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1.
Sci Adv ; 8(7): eabl6242, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1714333

ABSTRACT

Large segmental osseous defects heal poorly. Recombinant, human bone morphogenetic protein-2 (rhBMP-2) is used clinically to promote bone healing, but it is applied at very high doses that cause adverse side effects and raise costs while providing only incremental benefit. We describe a previously unexplored, alternative approach to bone regeneration using chemically modified messenger RNA (cmRNA). An optimized cmRNA encoding BMP-2 was delivered to critical-sized femoral osteotomies in rats. The cmRNA remained orthotopically localized and generated BMP locally for several days. Defects healed at doses ≥25 µg of BMP-2 cmRNA. By 4 weeks, all animals treated with 50 µg of BMP-2 cmRNA had bridged bone defects without forming the massive callus seen with rhBMP-2. Moreover, such defects recovered normal mechanical strength quicker and initiated bone remodeling faster. cmRNA regenerated bone via endochondral ossification, whereas rhBMP-2 drove intramembranous osteogenesis; cmRNA provides an innovative, safe, and highly translatable technology for bone healing.


Subject(s)
Bone Morphogenetic Protein 2 , Bone Regeneration , Animals , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 2/pharmacology , Femur , Osteogenesis , RNA, Messenger/genetics , Rats , Recombinant Proteins/pharmacology , Wound Healing
2.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Article in English | MEDLINE | ID: covidwho-1671749

ABSTRACT

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.


Subject(s)
COVID-19/drug therapy , Interferon-alpha/pharmacology , SARS-CoV-2/drug effects , Transcriptome , Virus Replication/drug effects , Animals , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Cloning, Molecular , Disease Models, Animal , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Profiling , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Interferon-alpha/genetics , Interferon-alpha/immunology , Mice , Protein Isoforms/classification , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/pharmacology , Recombinant Proteins/classification , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/pharmacology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Signal Transduction , Vero Cells
4.
Microbiol Spectr ; 10(1): e0052221, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1622001

ABSTRACT

Heme-containing peroxidases are widely distributed in the animal and plant kingdoms and play an important role in host defense by generating potent oxidants. Myeloperoxidase (MPO), the prototype of heme-containing peroxidases, exists in neutrophils and monocytes. MPO has a broad spectrum of microbial killing. The difficulty of producing MPO at a large scale hinders its study and utilization. This study aimed to overexpress recombinant human MPO and characterize its microbicidal activities in vitro and in vivo. A human HEK293 cell line stably expressing recombinant MPO (rMPO) was established as a component of this study. rMPO was overexpressed and purified for studies on its biochemical and enzymatic properties, as well as its microbicidal activities. In this study, rMPO was secreted into culture medium as a monomer. rMPO revealed enzymatic activity similar to that of native MPO. rMPO, like native MPO, was capable of killing a broad spectrum of microorganisms, including Gram-negative and -positive bacteria and fungi, at low nM levels. Interestingly, rMPO could kill antibiotic-resistant bacteria, making it very useful for treatment of nosocomial infections and mixed infections. The administration of rMPO significantly reduced the morbidity and mortality of murine lung infections induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. In animal safety tests, the administration of 100 nM rMPO via tail vein did not result in any sign of toxic effects. Taken together, the data suggest that rMPO purified from a stably expressing human cell line is a new class of antimicrobial agents with the ability to kill a broad spectrum of pathogens, including bacteria and fungi with or without drug resistance. IMPORTANCE Over the past 2 decades, more than 20 new infectious diseases have emerged. Unfortunately, novel antimicrobial therapeutics are discovered at much lower rates. Infections caused by resistant microorganisms often fail to respond to conventional treatment, resulting in prolonged illness, greater risk of death, and high health care costs. Currently, this is best seen with the lack of a cure for coronavirus disease 2019 (COVID-19). To combat such untreatable microorganisms, there is an urgent need to discover new classes of antimicrobial agents. Myeloperoxidase (MPO) plays an important role in host defense. The difficulty of producing MPO on a large scale hinders its study and utilization. We have produced recombinant MPO at a large scale and have characterized its antimicrobial activities. Most importantly, recombinant MPO significantly reduced the morbidity and mortality of murine pneumonia induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. Our data suggest that recombinant MPO from human cells is a new class of antimicrobials with a broad spectrum of activity.


Subject(s)
Anti-Infective Agents/pharmacology , Peroxidase/pharmacology , Acute Disease , Animals , Anti-Infective Agents/classification , Anti-Infective Agents/therapeutic use , Anti-Infective Agents/toxicity , Candida albicans/drug effects , Drug Resistance, Bacterial , Escherichia coli/drug effects , Female , HEK293 Cells , Humans , Hydrogen Peroxide/toxicity , Male , Methicillin-Resistant Staphylococcus aureus/drug effects , Mice , Mice, Inbred C57BL , Peroxidase/genetics , Peroxidase/therapeutic use , Peroxidase/toxicity , Pneumonia, Bacterial/drug therapy , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Recombinant Proteins/toxicity , Staphylococcal Infections/drug therapy , Staphylococcus aureus/drug effects
5.
Bull Exp Biol Med ; 172(1): 53-56, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1520385

ABSTRACT

The antiviral activity of recombinant human IFN-lambda type 1 (IFNλ-1) against culture strain of SARS-CoV-2 virus was determined by infecting a highly sensitive VeroE6 coronavirus cell culture after preincubation test (the cell monolayer was incubated with 4-fold dilutions of IFNλ-1 in a concentration range of 0.16-42,500 ng/ml in a culture medium for 12 h at 37°C) and without preincubation (simultaneous addition of different concentrations of IFNλ-1 and SARS-CoV-2 infection in a dose of 102 TCID50). The created recombinant human IFNλ-1 demonstrated obvious antiviral activity against SARS-CoV-2 virus in vitro. In the tests with and without preincubation, IFNλ-1 exhibited significant activity, although somewhat lower in variant with simultaneous addition of IFNλ-1 and virus to the cell culture. It should be noted that the antiviral effect of IFNλ-1 was observed in a wide range of concentrations.


Subject(s)
Antiviral Agents/pharmacology , Interferons/pharmacology , Recombinant Proteins/pharmacology , SARS-CoV-2/drug effects , Viral Load/drug effects , Virus Replication/drug effects , Animals , Antiviral Agents/isolation & purification , COVID-19/drug therapy , COVID-19/virology , Chlorocebus aethiops , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Interferons/biosynthesis , Interferons/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , Vero Cells , Viral Load/genetics
6.
Clin Immunol ; 233: 108888, 2021 12.
Article in English | MEDLINE | ID: covidwho-1517099

ABSTRACT

Human interferon alpha (hIFN-α) administration constitutes the current FDA approved therapy for chronic Hepatitis B and C virus infections. Additionally, hIFN-α treatment efficacy was recently demonstrated in patients with COVID-19. Thus, hIFN-α constitutes a therapeutic alternative for those countries where vaccination is inaccessible and for people who did not respond effectively to vaccination. However, hIFN-α2b exhibits a short plasma half-life resulting in the occurrence of severe side effects. To optimize the cytokine's pharmacokinetic profile, we developed a hyperglycosylated IFN, referred to as GMOP-IFN. Given the significant number of reports showing neutralizing antibodies (NAb) formation after hIFN-α administration, here we applied the DeFT (De-immunization of Functional Therapeutics) approach to develop functional, de-immunized versions of GMOP-IFN. Two GMOP-IFN variants exhibited significantly reduced ex vivo immunogenicity and null antiproliferative activity, while preserving antiviral function. The results obtained in this work indicate that the new de-immunized GMOP-IFN variants constitute promising candidates for antiviral therapy.


Subject(s)
Hepatitis B, Chronic/immunology , Hepatitis C, Chronic/immunology , Interferon-alpha/immunology , Recombinant Proteins/immunology , Adult , Amino Acid Sequence , Animals , Antibodies, Neutralizing/immunology , Antiviral Agents/immunology , Antiviral Agents/pharmacology , CHO Cells , COVID-19/drug therapy , COVID-19/immunology , COVID-19/virology , Cattle , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Cells, Cultured , Cricetinae , Cricetulus , Drug Stability , HEK293 Cells , Hepatitis B, Chronic/drug therapy , Hepatitis B, Chronic/virology , Hepatitis C, Chronic/drug therapy , Hepatitis C, Chronic/virology , Humans , Interferon-alpha/genetics , Interferon-alpha/pharmacology , Recombinant Proteins/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/physiology
7.
Int J Mol Sci ; 22(21)2021 Nov 08.
Article in English | MEDLINE | ID: covidwho-1512381

ABSTRACT

Tumor-associated cell-free DNAs (cfDNA) play an important role in the promotion of metastases. Previous studies proved the high antimetastatic potential of bovine pancreatic DNase I and identified short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)and fragments of oncogenes in cfDNA as the main molecular targets of enzyme in the bloodstream. Here, recombinant human DNase I (commercial name Pulmozyme®), which is used for the treatment of cystic fibrosis in humans, was repurposed for the inhibition of lung metastases in the B16 melanoma model in mice. We found that Pulmozyme® strongly reduced migration and induced apoptosis of B16 cells in vitro and effectively inhibited metastases in lungs and liver in vivo. Pulmozyme® was shown to be two times more effective when administered intranasally (i.n.) than bovine DNase I, but intramuscular (i.m.) administration forced it to exhibit as high an antimetastatic activity as bovine DNase I. Both DNases administered to mice either i.m. or i.n. enhanced the DNase activity of blood serum to the level of healthy animals, significantly decreased cfDNA concentrations, efficiently degraded SINE and LINE repeats and c-Myc fragments in the bloodstream and induced apoptosis and disintegration of neutrophil extracellular traps in metastatic foci; as a result, this manifested as the inhibition of metastases spread. Thus, Pulmozyme®, which is already an approved drug, can be recommended for use in the treatment of lung metastases.


Subject(s)
Cell-Free Nucleic Acids/blood , Deoxyribonuclease I/metabolism , Long Interspersed Nucleotide Elements/genetics , Lung Neoplasms/drug therapy , Melanoma, Experimental/drug therapy , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Short Interspersed Nucleotide Elements/genetics , Animals , Cell Line, Tumor , Deoxyribonuclease I/pharmacology , Disease Models, Animal , Drug Repositioning , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Male , Melanoma, Experimental/genetics , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Proto-Oncogene Proteins c-myc/blood , Proto-Oncogene Proteins c-myc/genetics , Recombinant Proteins/pharmacology
8.
Biochemistry (Mosc) ; 86(10): 1275-1287, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1476404

ABSTRACT

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.


Subject(s)
COVID-19 Vaccines , COVID-19 , Epitopes , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Animals , COVID-19/genetics , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , COVID-19 Vaccines/isolation & purification , COVID-19 Vaccines/pharmacology , Epitopes/genetics , Epitopes/immunology , Epitopes/isolation & purification , Epitopes/pharmacology , Female , Humans , Mice , Mice, Inbred BALB C , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/isolation & purification , Spike Glycoprotein, Coronavirus/pharmacology
9.
Protein Expr Purif ; 190: 106003, 2022 02.
Article in English | MEDLINE | ID: covidwho-1474960

ABSTRACT

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.


Subject(s)
COVID-19 Vaccines , Gene Expression , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Animals , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/genetics , COVID-19 Vaccines/pharmacology , Humans , Mice , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/pharmacology
10.
PLoS One ; 16(10): e0254985, 2021.
Article in English | MEDLINE | ID: covidwho-1448572

ABSTRACT

BACKGROUND: The goal of this study was to determine if IL-22:Fc would Acute Respiratory Distress Syndrome (ARDS). SUMMARY BACKGROUND DATA: No therapies exist for ARDS and treatment is purely supportive. Interleukin-22 (IL-22) plays an integral component in recovery of the lung from infection. IL-22:Fc is a recombinant protein with a human FC immunoglobulin that increases the half-life of IL-22. STUDY DESIGN: ARDS was induced in C57BL/6 mice with intra-tracheal lipopolysaccharide (LPS) at a dose of 33.3 or 100 ug. In the low-dose LPS group (LDG), IL-22:FC was administered via tail vein injection at 30 minutes (n = 9) and compared to sham (n = 9). In the high-dose LPS group (HDG), IL-22:FC was administered (n = 11) then compared to sham (n = 8). Euthanasia occurred after bronchioalveolar lavage (BAL) on post-injury day 4. RESULTS: In the LDG, IL-22:FC resulted in decreased protein leak (0.15 vs. 0.25 ug/uL, p = 0.02). BAL protein in animals receiving IL-22:Fc in the HDG was not different. For the HDG, animals receiving IL-22:Fc had lower BAL cell counts (539,636 vs 3,147,556 cells/uL, p = 0.02). For the HDG, IL-6 (110.6 vs. 527.1 pg/mL, p = 0.04), TNF-α (5.87 vs. 25.41 pg/mL, p = 0.04), and G-CSF (95.14 vs. 659.6, p = 0.01) levels were lower in the BAL fluid of IL-22:Fc treated animals compared to sham. CONCLUSIONS: IL-22:Fc decreases lung inflammation and lung capillary leak in ARDS. IL-22:Fc may be a novel therapy for ARDS.


Subject(s)
Immunoglobulin Fc Fragments/pharmacology , Interleukins/pharmacology , Lung Injury/drug therapy , Pneumonia/drug therapy , Respiratory Distress Syndrome/drug therapy , Animals , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Female , Lipopolysaccharides/toxicity , Lung Injury/pathology , Lymphocyte Count , Lymphocytes/immunology , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Neutrophils/immunology , Pneumonia/pathology , Receptors, Interleukin/metabolism , Recombinant Proteins/pharmacology , Respiratory Distress Syndrome/pathology , Respiratory Mucosa/pathology
11.
Viruses ; 13(8)2021 08 12.
Article in English | MEDLINE | ID: covidwho-1436097

ABSTRACT

Type III interferons (lambda IFNs) are a quite new, small family of three closely related cytokines with interferon-like activity. Attention to IFN-λ is mainly focused on direct antiviral activity in which, as with IFN-α, viral genome replication is inhibited without the participation of immune system cells. The heterodimeric receptor for lambda interferons is exposed mainly on epithelial cells, which limits its possible action on other cells, thus reducing the likelihood of developing undesirable side effects compared to type I IFN. In this study, we examined the antiviral potential of exogenous human IFN-λ1 in cellular models of viral infection. To study the protective effects of IFN-λ1, three administration schemes were used: 'preventive' (pretreatment); 'preventive/therapeutic' (pre/post); and 'therapeutic' (post). Three IFN-λ1 concentrations (from 10 to 500 ng/mL) were used. We have shown that human IFN-λ1 restricts SARS-CoV-2 replication in Vero cells with all three treatment schemes. In addition, we have shown a decrease in the viral loads of CHIKV and IVA with the 'preventive' and 'preventive/therapeutic' regimes. No significant antiviral effect of IFN-λ1 against AdV was detected. Our study highlights the potential for using IFN-λ as a broad-spectrum therapeutic agent against respiratory RNA viruses.


Subject(s)
Adenoviruses, Human/drug effects , Chikungunya virus/drug effects , Influenza A virus/drug effects , Interferons/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Adenoviruses, Human/physiology , Animals , Chikungunya virus/physiology , Chlorocebus aethiops , Dose-Response Relationship, Drug , Gene Expression Regulation , Humans , Influenza A virus/physiology , Interferons/therapeutic use , Interleukins , RNA Virus Infections/drug therapy , RNA Virus Infections/prevention & control , Recombinant Proteins/pharmacology , SARS-CoV-2/physiology , Vero Cells , Viral Load/drug effects , Virus Replication/drug effects
12.
World J Gastroenterol ; 27(32): 5404-5423, 2021 Aug 28.
Article in English | MEDLINE | ID: covidwho-1379993

ABSTRACT

BACKGROUND: Intestinal barrier breakdown, a frequent complication of intestinal ischemia-reperfusion (I/R) including dysfunction and the structure changes of the intestine, is characterized by a loss of tight junction and enhanced permeability of the intestinal barrier and increased mortality. To develop effective and novel therapeutics is important for the improvement of outcome of patients with intestinal barrier deterioration. Recombinant human angiopoietin-like protein 4 (rhANGPTL4) is reported to protect the blood-brain barrier when administered exogenously, and endogenous ANGPTL4 deficiency deteriorates radiation-induced intestinal injury. AIM: To identify whether rhANGPTL4 may protect intestinal barrier breakdown induced by I/R. METHODS: Intestinal I/R injury was elicited through clamping the superior mesenteric artery for 60 min followed by 240 min reperfusion. Intestinal epithelial (Caco-2) cells and human umbilical vein endothelial cells were challenged by hypoxia/ reoxygenation to mimic I/R in vitro. RESULTS: Indicators including fluorescein isothiocyanate-conjugated dextran (4 kilodaltons; FD-4) clearance, ratio of phosphorylated myosin light chain/total myosin light chain, myosin light chain kinase and loss of zonula occludens-1, claudin-2 and VE-cadherin were significantly increased after intestinal I/R or cell hypoxia/reoxygenation. rhANGPTL4 treatment significantly reversed these indicators, which were associated with inhibiting the inflammatory and oxidative cascade, excessive activation of cellular autophagy and apoptosis and improvement of survival rate. Similar results were observed in vitro when cells were challenged by hypoxia/reoxygenation, whereas rhANGPTL4 reversed the indicators close to normal level in Caco-2 cells and human umbilical vein endothelial cells significantly. CONCLUSION: rhANGPTL4 can function as a protective agent against intestinal injury induced by intestinal I/R and improve survival via maintenance of intestinal barrier structure and functions.


Subject(s)
/pharmacology , Intestines , Reperfusion Injury , Caco-2 Cells , Human Umbilical Vein Endothelial Cells , Humans , Intestinal Mucosa , Recombinant Proteins/pharmacology , Reperfusion Injury/prevention & control
13.
Int J Mol Sci ; 22(17)2021 Aug 25.
Article in English | MEDLINE | ID: covidwho-1376841

ABSTRACT

In recent years, enzymes have risen as promising therapeutic tools for different pathologies, from metabolic deficiencies, such as fibrosis conditions, ocular pathologies or joint problems, to cancer or cardiovascular diseases. Treatments based on the catalytic activity of enzymes are able to convert a wide range of target molecules to restore the correct physiological metabolism. These treatments present several advantages compared to established therapeutic approaches thanks to their affinity and specificity properties. However, enzymes present some challenges, such as short in vivo half-life, lack of targeted action and, in particular, patient immune system reaction against the enzyme. For this reason, it is important to monitor serum immune response during treatment. This can be achieved by conventional techniques (ELISA) but also by new promising tools such as microarrays. These assays have gained popularity due to their high-throughput analysis capacity, their simplicity, and their potential to monitor the immune response of patients during enzyme therapies. In this growing field, research is still ongoing to solve current health problems such as COVID-19. Currently, promising therapeutic alternatives using the angiotensin-converting enzyme 2 (ACE2) are being studied to treat COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/therapeutic use , COVID-19/drug therapy , Enzyme Therapy/methods , Recombinant Proteins/therapeutic use , Angiotensin-Converting Enzyme 2/pharmacology , Clinical Trials, Phase II as Topic , Drug Compounding/methods , Enzyme Stability , Enzyme Therapy/history , Enzyme Therapy/trends , Half-Life , History, 20th Century , History, 21st Century , Humans , Recombinant Proteins/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Treatment Outcome , Virus Internalization/drug effects
14.
Curr Top Med Chem ; 21(10): 920-927, 2021.
Article in English | MEDLINE | ID: covidwho-1374190

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), known as Coronavirus disease-2019 (COVID-19), has caused the sixth world's public health emergency. Healthcare staff, as the frontline population fighting the pandemic, are exposed to a high risk of infection. Therefore, developing a protective intervention for medical staff is of significant importance. OBJECTIVE: The aim of the study was to explore the effectiveness and safety of recombinant human interferon alpha (rhIFN-α) nasal drops for the prevention of coronavirus disease 2019 (COVID-19) through administering it to medical staff. METHODS: This was a prospective open-label clinical trial with parallel intervention assignment conducted on 2944 medical staff including both doctors and nurses from Taihe Hospital, Shiyan City, Hubei Province, China from January 21, 2020 to July 30, 2020. The participants were bifurcated into two groups of low risk and high risk groups according to the level of direct exposure to COVID-19 patients. The individuals of the low-risk group received rhIFN-α nasal drops for one month in addition to first level protection, and the high-risk group received a combination of rhIFN-α nasal drops coupled with thymosin-α1 with either second or third-level protection protocol. Moreover, the new-outset of COVID-19 pneumonia diagnosed by chest computed tomography (CT), after thirty days, was the primary outcome. The adverse reactions were recorded in all participants. RESULTS: 2415 of 2944 individuals belonged to the low-risk group, while 529 to the high-risk group. There was no COVID-19 pneumonia in either of the group after thirty days. The pulmonary CT scans were negative for COVID-19 pneumonia in both the groups with no new clinical symptoms. No serious adverse event was observed during the course of the intervention. CONCLUSION: The rhIFN-α nasal drops along with augmented safeguards based on standard physical isolation could effectively protect medical staff against COVID-19 pneumonia.


Subject(s)
COVID-19/prevention & control , Interferon-alpha/pharmacology , Administration, Intranasal , Adult , Anti-Infective Agents, Local/pharmacology , COVID-19/epidemiology , China/epidemiology , Female , Humans , Interferon-alpha/administration & dosage , Interferon-alpha/adverse effects , Male , Personnel, Hospital , Prospective Studies , Recombinant Proteins/administration & dosage , Recombinant Proteins/adverse effects , Recombinant Proteins/pharmacology
15.
J Gerontol A Biol Sci Med Sci ; 76(10): 1775-1783, 2021 09 13.
Article in English | MEDLINE | ID: covidwho-1358442

ABSTRACT

Aging and comorbidities make individuals at greatest risk of COVID-19 serious illness and mortality due to senescence-related events and deleterious inflammation. Long-living individuals (LLIs) are less susceptible to inflammation and develop more resiliency to COVID-19. As demonstrated, LLIs are characterized by high circulating levels of BPIFB4, a protein involved in homeostatic response to inflammatory stimuli. Also, LLIs show enrichment of homozygous genotype for the minor alleles of a 4 missense single-nucleotide polymorphism haplotype (longevity-associated variant [LAV]) in BPIFB4, able to counteract progression of diseases in animal models. Thus, the present study was designed to assess the presence and significance of BPIFB4 level in COVID-19 patients and the potential therapeutic use of LAV-BPIFB4 in fighting COVID-19. BPIFB4 plasma concentration was found significantly higher in LLIs compared to old healthy controls while it significantly decreased in 64 COVID-19 patients. Further, the drop in BPIFB4 values correlated with disease severity. Accordingly to the LAV-BPIFB4 immunomodulatory role, while lysates of SARS-CoV-2-infected cells induced an inflammatory response in healthy peripheral blood mononuclear cells in vitro, the co-treatment with recombinant protein (rh) LAV-BPIFB4 resulted in a protective and self-limiting reaction, culminating in the downregulation of CD69 activating-marker for T cells (both TCD4+ and TCD8+) and in MCP-1 reduction. On the contrary, rhLAV-BPIFB4 induced a rapid increase in IL-18 and IL-1b levels, shown largely protective during the early stages of the virus infection. This evidence, along with the ability of rhLAV-BPIFB4 to counteract the cytotoxicity induced by SARS-CoV-2 lysate in selected target cell lines, corroborates BPIFB4 prognostic value and open new therapeutic possibilities in more vulnerable people.


Subject(s)
COVID-19 , Intercellular Signaling Peptides and Proteins , Longevity/immunology , Aged, 80 and over , Biomarkers/blood , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19/immunology , Cell Line , Cytokines/blood , Cytotoxicity, Immunologic/drug effects , Female , Humans , Immunologic Factors/immunology , Immunologic Factors/pharmacology , Inflammation/blood , Inflammation/immunology , Intercellular Signaling Peptides and Proteins/blood , Intercellular Signaling Peptides and Proteins/immunology , Italy/epidemiology , Male , Prognosis , Recombinant Proteins/immunology , Recombinant Proteins/pharmacology , SARS-CoV-2/immunology , Severity of Illness Index
16.
Biomolecules ; 11(8)2021 07 28.
Article in English | MEDLINE | ID: covidwho-1334992

ABSTRACT

SARS-CoV-2 infection of host cells is driven by binding of the SARS-CoV-2 spike-(S)-protein to lung type II pneumocytes, followed by virus replication. Surfactant protein SP-D, member of the front-line immune defense of the lungs, binds glycosylated structures on invading pathogens such as viruses to induce their clearance from the lungs. The objective of this study is to measure the pulmonary SP-D levels in COVID-19 patients and demonstrate the activity of SP-D against SARS-CoV-2, opening the possibility of using SP-D as potential therapy for COVID-19 patients. Pulmonary SP-D concentrations were measured in bronchoalveolar lavage samples from patients with corona virus disease 2019 (COVID-19) by anti-SP-D ELISA. Binding assays were performed by ELISAs. Protein bridge and aggregation assays were performed by gel electrophoresis followed by silver staining and band densitometry. Viral replication was evaluated in vitro using epithelial Caco-2 cells. Results indicate that COVID-19 patients (n = 12) show decreased pulmonary levels of SP-D (median = 68.9 ng/mL) when compared to levels reported for healthy controls in literature. Binding assays demonstrate that SP-D binds the SARS-CoV-2 glycosylated spike-(S)-protein of different emerging clinical variants. Binding induces the formation of protein bridges, the critical step of viral aggregation to facilitate its clearance. SP-D inhibits SARS-CoV-2 replication in Caco-2 cells (EC90 = 3.7 µg/mL). Therefore, SP-D recognizes and binds to the spike-(S)-protein of SARS-CoV-2 in vitro, initiates the aggregation, and inhibits viral replication in cells. Combined with the low levels of SP-D observed in COVID-19 patients, these results suggest that SP-D is important in the immune response to SARS-CoV-2 and that rhSP-D supplementation has the potential to be a novel class of anti-viral that will target SARS-CoV-2 infection.


Subject(s)
COVID-19/metabolism , Pulmonary Surfactant-Associated Protein D/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Adult , Aged , COVID-19/virology , Caco-2 Cells , Female , Humans , Male , Middle Aged , Protein Binding , Pulmonary Surfactant-Associated Protein D/genetics , Pulmonary Surfactant-Associated Protein D/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Virus Replication
17.
J Virol Methods ; 295: 114221, 2021 09.
Article in English | MEDLINE | ID: covidwho-1284316

ABSTRACT

SARS-CoV-2 is the culprit causing Coronavirus Disease 2019 (COVID-19). For the study of SARS-CoV-2 infection in a BSL-2 laboratory, a SARS-CoV-2 pseudovirus particle (SARS2pp) production and infection system was constructed by using a lentiviral vector bearing dual-reporter genes eGFP and firefly luciferase (Luc2) for easy observation and analysis. Comparison of SARS2pp different production conditions revealed that the pseudovirus titer could be greatly improved by: 1) removing the last 19 amino acids of the spike protein and replacing the signal peptide with the mouse Igk signal sequence; 2) expressing the spike protein using CMV promoter other than CAG (a hybrid promoter consisting of a CMV enhancer, beta-actin promoter, splice donor, and a beta-globin splice acceptor); 3) screening better optimized spike protein sequences for SARS2pp production; and 4) adding 1 % BSA in the SARS2pp production medium. For infection, this SARS2pp system showed a good linear relationship between MOI 2-0.0002 and then was successfully used to evaluate SARS-CoV-2 infection inhibitors including recombinant human ACE2 proteins and SARS-CoV-2 neutralizing antibodies. The kidney, liver and small intestine-derived cell lines were also found to show different susceptibility to SARSpp and SARS2pp. Given its robustness and good performance, it is believed that this pseudovirus particle production and infection system will greatly promote future research for SARS-CoV-2 entry mechanisms and inhibitors and can be easily applied to study new emerging SARS-CoV-2 variants.


Subject(s)
Neutralization Tests/methods , SARS-CoV-2/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2/pharmacology , Animals , Antibodies, Neutralizing/pharmacology , Antiviral Agents/pharmacology , Cell Line , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Lentivirus/genetics , Luciferases, Firefly/genetics , Luciferases, Firefly/metabolism , Recombinant Proteins/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virion , Virus Internalization/drug effects
18.
Mol Cell Proteomics ; 20: 100113, 2021.
Article in English | MEDLINE | ID: covidwho-1275575

ABSTRACT

Severe coronavirus disease 2019 (COVID-19) can result in pneumonia and acute respiratory failure. Accumulation of mucus in the airways is a hallmark of the disease and can result in hypoxemia. Here, we show that quantitative proteome analysis of the sputum from severe patients with COVID-19 reveal high levels of neutrophil extracellular trap (NET) components, which was confirmed by microscopy. Extracellular DNA from excessive NET formation can increase sputum viscosity and lead to acute respiratory distress syndrome. Recombinant human DNase (Pulmozyme; Roche) has been shown to be beneficial in reducing sputum viscosity and improve lung function. We treated five patients pwith COVID-19 resenting acute symptoms with clinically approved aerosolized Pulmozyme. No adverse reactions to the drug were seen, and improved oxygen saturation and recovery in all severely ill patients with COVID-19 was observed after therapy. Immunofluorescence and proteome analysis of sputum and blood plasma samples after treatment revealed a marked reduction of NETs and a set of statistically significant proteome changes that indicate reduction of hemorrhage, plasma leakage and inflammation in the airways, and reduced systemic inflammatory state in the blood plasma of patients. Taken together, the results indicate that NETs contribute to acute respiratory failure in COVID-19 and that degrading NETs may reduce dependency on external high-flow oxygen therapy in patients. Targeting NETs using recombinant human DNase may have significant therapeutic implications in COVID-19 disease and warrants further studies.


Subject(s)
COVID-19/drug therapy , Deoxyribonuclease I/pharmacology , Extracellular Traps/metabolism , Proteome/analysis , Aged , Blood Proteins/analysis , COVID-19/metabolism , COVID-19/therapy , Female , Fluorescent Antibody Technique , Humans , Male , Middle Aged , Recombinant Proteins/pharmacology , Severity of Illness Index , Sputum/drug effects , Sputum/metabolism , Sputum/virology , Systemic Inflammatory Response Syndrome/drug therapy , Systemic Inflammatory Response Syndrome/metabolism , Systemic Inflammatory Response Syndrome/virology
19.
Biochem Biophys Res Commun ; 546: 97-102, 2021 03 26.
Article in English | MEDLINE | ID: covidwho-1062239

ABSTRACT

The SARS-CoV-2 virus causes elevated production of senescence-associated secretory phenotype (SASP) markers by macrophages. SARS-CoV-2 enters macrophages through its Spike-protein aided by cathepsin (Cat) B and L, which also mediate SASP production. Since M-CSF and IL-34 control macrophage differentiation, we investigated the age-dependent effects of the Spike-protein on SASP-related pro-inflammatory-cytokines and nuclear-senescence-regulatory-factors, and CatB, L and K, in mouse M-CSF- and IL-34-differentiated macrophages. The Spike-protein upregulated SASP expression in young and aged male M-CSF-macrophages. In contrast, only young and aged male IL-34-macrophages demonstrated significantly reduced pro-inflammatory cytokine expression in response to the Spike-protein in vitro. Furthermore, the S-protein elevated CatB expression in young male M-CSF-macrophages and young female IL-34-macrophages, whereas CatL was overexpressed in young male IL-34- and old male M-CSF-macrophages. Surprisingly, the S-protein increased CatK activity in young and aged male M-CSF-macrophages, indicating that CatK may be also involved in the COVID-19 pathology. Altogether, we demonstrated the age- and sex-dependent effects of the Spike-protein on M-CSF and IL-34-macrophages using a novel in vitro mouse model of SARS-CoV-2/COVID-19.


Subject(s)
Age Factors , Macrophages/virology , Spike Glycoprotein, Coronavirus/pharmacology , Animals , Cathepsins/metabolism , Cell Differentiation , Cellular Senescence , Cytokines/metabolism , Female , Interleukins , Macrophage Colony-Stimulating Factor , Macrophages/cytology , Male , Mice , Mice, Inbred C57BL , Recombinant Proteins/pharmacology , SARS-CoV-2 , Sex Factors
20.
J Cell Mol Med ; 25(3): 1342-1349, 2021 02.
Article in English | MEDLINE | ID: covidwho-1030565

ABSTRACT

SARS-CoV-2, the virus responsible for the global coronavirus disease (COVID-19) pandemic, attacks multiple organs of the human body by binding to angiotensin-converting enzyme 2 (ACE2) to enter cells. More than 20 million people have already been infected by the virus. ACE2 is not only a functional receptor of COVID-19 but also an important endogenous antagonist of the renin-angiotensin system (RAS). A large number of studies have shown that ACE2 can reverse myocardial injury in various cardiovascular diseases (CVDs) as well as is exert anti-inflammatory, antioxidant, anti-apoptotic and anticardiomyocyte fibrosis effects by regulating transforming growth factor beta, mitogen-activated protein kinases, calcium ions in cells and other major pathways. The ACE2/angiotensin-(1-7)/Mas receptor axis plays a decisive role in the cardiovascular system to combat the negative effects of the ACE/angiotensin II/angiotensin II type 1 receptor axis. However, the underlying mechanism of ACE2 in cardiac protection remains unclear. Some approaches for enhancing ACE2 expression in CVDs have been suggested, which may provide targets for the development of novel clinical therapies. In this review, we aimed to identify and summarize the role of ACE2 in CVDs.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/complications , Cardiovascular Diseases/metabolism , Angiotensin-Converting Enzyme 2/pharmacology , Animals , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/physiopathology , COVID-19/drug therapy , COVID-19/metabolism , Cardiovascular Diseases/complications , Cardiovascular Diseases/physiopathology , Diminazene/pharmacology , Heart Failure/etiology , Humans , Hypertension/metabolism , Hypertension/physiopathology , Myocardial Infarction/drug therapy , Myocardial Infarction/metabolism , Myocardial Infarction/physiopathology , Recombinant Proteins/pharmacology
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