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1.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-308709

ABSTRACT

Background: The use of cytokine-blocking agents has been proposed to modulate the inflammatory response in patients with COVID19. Tocilizumab and Anakinra were included in the local protocol as an optional treatment in critically ill patients with acute respiratory distress syndrome (ARDS) by SARS-CoV2 infection. This cohort study evaluated the effects of therapy with cytokine blocking agents on in-hospital mortality in COVID19 patients requiring mechanical ventilation and admitted to intensive care unit. Methods The association between therapy with Tocilizumab or Anakinra and in-hospital mortality was assessed in consecutive adult COVID19 patients admitted to our ICU with moderate to severe ARDS. The association was evaluated by comparing patients who receive to those who did not receive Tocilizumab or Anakinra and by using different multivariable Cox models adjusted for variables related to poor outcome, for the propensity to be treated with Tocilizumab or Anakinra and after patient matching. Results Sixty-six patients who received immunotherapy (49 Tocilizumab, 17 Anakinra) and 28 patients who did not receive immunotherapy were included. The in-hospital crude mortality was 30,3% in treated patients and 50% in non-treated (OR 0,77, 95% CI 0,56-1,05, p=0,069). The adjusted Cox model showed an association between therapy with immunotherapy and in-hospital mortality (HR 0,35, 95% CI 0,16-0,77, p=0,009). This protective effect was further confirmed in the analysis adjusted for propensity score, in the propensity-matched cohort and in the cohort of patients with invasive mechanical ventilation within 2 hours after ICU admission. Conclusions Although important limitations, our study showed that cytokine-blocking agents seem to be safe and to improve survival in COVID-19 patients admitted to ICU with ARDS and the need of mechanical ventilation.

2.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-306884

ABSTRACT

We have deeply investigated T cell compartment, plasma cytokines and cells producing cytokines in patients affected by Covid-19. At admission, patients were lymphopenic;in all of them SARS-CoV-2 was detected in a nasopharyngeal swab specimen by real-time RT-PCR, and pneumonia was subsequently confirmed by X-rays.Detailed 18-parameter flow cytometry was performed in 21 patients and 13 controls. Coupling polychromatic cytometry with unsupervised data analysis, we found that patients show an increased amount of CD4+ T lymphocytes that were activated, exhausted, stem memory or Treg. Similar results concerning activation and exhaustion were found in the CD8+ T cell compartment, within which the differences were even greater.Measuring plasma level of 31 cytokines linked to inflammation revealed that Covid-19 showed a dramatic increase of several molecules, such as TH1 and TH2 cytokines, chemokines, galectins, pro- and anti-inflammatory mediators, confirming the importance of a massive immune activation causing the cytokine storm. Then, intracellular staining detecting the simultaneous production of different cytokines after a para-physiologic stimulus given by anti-CD3/CD28 mAbs revealed not only a high capacity to produce a variety of molecules, including TNF-a, IFN-g and IL-2, but also a significant skewing of CD4+ T cells towards the TH17 phenotype.A therapeutic approach now exists based on the administration of drugs that block IL-6 pathway, and is now consistently improving the course of the disease. IL-17 is crucial in recruiting and activating neutrophils, cells that can migrate to the lung and are heavily involved in the pathogenesis of Covid-19. We show here that a significant skewing of activated T cells towards TH17 functional phenotype exists in Covid-19 patients. Thus, we suggest that blocking IL-17 pathway by already available biological drugs that are used to treat different pathologies could be a novel, additional strategy to improve the health of patients infected by SARS-CoV-2.

3.
Eur J Immunol ; 52(3): 484-502, 2022 03.
Article in English | MEDLINE | ID: covidwho-1555185

ABSTRACT

To better understand the mechanisms at the basis of neutrophil functions during SARS-CoV-2, we studied patients with severe COVID-19 pneumonia. They had high blood proportion of degranulated neutrophils and elevated plasma levels of myeloperoxidase (MPO), elastase, and MPO-DNA complexes, which are typical markers of neutrophil extracellular traps (NET). Their neutrophils display dysfunctional mitochondria, defective oxidative burst, increased glycolysis, glycogen accumulation in the cytoplasm, and increase glycogenolysis. Hypoxia-inducible factor 1α (ΗΙF-1α) is stabilized in such cells, and it controls the level of glycogen phosphorylase L (PYGL), a key enzyme in glycogenolysis. Inhibiting PYGL abolishes the ability of neutrophils to produce NET. Patients displayed significant increases of plasma levels of molecules involved in the regulation of neutrophils' function including CCL2, CXCL10, CCL20, IL-18, IL-3, IL-6, G-CSF, GM-CSF, IFN-γ. Our data suggest that metabolic remodelling is vital for the formation of NET and for boosting neutrophil inflammatory response, thus, suggesting that modulating ΗΙF-1α or PYGL could represent a novel approach for innovative therapies.


Subject(s)
COVID-19/immunology , COVID-19/metabolism , Neutrophils/immunology , Neutrophils/metabolism , SARS-CoV-2 , Adult , Aged , Aged, 80 and over , COVID-19/blood , Case-Control Studies , Cohort Studies , Cytokines/blood , Extracellular Traps/immunology , Extracellular Traps/metabolism , Female , Glycogen Phosphorylase, Liver Form/blood , Granulocytes/immunology , Granulocytes/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/blood , Male , Metabolic Networks and Pathways/genetics , Metabolic Networks and Pathways/immunology , Middle Aged , Neutrophil Activation , Peroxidase/blood , Respiratory Burst , Severity of Illness Index
4.
Eur J Immunol ; 50(9): 1283-1294, 2020 09.
Article in English | MEDLINE | ID: covidwho-670172

ABSTRACT

Studies on the interactions between SARS-CoV-2 and humoral immunity are fundamental to elaborate effective therapies including vaccines. We used polychromatic flow cytometry, coupled with unsupervised data analysis and principal component analysis (PCA), to interrogate B cells in untreated patients with COVID-19 pneumonia. COVID-19 patients displayed normal plasma levels of the main immunoglobulin classes, of antibodies against common antigens or against antigens present in common vaccines. However, we found a decreased number of total and naïve B cells, along with decreased percentages and numbers of memory switched and unswitched B cells. On the contrary, IgM+ and IgM- plasmablasts were significantly increased. In vitro cell activation revealed that B lymphocytes showed a normal proliferation index and number of dividing cells per cycle. PCA indicated that B-cell number, naive and memory B cells but not plasmablasts clustered with patients who were discharged, while plasma IgM level, C-reactive protein, D-dimer, and SOFA score with those who died. In patients with pneumonia, the derangement of the B-cell compartment could be one of the causes of the immunological failure to control SARS-Cov2, have a relevant influence on several pathways, organs and systems, and must be considered to develop vaccine strategies.


Subject(s)
Antibodies, Viral/blood , B-Lymphocytes/immunology , Betacoronavirus/pathogenicity , Coronavirus Infections/immunology , Immunoglobulin Isotypes/blood , Lung/immunology , Pneumonia, Viral/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Viral/classification , B-Lymphocytes/virology , Betacoronavirus/immunology , C-Reactive Protein/immunology , COVID-19 , Case-Control Studies , Cell Proliferation , Coronavirus Infections/mortality , Coronavirus Infections/pathology , Coronavirus Infections/virology , Cross-Sectional Studies , Cytokines/genetics , Cytokines/immunology , Female , Fibrin Fibrinogen Degradation Products/immunology , Humans , Immunity, Humoral , Immunologic Memory , Lung/pathology , Lung/virology , Lymphocyte Activation , Lymphocyte Count , Male , Middle Aged , Organ Dysfunction Scores , Pandemics , Pneumonia, Viral/mortality , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Primary Cell Culture , SARS-CoV-2 , Severity of Illness Index , Survival Analysis
5.
Nat Commun ; 11(1): 3434, 2020 07 06.
Article in English | MEDLINE | ID: covidwho-631255

ABSTRACT

The immune system of patients infected by SARS-CoV-2 is severely impaired. Detailed investigation of T cells and cytokine production in patients affected by COVID-19 pneumonia are urgently required. Here we show that, compared with healthy controls, COVID-19 patients' T cell compartment displays several alterations involving naïve, central memory, effector memory and terminally differentiated cells, as well as regulatory T cells and PD1+CD57+ exhausted T cells. Significant alterations exist also in several lineage-specifying transcription factors and chemokine receptors. Terminally differentiated T cells from patients proliferate less than those from healthy controls, whereas their mitochondria functionality is similar in CD4+ T cells from both groups. Patients display significant increases of proinflammatory or anti-inflammatory cytokines, including T helper type-1 and type-2 cytokines, chemokines and galectins; their lymphocytes produce more tumor necrosis factor (TNF), interferon-γ, interleukin (IL)-2 and IL-17, with the last observation implying that blocking IL-17 could provide a novel therapeutic strategy for COVID-19.


Subject(s)
Betacoronavirus/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , T-Lymphocyte Subsets/immunology , Adult , Aged , Aged, 80 and over , Biomarkers/metabolism , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , COVID-19 , Cellular Senescence , Coronavirus Infections/blood , Coronavirus Infections/pathology , Cytokine Release Syndrome , Cytokines/immunology , Cytokines/metabolism , Female , Humans , Immunologic Memory , Italy/epidemiology , Lymphocyte Activation , Lymphocyte Count , Male , Middle Aged , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/pathology , SARS-CoV-2 , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/pathology , Th17 Cells/immunology , Th17 Cells/metabolism , Th17 Cells/pathology
6.
Cytometry A ; 97(7): 668-673, 2020 07.
Article in English | MEDLINE | ID: covidwho-47563

ABSTRACT

The pandemic caused by severe acute respiratory syndrome coronavirus 2 heavily involves all those working in a laboratory. Samples from known infected patients or donors who are considered healthy can arrive, and a colleague might be asymptomatic but able to transmit the virus. Working in a clinical laboratory is posing several safety challenges. Few years ago, International Society for Advancement of Cytometry published guidelines to safely analyze and sort human samples that were revised in these days. We describe the procedures that we have been following since the first patient appeared in Italy, which have only slightly modified our standard one, being all human samples associated with risks. © 2020 International Society for Advancement of Cytometry.


Subject(s)
Betacoronavirus , Coronavirus Infections/blood , Medical Laboratory Personnel , Occupational Health , Pneumonia, Viral/blood , Specimen Handling/methods , COVID-19 , Cytokine Release Syndrome/blood , Flow Cytometry/methods , Humans , Italy , Masks , Occupational Exposure/prevention & control , Pandemics , Protective Clothing , SARS-CoV-2
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