Protein Kinase Inhibitors/pharmacology , Sex Factors , Transcriptome/genetics
8.
J Clin Microbiol ; 60(1): e0168921, 2022 01 19.
Article in English | MEDLINE | ID: covidwho-1630019

ABSTRACT

This multicenter study evaluated the IMMY Aspergillus Galactomannan Lateral Flow Assay (LFA) with automated reader for diagnosis of pulmonary aspergillosis in patients with COVID-19-associated acute respiratory failure (ARF) requiring intensive care unit (ICU) admission between 03/2020 and 04/2021. A total of 196 respiratory samples and 148 serum samples (n = 344) from 238 patients were retrospectively included, with a maximum of one of each sample type per patient. Cases were retrospectively classified for COVID-19-associated pulmonary aspergillosis (CAPA) status following the 2020 consensus criteria, with the exclusion of LFA results as a mycological criterion. At the 1.0 cutoff, sensitivity of LFA for CAPA (proven/probable/possible) was 52%, 80% and 81%, and specificity was 98%, 88% and 67%, for bronchoalveolar lavage fluid (BALF), nondirected bronchoalveolar lavage (NBL), and tracheal aspiration (TA), respectively. At the 0.5 manufacturer's cutoff, sensitivity was 72%, 90% and 100%, and specificity was 79%, 83% and 44%, for BALF, NBL and TA, respectively. When combining all respiratory samples, the receiver operating characteristic (ROC) area under the curve (AUC) was 0.823, versus 0.754, 0.890 and 0.814 for BALF, NBL and TA, respectively. Sensitivity and specificity of serum LFA were 20% and 93%, respectively, at the 0.5 ODI cutoff. Overall, the Aspergillus Galactomannan LFA showed good performances for CAPA diagnosis, when used from respiratory samples at the 1.0 cutoff, while sensitivity from serum was limited, linked to weak invasiveness during CAPA. As some false-positive results can occur, isolated results slightly above the recommended cutoff should lead to further mycological investigations.


Subject(s)
COVID-19 , Invasive Pulmonary Aspergillosis , Pulmonary Aspergillosis , Aspergillus , Bronchoalveolar Lavage Fluid , Humans , Invasive Pulmonary Aspergillosis/diagnosis , Mannans , Pulmonary Aspergillosis/diagnosis , Retrospective Studies , SARS-CoV-2 , Sensitivity and Specificity
9.
J Immunol ; 208(4): 979-990, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1631932

ABSTRACT

Calprotectin is released by activated neutrophils along with myeloperoxidase (MPO) and proteases. It plays numerous roles in inflammation and infection, and is used as an inflammatory biomarker. However, calprotectin is readily oxidized by MPO-derived hypohalous acids to form covalent dimers of its S100A8 and S100A9 subunits. The dimers are susceptible to degradation by proteases. We show that detection of human calprotectin by ELISA declines markedly because of its oxidation by hypochlorous acid and subsequent degradation. Also, proteolysis liberates specific peptides from oxidized calprotectin that is present at inflammatory sites. We identified six calprotectin-derived peptides by mass spectrometry and detected them in the bronchoalveolar lavage fluid of children with cystic fibrosis (CF). We assessed the peptides as biomarkers of neutrophilic inflammation and infection. The content of the calprotectin peptide ILVI was related to calprotectin (r = 0.72, p = 0.01, n = 10). Four of the peptides were correlated with the concentration of MPO (r > 0.7, p ≤ 0.01, n = 21), while three were higher (p < 0.05) in neutrophil elastase-positive (n = 14) than -negative samples (n = 7). Also, five of the peptides were higher (p < 0.05) in the bronchoalveolar lavage fluid from children with CF with infections (n = 21) than from non-CF children without infections (n = 6). The specific peptides liberated from calprotectin will signal uncontrolled activity of proteases and MPO during inflammation. They may prove useful in tracking inflammation in respiratory diseases dominated by neutrophils, including coronavirus disease 2019.


Subject(s)
Bronchoalveolar Lavage Fluid/immunology , Cystic Fibrosis/immunology , Inflammation/immunology , Leukocyte L1 Antigen Complex/metabolism , Neutrophils/immunology , Peptides/metabolism , Respiratory System/metabolism , Child , Child, Preschool , Cystic Fibrosis/diagnosis , Female , Humans , Inflammation/diagnosis , Leukocyte L1 Antigen Complex/genetics , Leukocyte L1 Antigen Complex/immunology , Male , Neutrophil Activation , Oxidation-Reduction , Peptides/genetics , Peptides/immunology , Proteolysis
10.
Life Sci Alliance ; 5(4)2022 04.
Article in English | MEDLINE | ID: covidwho-1637974

ABSTRACT

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, we used the rhesus macaque model of SARS-CoV-2 infection. Eight older and eight younger macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at predefined time points in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication were limited. Transcriptional signatures of inflammation-associated genes in bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both cohorts. However, age-specific divergence of immune responses emerged during the post-acute phase. Older animals exhibited sustained local inflammatory innate responses, whereas local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, and persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling suggests that age may delay or impair antiviral cellular immune responses and delay efficient return to immune homeostasis.


Subject(s)
Aging/immunology , COVID-19/immunology , COVID-19/veterinary , SARS-CoV-2/immunology , Acute Disease , Animals , Antibody Formation/immunology , Bronchoalveolar Lavage Fluid , COVID-19/complications , COVID-19/genetics , Cytokines/blood , Gene Expression Regulation , Gene Regulatory Networks , Genomics , Immunity, Cellular/genetics , Immunomodulation , Inflammation/complications , Inflammation/pathology , Lung/immunology , Lung/pathology , Lung/virology , Lymphoid Tissue/pathology , Macaca mulatta/immunology , Macaca mulatta/virology , Models, Biological , Single-Cell Analysis , T-Lymphocytes/immunology , Transcription, Genetic
11.
Int J Mol Sci ; 23(2)2022 Jan 17.
Article in English | MEDLINE | ID: covidwho-1624942

ABSTRACT

Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019, and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes into close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic, innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb-host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional, with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay shows that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows a lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status, determined by factors such as age, might play an important role in determining infection outcome.


Subject(s)
Bacterial Capsules/genetics , Bacterial Capsules/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Adolescent , Adult , Age Factors , Aged , Bronchoalveolar Lavage Fluid , Cellular Structures , Female , Gene Expression Regulation, Bacterial , Humans , Lipopolysaccharides/biosynthesis , Lipopolysaccharides/genetics , Male , Mannosides/biosynthesis , Mannosides/genetics , Mannosyltransferases/biosynthesis , Mannosyltransferases/genetics , Middle Aged , Young Adult
12.
Inflamm Res ; 71(2): 183-185, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1611373

ABSTRACT

Growth Hormone-Releasing Hormone (GHRH) is a neuropeptide regulating the release of Growth Hormone (GH) from the anterior pituitary gland, and acts as a growth factor in a diverse variety of tissues. GHRH antagonists (GHRHAnt) have been developed to counteract those events, and the beneficial effects of those peptides toward homeostasis have been associated with anti-inflammatory activities. Our lab is interested in delineating the mechanisms governing endothelial barrier function. Our goal is to establish new grounds on the development of efficient countermeasures against Acute Respiratory Distress Syndrome (ARDS), which has been associated with thousands of deaths worldwide due to COVID-19. Herein we demonstrate in vivo that GHRHAnt suppresses LPS-induced increase in bronchoalveolar lavage fluid (BALF) protein concentration, thus protecting the lungs against edema and inflammation.


Subject(s)
Bronchoalveolar Lavage Fluid/chemistry , Gonadotropin-Releasing Hormone/antagonists & inhibitors , Lipopolysaccharides , Animals , COVID-19/complications , Growth Hormone-Releasing Hormone , Inflammation/etiology , Inflammation/prevention & control , Male , Mice , Mice, Inbred C57BL , Proteins/chemistry , Pulmonary Edema/etiology , Pulmonary Edema/prevention & control , Reactive Oxygen Species , Respiratory Distress Syndrome/drug therapy , Respiratory Distress Syndrome/etiology , SARS-CoV-2
13.
Sci Rep ; 11(1): 24432, 2021 12 24.
Article in English | MEDLINE | ID: covidwho-1585772

ABSTRACT

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1ß and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1ß, levels of IL-1ß and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.


Subject(s)
Coronavirus Envelope Proteins/metabolism , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , SARS-CoV-2/metabolism , Animals , Bronchoalveolar Lavage Fluid/chemistry , COVID-19/pathology , COVID-19/virology , Coronavirus Envelope Proteins/genetics , Down-Regulation/drug effects , Endoplasmic Reticulum Stress , Humans , Inflammasomes/drug effects , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Janus Kinases/genetics , Janus Kinases/metabolism , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/deficiency , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Poly I-C/pharmacology , RNA, Viral/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/isolation & purification
14.
Adv Sci (Weinh) ; 8(23): e2102593, 2021 12.
Article in English | MEDLINE | ID: covidwho-1559092

ABSTRACT

Fast and accurate identification of microbial pathogens is critical for the proper treatment of infections. Traditional culture-based diagnosis in clinics is increasingly supplemented by metagenomic next-generation-sequencing (mNGS). Here, RNA/cDNA-targeted sequencing (meta-transcriptomics using NGS (mtNGS)) is established to reduce the host nucleotide percentage in clinic samples and by combining with Oxford Nanopore Technology (ONT) platforms (meta-transcriptomics using third-generation sequencing, mtTGS) to improve the sequencing time. It shows that mtNGS improves the ratio of microbial reads, facilitates bacterial identification using multiple-strategies, and discovers fungi, viruses, and antibiotic resistance genes, and displaying agreement with clinical findings. Furthermore, longer reads in mtTGS lead to additional improvement in pathogen identification and also accelerate the clinical diagnosis. Additionally, primary tests utilizing direct-RNA sequencing and targeted sequencing of ONT show that ONT displays important potential but must be further developed. This study presents the potential of RNA-targeted pathogen identification in clinical samples, especially when combined with the newest developments in ONT.


Subject(s)
Bronchoalveolar Lavage Fluid/microbiology , High-Throughput Nucleotide Sequencing/methods , Infections/genetics , Metagenomics/methods , RNA/genetics , Sequence Analysis, RNA/methods , Aged , Bronchoalveolar Lavage/methods , Female , Humans , Male , Metagenome/genetics , Middle Aged
15.
Viruses ; 13(11)2021 11 03.
Article in English | MEDLINE | ID: covidwho-1542790

ABSTRACT

The detailed characterization of human γδ T lymphocyte differentiation at the single-cell transcriptomic (scRNAseq) level in tumors and patients with coronavirus disease 2019 (COVID-19) requires both a reference differentiation trajectory of γδ T cells and a robust mapping method for additional γδ T lymphocytes. Here, we incepted such a method to characterize thousands of γδ T lymphocytes from (n = 95) patients with cancer or adult and pediatric COVID-19 disease. We found that cancer patients with human papillomavirus-positive head and neck squamous cell carcinoma and Epstein-Barr virus-positive Hodgkin's lymphoma have γδ tumor-infiltrating T lymphocytes that are more prone to recirculate from the tumor and avoid exhaustion. In COVID-19, both TCRVγ9 and TCRVγnon9 subsets of γδ T lymphocytes relocalize from peripheral blood mononuclear cells (PBMC) to the infected lung tissue, where their advanced differentiation, tissue residency, and exhaustion reflect T cell activation. Although severe COVID-19 disease increases both recruitment and exhaustion of γδ T lymphocytes in infected lung lesions but not blood, the anti-IL6R therapy with Tocilizumab promotes γδ T lymphocyte differentiation in patients with COVID-19. PBMC from pediatric patients with acute COVID-19 disease display similar γδ T cell lymphopenia to that seen in adult patients. However, blood γδ T cells from children with the COVID-19-related multisystem inflammatory syndrome are not lymphodepleted, but they are differentiated as in healthy PBMC. These findings suggest that some virus-induced memory γδ T lymphocytes durably persist in the blood of adults and could subsequently infiltrate and recirculate in tumors.


Subject(s)
COVID-19/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Neoplasms/immunology , RNA-Seq , Receptors, Antigen, T-Cell, gamma-delta/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Adult , Bronchoalveolar Lavage Fluid/immunology , COVID-19/complications , Cell Differentiation , Child , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/virology , Herpesvirus 4, Human/isolation & purification , Hodgkin Disease/immunology , Hodgkin Disease/virology , Humans , Lung/immunology , Lymphocyte Activation , Lymphocyte Count , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/physiology , Neoplasms/virology , Papillomaviridae/isolation & purification , Severity of Illness Index , Single-Cell Analysis , Systemic Inflammatory Response Syndrome/immunology , T-Lymphocyte Subsets/physiology
16.
Clin Sci (Lond) ; 135(22): 2559-2573, 2021 11 26.
Article in English | MEDLINE | ID: covidwho-1541262

ABSTRACT

Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor ß subunit (GMCSFRß) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRß KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.


Subject(s)
Arthritis, Rheumatoid/metabolism , COVID-19/therapy , Pulmonary Alveolar Proteinosis/immunology , Pulmonary Surfactants/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Arthritis, Rheumatoid/therapy , Autoantibodies/chemistry , Bronchoalveolar Lavage Fluid , COVID-19/immunology , Choline/analogs & derivatives , Female , Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Inflammation , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Pulmonary Alveolar Proteinosis/genetics , SARS-CoV-2/immunology , Surface-Active Agents
17.
J Infect ; 83(5): e6-e9, 2021 11.
Article in English | MEDLINE | ID: covidwho-1527752

ABSTRACT

PURPOSE: To describe the relationship between the severity of lung damage and cytokine levels in sputum, bronchoalveolar lavage fluid (BALF), serum. METHOD: Eight severe patients infected with coronavirus disease 2019 (COVID-19) were admitted and their cytokines and chest computed tomography (CT) were analyzed. RESULTS: Compared with in serum, IL-6 and TNF-α in sputum and in BALF show more directly reflect the severity of COVID-19 critical patients. The gradient ratio of IL-6 levels may predict the prognosis of severe patients. CONCLUSION: Cytokine levels in the sputum may be more helpful for indicating lung damage. Local intervention through the respiratory tract is expected to benefit patients with severe COVID-19.


Subject(s)
COVID-19 , Cytokines , Sputum/chemistry , Bronchoalveolar Lavage Fluid , COVID-19/diagnosis , COVID-19/pathology , Cytokines/analysis , Humans , Lung/pathology , Lung/virology , Prognosis
18.
JCI Insight ; 7(1)2022 01 11.
Article in English | MEDLINE | ID: covidwho-1523122

ABSTRACT

Neutrophils are recognized as important circulating effector cells in the pathophysiology of severe coronavirus disease 2019 (COVID-19). However, their role within the inflamed lungs is incompletely understood. Here, we collected bronchoalveolar lavage (BAL) fluids and parallel blood samples of critically ill COVID-19 patients requiring invasive mechanical ventilation and compared BAL fluid parameters with those of mechanically ventilated patients with influenza, as a non-COVID-19 viral pneumonia cohort. Compared with those of patients with influenza, BAL fluids of patients with COVID-19 contained increased numbers of hyperactivated degranulating neutrophils and elevated concentrations of the cytokines IL-1ß, IL-1RA, IL-17A, TNF-α, and G-CSF; the chemokines CCL7, CXCL1, CXCL8, CXCL11, and CXCL12α; and the protease inhibitors elafin, secretory leukocyte protease inhibitor, and tissue inhibitor of metalloproteinases 1. In contrast, α-1 antitrypsin levels and net proteolytic activity were comparable in COVID-19 and influenza BAL fluids. During antibiotic treatment for bacterial coinfections, increased BAL fluid levels of several activating and chemotactic factors for monocytes, lymphocytes, and NK cells were detected in patients with COVID-19 whereas concentrations tended to decrease in patients with influenza, highlighting the persistent immunological response to coinfections in COVID-19. Finally, the high proteolytic activity in COVID-19 lungs suggests considering protease inhibitors as a treatment option.


Subject(s)
Bacterial Infections , Bronchoalveolar Lavage Fluid , COVID-19 , Coinfection , Influenza, Human , Adult , Aged , Bacterial Infections/complications , Bacterial Infections/immunology , Bacterial Infections/metabolism , Bacterial Infections/pathology , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , COVID-19/complications , COVID-19/diagnosis , COVID-19/immunology , COVID-19/pathology , Coinfection/immunology , Coinfection/metabolism , Coinfection/pathology , Cytokines/analysis , Female , Humans , Inflammation , Influenza, Human/complications , Influenza, Human/diagnosis , Influenza, Human/immunology , Influenza, Human/pathology , Lung/immunology , Lung/metabolism , Lung/pathology , Male , Middle Aged
19.
Clin Sci (Lond) ; 135(22): 2559-2573, 2021 11 26.
Article in English | MEDLINE | ID: covidwho-1517650

ABSTRACT

Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor ß subunit (GMCSFRß) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRß KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.


Subject(s)
Arthritis, Rheumatoid/metabolism , COVID-19/therapy , Pulmonary Alveolar Proteinosis/immunology , Pulmonary Surfactants/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Arthritis, Rheumatoid/therapy , Autoantibodies/chemistry , Bronchoalveolar Lavage Fluid , COVID-19/immunology , Choline/analogs & derivatives , Female , Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Inflammation , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Pulmonary Alveolar Proteinosis/genetics , SARS-CoV-2/immunology , Surface-Active Agents
20.
Nat Immunol ; 23(1): 62-74, 2022 01.
Article in English | MEDLINE | ID: covidwho-1514418

ABSTRACT

The molecular mechanisms governing orderly shutdown and retraction of CD4+ type 1 helper T (TH1) cell responses remain poorly understood. Here we show that complement triggers contraction of TH1 responses by inducing intrinsic expression of the vitamin D (VitD) receptor and the VitD-activating enzyme CYP27B1, permitting T cells to both activate and respond to VitD. VitD then initiated the transition from pro-inflammatory interferon-γ+ TH1 cells to suppressive interleukin-10+ cells. This process was primed by dynamic changes in the epigenetic landscape of CD4+ T cells, generating super-enhancers and recruiting several transcription factors, notably c-JUN, STAT3 and BACH2, which together with VitD receptor shaped the transcriptional response to VitD. Accordingly, VitD did not induce interleukin-10 expression in cells with dysfunctional BACH2 or STAT3. Bronchoalveolar lavage fluid CD4+ T cells of patients with COVID-19 were TH1-skewed and showed de-repression of genes downregulated by VitD, from either lack of substrate (VitD deficiency) and/or abnormal regulation of this system.


Subject(s)
Interferon-gamma/immunology , Interleukin-10/immunology , SARS-CoV-2/immunology , Th1 Cells/immunology , Vitamin D/metabolism , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , Bronchoalveolar Lavage Fluid/cytology , COVID-19/immunology , COVID-19/pathology , Complement C3a/immunology , Complement C3b/immunology , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Lymphocyte Activation/immunology , Receptors, Calcitriol/metabolism , Respiratory Distress Syndrome/immunology , Respiratory Distress Syndrome/pathology , Respiratory Distress Syndrome/virology , STAT3 Transcription Factor/metabolism , Signal Transduction/immunology , Transcription, Genetic/genetics
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