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1.
Immunogenetics ; 73(6): 459-477, 2021 12.
Article in English | MEDLINE | ID: covidwho-1427234

ABSTRACT

Since 2019, the world was involved with SARS-CoV-2 and consequently, with the announcement by the World Health Organization that COVID-19 was a pandemic, scientific were an effort to obtain the best approach to combat this global dilemma. The best way to prevent the pandemic from spreading further is to use a vaccine against COVID-19. Here, we report the design of a recombinant multi-epitope vaccine against the four proteins spike or crown (S), membrane (M), nucleocapsid (N), and envelope (E) of SARS-CoV-2 using immunoinformatics tools. We evaluated the most antigenic epitopes that bind to HLA class 1 subtypes, along with HLA class 2, as well as B cell epitopes. Beta-defensin 3 and PADRE sequence were used as adjuvants in the structure of the vaccine. KK, GPGPG, and AAY linkers were used to fuse the selected epitopes. The nucleotide sequence was cloned into pET26b(+) vector using restriction enzymes XhoI and NdeI, and HisTag sequence was considered in the C-terminal of the construct. The results showed that the proposed candidate vaccine is a 70.87 kDa protein with high antigenicity and immunogenicity as well as non-allergenic and non-toxic. A total of 95% of the selected epitopes have conservancy with similar sequences. Molecular docking showed a strong binding between the vaccine structure and tool-like receptor (TLR) 7/8. The docking, molecular dynamics, and MM/PBSA analysis showed that the vaccine established a stable interaction with both structures of TLR7 and TLR8. Simulation of immune stimulation by this vaccine showed that it evokes immune responses related to humoral and cellular immunity.


Subject(s)
COVID-19 Vaccines/immunology , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , SARS-CoV-2/immunology , Amino Acid Sequence , Base Sequence , COVID-19/prevention & control , COVID-19 Vaccines/genetics , COVID-19 Vaccines/metabolism , Computational Biology , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/genetics , HLA Antigens/immunology , Humans , Immunogenicity, Vaccine , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Weight , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Toll-Like Receptor 7/chemistry , Toll-Like Receptor 8/chemistry , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccines, Subunit/metabolism , Vaccinology , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/immunology
2.
J Gen Virol ; 102(8)2021 08.
Article in English | MEDLINE | ID: covidwho-1368372

ABSTRACT

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein's surface. Here we used IBV propagated in embryonated hens' eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.


Subject(s)
Coronavirus/physiology , Polysaccharides/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Alkaloids/chemistry , Alkaloids/pharmacology , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Chromatography, Liquid , Computational Biology/methods , Coronavirus/drug effects , Coronavirus Infections/veterinary , Gene Expression Regulation, Viral , Glycosylation/drug effects , Infectious bronchitis virus/physiology , Models, Molecular , Molecular Conformation , Molecular Weight , Neutralization Tests , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Polysaccharides/chemistry , Poultry Diseases/virology , Protein Transport , Spectrometry, Mass, Electrospray Ionization , Spike Glycoprotein, Coronavirus/genetics , Structure-Activity Relationship , Virus Replication/drug effects
3.
Carbohydr Polym ; 273: 118567, 2021 Dec 01.
Article in English | MEDLINE | ID: covidwho-1363900

ABSTRACT

Diffuse alveolar injury and pulmonary fibrosis (PF) are the main causes of death of Covid-19 cases. In this study a low molecular weight fucoidan (LMWF) with unique structural was obtained from Laminaria japonica, and its anti- PF and anti-epithelial-mesenchymal transition (EMT) bioactivity were investigated both in vivo and in vitro. After LWMF treatment the fibrosis and inflammatory factors stimulated by Bleomycin (BLM) were in lung tissue. Immunohistochemical and Western-blot results found the expression of COL2A1, ß-catenin, TGF-ß, TNF-α and IL-6 were declined in mice lung tissue. Besides, the phosphorylation of PI3K and Akt were inhibited by LMWF. In addition, the progression of EMT induced by TGF-ß1 was inhibited by LMWF through down-regulated both TGF-ß/Smad and PI3K/AKT signaling pathways. These data indicate that unique LMWF can protect the lung from fibrosis by weakening the process of inflammation and EMT, and it is a promising therapeutic option for the treatment of PF.


Subject(s)
COVID-19/complications , Epithelial-Mesenchymal Transition/drug effects , Polysaccharides/administration & dosage , Polysaccharides/chemistry , Pulmonary Fibrosis/complications , Pulmonary Fibrosis/drug therapy , SARS-CoV-2 , A549 Cells , Animals , Bleomycin/adverse effects , COVID-19/virology , Cell Survival/drug effects , Cytokines/antagonists & inhibitors , Cytokines/metabolism , Cytokines/pharmacology , Disease Models, Animal , Humans , Inflammation/drug therapy , Lung/immunology , Male , Mice , Mice, Inbred C57BL , Molecular Weight , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/mortality , Signal Transduction/drug effects
4.
Nat Prod Res ; 34(16): 2249-2254, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-1343564

ABSTRACT

The enhanced osteoblast differentiation is beneficial to the prevention of osteoporosis. In this study, a homogeneous polysaccharide (LRP-S2A) with the potential of promoting osteoblast differentiation was obtained from the fruits of Lycium ruthenicum, a traditional herb for treatment of postmenopausal metabolic disorders. Structural identification indicated that LRP-S2A, with a relative molecular weight of 2.65 × 106 Da and an uronic acid content of 41.8%, contained Rha, Ara, Gal, Glc and GlcA in a molar ratio of 1.00 : 2.07 : 0.57 : 2.59 : 4.33 and was composed of a backbone consisting of 6-O-Me-α-(1→4)-D-GlcpA, 2-O-acetyl-α-(1→4)-D-Glcp, α-(1→2,4)-L-Rhap, ß-(1→3)-D-Galp andα-(1→3,5)-L-Araf, and some branches consisting of 6-O-Me-α-(1→4)-D-GlcpA and terminal α-L-Araf. These results suggested that LRP-S2A with the potential of promoting osteoblast differentiation was a new acidic polysaccharide.


Subject(s)
Cell Differentiation/drug effects , Lycium/chemistry , Osteoblasts/cytology , Polysaccharides/chemistry , Animals , Cells, Cultured , Fruit/chemistry , Humans , Molecular Weight , Polysaccharides/pharmacology , Uronic Acids/analysis
5.
Bratisl Lek Listy ; 122(8): 582-589, 2021.
Article in English | MEDLINE | ID: covidwho-1318438

ABSTRACT

OBJECTIVES: Low molecular weight heparin (LMWH) may provide beneficial effects on outcomes of COVID-19. We aimed to examine the impact of LMWH treatment on clinical outcomes (duration of hospitalization, admission to intensive care unit, the requirement for mechanical ventilation, and death) of COVID-19 patients with normal D-dimer levels at admission. BACKGROUND: Coronavirus disease-2019 (COVID-19) predisposes patients to arterial and venous thrombosis. METHODS: In this retrospective, multicentre and observational study we analysed the data of 308 confirmed COVID-19 patients with normal D-dimer levels at initial admission. After propensity score matching (PSM) patients were grouped; Group 1; patients who received LMWH with D-dimer ≤0.5 mg/L, Group 2; patients who received LMWH after D-dimer levels exceeded 0.5 mg/L, and Group 3; patients who did not receive LMWH. RESULTS: After PSM, each group comprised 40 patients. The patients in Group1 had the best clinical outcomes compared to the other groups. Group 3 had the worst clinical outcomes (p<0.005). The benefit of LMWH increased with early prophylactic therapy especially when started while the D-dimer levels were ≤0.5 mg/L. CONCLUSION: Our results strongly suggest that proactive LMWH therapy improves clinical outcomes in hospitalized COVID-19 patients even with normal D-dimer levels (≤ 0.5 mg/L) (Tab. 3, Fig. 2, Ref. 34).


Subject(s)
COVID-19 , Heparin, Low-Molecular-Weight , Anticoagulants , Heparin , Humans , Molecular Weight , Retrospective Studies , SARS-CoV-2
7.
Nanoscale ; 13(15): 7285-7293, 2021 Apr 21.
Article in English | MEDLINE | ID: covidwho-1199322

ABSTRACT

Interest in cryo-Electron Microscopy (EM) imaging has skyrocketed in recent years due to its pristine views of macromolecules and materials. As advances in instrumentation and computing algorithms spurred this progress, there is renewed focus to address specimen-related challenges. Here we contribute a microchip-based toolkit to perform complementary structural and biochemical analysis on low-molecular weight proteins. As a model system, we used the SARS-CoV-2 nucleocapsid (N) protein (48 kDa) due to its stability and important role in therapeutic development. Cryo-EM structures of the N protein monomer revealed a flexible N-terminal "top hat" motif and a helical-rich C-terminal domain. To complement our structural findings, we engineered microchip-based immunoprecipitation assays that led to the discovery of the first antibody binding site on the N protein. The data also facilitated molecular modeling of a variety of pandemic and common cold-related coronavirus proteins. Such insights may guide future pandemic-preparedness protocols through immuno-engineering strategies to mitigate viral outbreaks.


Subject(s)
Coronavirus Nucleocapsid Proteins/chemistry , Cryoelectron Microscopy , SARS-CoV-2/chemistry , Molecular Weight , Phosphoproteins/chemistry , Protein Structure, Secondary
8.
Ter Arkh ; 93(1): 108-113, 2021 Jan 10.
Article in Russian | MEDLINE | ID: covidwho-1134694

ABSTRACT

During a pandemic, nonspecific immunoprophylaxis of SARS-CoV-2 infection and other acute respiratory infections (ARI), which can worsen the course of COVID-19, is increasingly in demand in addition to specific immunization. BCG vaccine appears to be one of the candidate immunostimulants in this regard. At the same time, other microbe-derived preparations capable of inducing a state of trained immunity deserve attention. BCG and other bacterial immunostimulatory agents containing a large number of biologically active subunits have long been considered as objects of search for promising pharmacological substances. The review analyzes the linkages between BCG, mycobacterial adjuvants, bacterial lysates, trained immunity, muramylpeptides (MPs) and NOD2 receptors in light of the choice of a low molecular weight alternative to multicomponent bacterial immunostimulants for ARI prevention during the COVID-19 pandemic. The search for key molecules by which bacteria stimulate innate and adaptive immune responses proceeds in a spiral. On different loops of this spiral, MPs have repeatedly reproduced the nonspecific effects of multicomponent bacterial adjuvants, vaccines and immunostimulants. MPs and peptidoglycans containing MPs determine the adjuvant properties of the cell walls of mycobacteria and their peptide-glycolipid fraction (wax D). MPs were able to replace Mycobacterium tuberculosis in complete Freunds adjuvant. MPs determine the NOD2-dependent ability of BCG to induce trained immunity. Probably, MPs provide NOD2-mediated long-term prophylactic action of bacterial lysates. All of the above has prompted revisiting the previously obtained evidence of the efficacy of glucosaminylmuramyl dipeptide (GMDP) as a NOD2 agonist in treatment/prevention of respiratory infections. We speculate here that MPs, in particular GMDP, at rational dosing regimens will be able to reproduce many aspects of the nonspecific effects of BCG and multicomponent bacterial immunostimulants in preventing ARI during the COVID-19 pandemic and in the post-pandemic period.


Subject(s)
COVID-19 , Pandemics , BCG Vaccine , Cell Extracts , Humans , Immunity, Innate , Molecular Weight , SARS-CoV-2
9.
Carbohydr Polym ; 260: 117797, 2021 May 15.
Article in English | MEDLINE | ID: covidwho-1084646

ABSTRACT

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has resulted in a pandemic and continues to spread at an unprecedented rate around the world. Although a vaccine has recently been approved, there are currently few effective therapeutics to fight its associated disease in humans, COVID-19. SARS-CoV-2 and the related severe acute respiratory syndrome (SARS-CoV-1), and Middle East respiratory syndrome (MERS-CoV) result from zoonotic respiratory viruses that have bats as the primary host and an as yet unknown secondary host. While each of these viruses has different protein-based cell-surface receptors, each rely on the glycosaminoglycan, heparan sulfate as a co-receptor. In this study we compare, for the first time, differences and similarities in the structure of heparan sulfate in human and bat lungs. Furthermore, we show that the spike glycoprotein of COVID-19 binds 3.5 times stronger to human lung heparan sulfate than bat lung heparan sulfate.


Subject(s)
Heparitin Sulfate/metabolism , Lung/chemistry , Receptors, Virus/metabolism , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Animals , Chiroptera , Female , Heparitin Sulfate/chemistry , Heparitin Sulfate/isolation & purification , Humans , Male , Molecular Structure , Molecular Weight , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/isolation & purification
10.
Int J Nanomedicine ; 16: 715-724, 2021.
Article in English | MEDLINE | ID: covidwho-1067512

ABSTRACT

Objective: The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is now rapidly spreading globally. Serological tests are an important method to assist in the diagnosis of COVID-19, used for epidemiological investigations. In this study, we aimed to investigate the impact of different types of vacuum collection tubes on the detection of SARS-CoV-2 IgM and IgG antibodies, using the colloidal gold immunochromatographic assay (GICA). Patients and Methods: A total of 112 patients with COVID-19 and 200 healthy control subjects with no infection were enrolled in this study. Their serum and plasma were collected into four different types of vacuum blood collection tubes. SARS-CoV-2 IgM and IgG specific antibodies in the plasma and serum were then detected by GICA and chemiluminescence assay (CA), respectively. In addition, the particle sizes of different colloidal gold solutions in the presence of different anticoagulants and coagulants were evaluated by both laser diffraction (Malvern) and confocal laser microscope, respectively. Results: Our results revealed that anticoagulated plasma with EDTA-K2 improved the positive detection rate of SARS-CoV-2 IgM antibodies. Furthermore, our results shown that the detection results by GICA and CA were highly consistent, especially, the results of EDTA-K2 anticoagulated plasma detected by GICA was more consistent with CA results. We confirmed that EDTA-K2 could improve the detection sensitivity of SARS-CoV-2 IgG antibodies by chelating excessive colloidal gold compared with sodium citrate or lithium heparin, these methodologies did not appear to cause false positives. Colloidal gold particles could be chelated and aggregated by EDTA-K2, but not by sodium citrate, lithium heparin and coagulants. Conclusion: GICA is widely used to detect antibodies for the advantages of convenient, fast, low cost, suitable for screening large sample and require minimal equipment. In this study, we found that EDTA-K2 amplified the positive antibody signal by chelating colloidal gold and improved the detection sensitivity of SARS-CoV-2 IgM and IgG antibodies when using the GICA. Therefore, we suggested that EDTA-K2 anticoagulated plasma was more suitable for the detection of SARS-CoV-2 antibodies.


Subject(s)
Antibodies, Viral/isolation & purification , Chelating Agents/chemistry , Edetic Acid/chemistry , Gold Colloid/chemistry , Immunoassay/methods , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , SARS-CoV-2/immunology , Adult , Antibodies, Viral/blood , Antibody Specificity/immunology , COVID-19/blood , COVID-19/immunology , COVID-19/virology , Female , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Male , Middle Aged , Molecular Weight , Particle Size , Polymers/chemistry , Sensitivity and Specificity
11.
Crit Rev Immunol ; 40(6): 465-473, 2020.
Article in English | MEDLINE | ID: covidwho-1050517

ABSTRACT

Coronavirus disease 2019 (COVID-19) consists of a severe involvement of the lower respiratory tract leading to an acute respiratory syndrome. But there exist other infectious respiratory syndromes that have the same initial respiratory symptoms, show similar pattern in the size of the antigenic proteins and release comparable cytokines pathways, but with an unlike response magnitude. Here we propose that COVID-19 disease wrong response in the host immune system can be explained in the perspective of the antigen viral size. In COVID-19 sepsis, the < 70 kDa antigens activate the B-cell receptor (BCR), which modulates the shift in the pattern of T-helper 1 (Th1) to Th2 cytokines, increases the release of interleukin-10 (IL-10) and the up-regulation of the membrane form of tumor necrosis factor alpha (TNF-α), promoting the production of immunoglobulin G1 (IgG1)- and IgG3-neutralizing antibodies, but failing in IgG2a production and in developing long-lasting B-cell immune memory. The sustained infected cells lysis overfeeds high levels of viral proteins < 70 kDa, increases B-cell activation and, in the shift from a Th1 to a Th2 immune response, can trigger a cytokine storm. The continuous BCR activation increases IL-10 release that can lead to cytokine storm, apoptosis, and immune paralysis. Here, we propose a new vaccine design using the polymerization of viral antigens that could be ready in short time, would be cheap and easy to develop because it is based on classic technologies available in every country, is safe because it does not employ genetic material, and would able to promote long-lasting B-cell immune memory and IgG2a production.


Subject(s)
Antigens, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , Immunoglobulin G/immunology , Immunologic Memory , SARS-CoV-2/immunology , Viral Proteins/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/chemistry , B-Lymphocytes/immunology , COVID-19/prevention & control , Cytokines/metabolism , Humans , Immunity, Humoral , Lymphocyte Activation , Molecular Weight , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Viral Proteins/chemistry
12.
J Virol ; 95(3)2021 01 13.
Article in English | MEDLINE | ID: covidwho-1048660

ABSTRACT

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH, and 6-O-desulfated enoxaparin with 50% inhibitory concentrations (IC50s) of 5.99 µg/liter, 1.08 mg/liter, 1.77 µg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.


Subject(s)
Antiviral Agents/pharmacology , Heparin/pharmacology , SARS-CoV-2/drug effects , Virus Internalization/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Drug Evaluation, Preclinical , Enoxaparin/chemistry , Enoxaparin/metabolism , Enoxaparin/pharmacology , Genetic Vectors/genetics , HEK293 Cells , Heparin/chemistry , Heparin/metabolism , Heparitin Sulfate/metabolism , Humans , Inhibitory Concentration 50 , Lentivirus/genetics , Molecular Structure , Molecular Weight , Polysaccharides/chemistry , Polysaccharides/metabolism , Polysaccharides/pharmacology , Protein Binding , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Transduction, Genetic , Virus Attachment/drug effects
13.
Angiogenesis ; 24(3): 505-517, 2021 08.
Article in English | MEDLINE | ID: covidwho-1032491

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a respiratory disease associated with endotheliitis and microthrombosis. OBJECTIVES: To correlate endothelial dysfunction to in-hospital mortality in a bi-centric cohort of COVID-19 adult patients. METHODS: Consecutive ambulatory and hospitalized patients with laboratory-confirmed COVID-19 were enrolled. A panel of endothelial biomarkers and von Willebrand factor (VWF) multimers were measured in each patient ≤ 48 h following admission. RESULTS: Study enrolled 208 COVID-19 patients of whom 23 were mild outpatients and 189 patients hospitalized after admission. Most of endothelial biomarkers tested were found increased in the 89 critical patients transferred to intensive care unit. However, only von Willebrand factor antigen (VWF:Ag) scaled according to clinical severity, with levels significantly higher in critical patients (median 507%, IQR 428-596) compared to non-critical patients (288%, 230-350, p < 0.0001) or COVID-19 outpatients (144%, 133-198, p = 0.007). Moreover, VWF high molecular weight multimers (HMWM) were significantly higher in critical patients (median ratio 1.18, IQR 0.86-1.09) compared to non-critical patients (0.96, 1.04-1.39, p < 0.001). Among all endothelial biomarkers measured, ROC curve analysis identified a VWF:Ag cut-off of 423% as the best predictor for in-hospital mortality. The accuracy of VWF:Ag was further confirmed in a Kaplan-Meier estimator analysis and a Cox proportional Hazard model adjusted on age, BMI, C-reactive protein and D-dimer levels. CONCLUSION: VWF:Ag is a relevant predictive factor for in-hospital mortality in COVID-19 patients. More than a biomarker, we hypothesize that VWF, including excess of HMWM forms, drives microthrombosis in COVID-19.


Subject(s)
COVID-19/blood , COVID-19/mortality , Pandemics , SARS-CoV-2 , von Willebrand Factor/metabolism , Adult , Aged , Biomarkers/blood , Biomarkers/chemistry , COVID-19/physiopathology , Cross-Sectional Studies , Endothelium, Vascular/physiopathology , Female , Hospital Mortality , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Molecular Weight , Paris/epidemiology , Proportional Hazards Models , Protein Multimerization , Severity of Illness Index , Thrombosis/blood , Thrombosis/etiology , von Willebrand Factor/chemistry
14.
J Transl Med ; 18(1): 452, 2020 11 30.
Article in English | MEDLINE | ID: covidwho-948411

ABSTRACT

BACKGROUND: Dysregulation of transcription and cytokine expression has been implicated in the pathogenesis of a variety inflammatory diseases. The resulting imbalance between inflammatory and resolving transcriptional programs can cause an overabundance of pro-inflammatory, classically activated macrophage type 1 (M1) and/or helper T cell type 1 (Th1) products, such as IFNγ, TNFα, IL1-ß, and IL12, that prevent immune switching to resolution and healing. The low molecular weight fraction of human serum albumin (LMWF5A) is a novel biologic drug that is currently under clinical investigation for the treatment of osteoarthritis and the hyper-inflammatory response associated with COVID-19. This study aims to elucidate transcriptional mechanisms of action involved with the ability of LMWF5A to reduce pro-inflammatory cytokine release. METHODS: ELISA arrays were used to identify cytokines and chemokines influenced by LMWF5A treatment of LPS-stimulated peripheral blood mononuclear cells (PBMC). The resulting profiles were analyzed by gene enrichment to gain mechanistic insight into the biologic processes and transcription factors (TFs) underlying the identified differentially expressed cytokines. DNA-binding ELISAs, luciferase reporter assays, and TNFα or IL-1ß relative potency were then employed to confirm the involvement of enriched pathways and TFs. RESULTS: LMWF5A was found to significantly inhibit a distinct set of pro-inflammatory cytokines (TNFα, IL-1ß, IL-12, CXCL9, CXCL10, and CXCL11) associated with pro-inflammatory M1/Th1 immune profiles. Gene enrichment analysis also suggests these cytokines are, in part, regulated by NF-κB and STAT transcription factors. Data from DNA-binding and reporter assays support this with LMWF5A inhibition of STAT1α DNA-binding activity as well as a reduction in overall NF-κB-driven luciferase expression. Experiments using antagonists specific for the immunomodulatory and NF-κB/STAT-repressing transcription factors, peroxisome proliferator-activated receptor (PPAR)γ and aryl hydrocarbon receptor (AhR), indicate these pathways are involved in the LMWF5A mechanisms of action by reducing LMWF5A drug potency as measured by TNFα and IL-1ß release. CONCLUSION: In this report, we provide evidence that LMWF5A reduces pro-inflammatory cytokine release by activating the immunoregulatory transcription factors PPARγ and AhR. In addition, our data indicate that LMWF5A suppresses NF-κB and STAT1α pro-inflammatory pathways. This suggests that LMWF5A acts through these mechanisms to decrease pro-inflammatory transcription factor activity and subsequent inflammatory cytokine production.


Subject(s)
Cytokines/metabolism , Inflammation/prevention & control , Leukocytes, Mononuclear/drug effects , Serum Albumin, Human/pharmacology , Anti-Inflammatory Agents/pharmacology , COVID-19/drug therapy , COVID-19/immunology , COVID-19/pathology , Cells, Cultured , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation Mediators/metabolism , Interferon-Stimulated Gene Factor 3/metabolism , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides , Lymphocyte Activation/drug effects , Molecular Weight , NF-kappa B/metabolism , Serum Albumin, Human/chemistry , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunology , Transcription Factors/metabolism
15.
J Phys Chem Lett ; 11(17): 7267-7272, 2020 Sep 03.
Article in English | MEDLINE | ID: covidwho-678527

ABSTRACT

The coronavirus disease pandemic caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected the global healthcare system. As low-molecular-weight drugs have high potential to completely match interactions with essential SARS-CoV-2 targets, we propose a strategy to identify such drugs using the fragment-based approach. Herein, using ligand- and protein-observed fragment screening approaches, we identified niacin and hit 1 binding to the catalytic pocket of the main protease (Mpro) of SARS-CoV-2, thereby modestly inhibiting the enzymatic activity of Mpro. We further searched for low-molecular-weight drugs containing niacin or hit 1 pharmacophores with enhanced inhibiting activity, e.g., carmofur, bendamustine, triclabendazole, emedastine, and omeprazole, in which omeprazole is the only one binding to the C-terminal domain of SARS-CoV-2 Mpro. Our study demonstrates that the fragment-based approach is a feasible strategy for identifying low-molecular-weight drugs against the SARS-CoV-2 and other potential targets lacking specific drugs.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Drug Repositioning , Peptide Hydrolases/metabolism , Dose-Response Relationship, Drug , Models, Molecular , Molecular Weight , Peptide Hydrolases/chemistry , Protein Domains , SARS-CoV-2
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