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1.
Toxicol In Vitro ; 84: 105449, 2022 Oct.
Article in English | MEDLINE | ID: covidwho-1956358

ABSTRACT

Biocidal disinfectants (BDs) that kill microorganisms or pathogens are widely used in hospitals and other healthcare fields. Recently, the use of BDs has rapidly increased as personal hygiene has become more apparent owing to the pandemic, namely the coronavirus outbreak. Despite frequent exposure to BDs, toxicity data of their potential neurotoxicity (NT) are lacking. In this study, a human-derived SH-SY5Y/astrocyte was used as a co-culture model to evaluate the chemical effects of BDs. Automated high-content screening was used to evaluate the potential NT of BDs through neurite growth analysis. A set of 12 BD substances classified from previous reports were tested. Our study confirms the potential NT of benzalkonium chloride (BKC) and provides the first evidence of the potential NT of poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride (PHMB). BKC and PHMB showed significant NT at concentrations without cytotoxicity. This test system for analyzing the potential NT of BDs may be useful in early screening studies for NT prior to starting in vivo studies.


Subject(s)
Disinfectants , Neuroblastoma , Neurotoxicity Syndromes , Astrocytes , Benzalkonium Compounds/toxicity , Coculture Techniques , Disinfectants/toxicity , Humans , Neurons
2.
Biomolecules ; 12(4)2022 03 31.
Article in English | MEDLINE | ID: covidwho-1776123

ABSTRACT

Intercellular communication between monocytes/macrophages and cells involved in tissue regeneration, such as mesenchymal stromal cells (MSCs) and primary tissue cells, is essential for tissue regeneration and recovery of homeostasis. Typically, in the final phase of the inflammation-resolving process, this intercellular communication drives an anti-inflammatory immunomodulatory response. To obtain a safe and effective treatment to counteract the cytokine storm associated with a disproportionate immune response to severe infections, including that associated with COVID-19, by means of naturally balanced immunomodulation, our group has standardized the production under GMP-like conditions of a secretome by coculture of macrophages and MSCs. To characterize this proteome, we determined the expression of molecules related to cellular immune response and tissue regeneration, as well as its possible toxicity and anti-inflammatory potency. The results show a specific molecular pattern of interaction between the two cell types studied, with an anti-inflammatory and regenerative profile. In addition, the secretome is not toxic by itself on human PBMC or on THP-1 monocytes and prevents lipopolysaccharide (LPS)-induced growth effects on those cell types. Finally, PRS CK STORM prevents LPS-induced TNF-A and IL-1Β secretion from PBMC and from THP-1 cells at the same level as hydrocortisone, demonstrating its anti-inflammatory potency.


Subject(s)
COVID-19 , Mesenchymal Stem Cells , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Coculture Techniques , Culture Media, Conditioned/pharmacology , Humans , Leukocytes, Mononuclear , Lipopolysaccharides/pharmacology , Monocytes
3.
Cells ; 11(6)2022 03 17.
Article in English | MEDLINE | ID: covidwho-1760409

ABSTRACT

Cancer therapy is an emergent application for mRNA therapeutics. While in tumor immunotherapy, mRNA encoding for tumor-associated antigens is delivered to antigen-presenting cells in spleen and lymph nodes, other therapeutic options benefit from immediate delivery of mRNA nanomedicines directly to the tumor. However, tumor targeting of mRNA therapeutics is still a challenge, since, in addition to delivery of the cargo to the tumor, specifics of the targeted cell type as well as its interplay with the tumor microenvironment are crucial for successful intervention. This study investigated lipoplex nanoparticle-mediated mRNA delivery to spheroid cell culture models of melanoma. Insights into cell-type specific targeting, non-cell-autonomous effects, and penetration capacity in tumor and stroma cells of the mRNA lipoplex nanoparticles were obtained. It was shown that both coculture of different cell types as well as three-dimensional cell growth characteristics can modulate distribution and transfection efficiency of mRNA lipoplex formulations. The results demonstrate that three-dimensional coculture spheroids can provide a valuable surplus of information in comparison to adherent cells. Thus, they may represent in vitro models with enhanced predictivity for the in vivo activity of cancer nanotherapeutics.


Subject(s)
Melanoma , Nanoparticles , Coculture Techniques , Humans , Melanoma/therapy , Nanoparticles/therapeutic use , RNA , RNA, Messenger/genetics , Tumor Microenvironment
4.
Front Immunol ; 13: 821595, 2022.
Article in English | MEDLINE | ID: covidwho-1686485

ABSTRACT

Heterologous immunity, when the memory T cell response elicited by one pathogen recognizes another pathogen, has been offered as a contributing factor for the high variability in coronavirus disease 2019 (COVID-19) severity outcomes. Here we demonstrate that sensitization with bacterial peptides can induce heterologous immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) derived peptides and that vaccination with the SARS-CoV-2 spike protein can induce heterologous immunity to bacterial peptides. Using in silico prediction methods, we identified 6 bacterial peptides with sequence homology to either the spike protein or non-structural protein 3 (NSP3) of SARS-CoV-2. Notwithstanding the effects of bystander activation, in vitro co-cultures showed that all individuals tested (n=18) developed heterologous immunity to SARS-CoV-2 peptides when sensitized with the identified bacterial peptides. T cell recall responses measured included cytokine production (IFN-γ, TNF, IL-2), activation (CD69) and proliferation (CellTrace). As an extension of the principle of heterologous immunity between bacterial pathogens and COVID-19, we tracked donor responses before and after SARS-CoV-2 vaccination and measured the cross-reactive T cell responses to bacterial peptides with similar sequence homology to the spike protein. We found that SARS-CoV-2 vaccination could induce heterologous immunity to bacterial peptides. These findings provide a mechanism for heterologous T cell immunity between common bacterial pathogens and SARS-CoV-2, which may explain the high variance in COVID-19 outcomes from asymptomatic to severe. We also demonstrate proof-of-concept that SARS-CoV-2 vaccination can induce heterologous immunity to pathogenic bacteria derived peptides.


Subject(s)
Bacterial Infections/immunology , COVID-19/immunology , Immunity, Heterologous/immunology , SARS-CoV-2/immunology , T-Lymphocytes/immunology , Adult , COVID-19 Vaccines/immunology , Cells, Cultured , Coculture Techniques , Female , Humans , Immunity, Cellular/immunology , Male , Spike Glycoprotein, Coronavirus/immunology
5.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Article in English | MEDLINE | ID: covidwho-1655773

ABSTRACT

SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography-tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines.


Subject(s)
Endothelial Cells/virology , Extracellular Space/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Vimentin/metabolism , Virus Internalization , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Coculture Techniques , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Endothelium, Vascular/virology , HEK293 Cells , Humans , Protein Binding
6.
Proc Natl Acad Sci U S A ; 119(1)2022 01 04.
Article in English | MEDLINE | ID: covidwho-1595265

ABSTRACT

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.


Subject(s)
COVID-19/immunology , Epithelial Cells/immunology , Monocytes/immunology , SARS-CoV-2/pathogenicity , Adult , B-Lymphocytes/immunology , COVID-19/pathology , Child , Coculture Techniques , Ebolavirus/pathogenicity , Epithelial Cells/virology , Gene Expression Profiling , Humans , Inflammation , Influenza A virus/pathogenicity , Lung/immunology , Myeloid Cells/immunology , Species Specificity , Viral Proteins/immunology
7.
Cell Transplant ; 30: 9636897211054481, 2021.
Article in English | MEDLINE | ID: covidwho-1511642

ABSTRACT

Biological and cellular interleukin-6 (IL-6)-related therapies have been used to treat severe COVID-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure, which prompted further exploration of the role of IL-6 in human umbilical cord mesenchymal stem cell (hUCMSC) therapy. Peripheral blood mononuclear cells (PBMCs) were responders cocultured with hUCMSCs or exogenous IL-6. A PBMC suppression assay was used to analyze the anti-inflammatory effects via MTT assay. The IL-6 concentration in the supernatant was measured using ELISA. The correlation between the anti-inflammatory effect of hUCMSCs and IL-6 levels and the relevant roles of IL-6 and IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was further evaluated. We reported that hUCMSCs did not initially exert any inhibitory effect on PHA-stimulated proliferation; however, a potent inhibitory effect on PHA-stimulated proliferation was observed, and the IL-6 concentration reached approximately 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 inhibited PHA-stimulated inflammation but less so than hUCMSCs. The inhibitory effects of hUCMSCs on PHA-stimulated PBMCs disappeared after adding an IL-6 neutralizing antibody or pretreatment with tocilizumab (TCZ), an IL-6R antagonist. hUCMSCs exert excellent anti-inflammatory effects by inducing higher IL-6 levels, which is different from TCZ. High concentration of IL-6 cytokine secretion plays an important role in the anti-inflammatory effect of hUCMSC therapy. Initial hUCMSC therapy, followed by TCZ, seems to optimize the therapeutic potential to treat COVID-19-related acute respiratory distress syndrome (ARDS).


Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , COVID-19/complications , Interleukin-6/biosynthesis , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Respiratory Distress Syndrome/therapy , SARS-CoV-2 , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Neutralizing/immunology , Cells, Cultured , Coculture Techniques , Combined Modality Therapy , DNA, Complementary/genetics , Gene Expression Regulation/drug effects , Humans , Inflammation , Interleukin-6/genetics , Interleukin-6/pharmacology , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation/drug effects , Phytohemagglutinins/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Interleukin-6/antagonists & inhibitors , Receptors, Interleukin-6/biosynthesis , Receptors, Interleukin-6/genetics , Respiratory Distress Syndrome/drug therapy , Respiratory Distress Syndrome/etiology , Umbilical Cord/cytology
8.
Viruses ; 13(11)2021 10 28.
Article in English | MEDLINE | ID: covidwho-1488759

ABSTRACT

We aimed to explore whether variants of SARS-CoV-2 (Chinese-derived strain (D614, lineage A), Italian strain PV10734 (D614G, lineage B.1.1) and Alpha strain (lineage B.1.1.7)) were able to infect monocytes (MN) and monocyte-derived macrophages (MDM) and whether these infected cells may, in turn, be vectors of infection. For this purpose, we designed an in vitro study following the evolution of MN and MDM infection at different time points in order to confirm whether these cells were permissive for SARS-CoV-2 replication. Finally, we investigated whether, regardless of viral replication, the persistent virus can be transferred to non-infected cells permissive for viral replication. Thus, we co-cultured the infected MN/MDM with permissive VERO E6 cells verifying the viral transmission. This is a further in vitro demonstration of the important role of MN and MDM in the dissemination of SARS-CoV-2 and evolution of the COVID-19 disease.


Subject(s)
Macrophages/virology , Monocytes/virology , SARS-CoV-2/physiology , Animals , Chlorocebus aethiops , Coculture Techniques , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Macrophages/ultrastructure , Monocytes/ultrastructure , Phosphoproteins/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Internalization , Virus Replication
9.
Cells ; 10(10)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470800

ABSTRACT

Pulmonary epithelial cells are widely considered to be the first line of defence in the lung and are responsible for coordinating the innate immune response to injury and subsequent repair. Consequently, epithelial cells communicate with multiple cell types including immune cells and fibroblasts to promote acute inflammation and normal wound healing in response to damage. However, aberrant epithelial cell death and damage are hallmarks of pulmonary disease, with necrotic cell death and cellular senescence contributing to disease pathogenesis in numerous respiratory diseases such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and coronavirus disease (COVID)-19. In this review, we summarise the literature that demonstrates that epithelial damage plays a pivotal role in the dysregulation of the immune response leading to tissue destruction and abnormal remodelling in several chronic diseases. Specifically, we highlight the role of epithelial-derived damage-associated molecular patterns (DAMPs) and senescence in shaping the immune response and assess their contribution to inflammatory and fibrotic signalling pathways in the lung.


Subject(s)
COVID-19/immunology , Epithelium/immunology , Idiopathic Pulmonary Fibrosis/immunology , Lung/immunology , Alarmins , Animals , Cellular Senescence , Coculture Techniques , Epithelial Cells/cytology , Epithelial Cells/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Fibrosis/metabolism , Humans , Idiopathic Pulmonary Fibrosis/metabolism , Immunity , Inflammation/metabolism , Ligands , Necroptosis , Necrosis/pathology , Pulmonary Disease, Chronic Obstructive , SARS-CoV-2 , Signal Transduction
10.
Cells ; 10(10)2021 10 03.
Article in English | MEDLINE | ID: covidwho-1444118

ABSTRACT

The PD-L1/PD-1 immune checkpoint axis is the strongest T cell exhaustion inducer. As immune dysfunction occurs during obesity, we analyzed the impact of obesity on PD-L1/PD-1 expression in white adipose tissue (WAT) in mice and in human white adipocytes. We found that PD-L1 was overexpressed in WAT of diet-induced obese mice and was associated with increased expression of PD-1 in visceral but not subcutaneous WAT. Human in vitro cocultures with adipose-tissue-derived mesenchymal stem cells (ASC) and mononuclear cells demonstrated that the presence of ASC harvested from obese WAT (i) enhanced PD-L1 expression as compared with ASC from lean WAT, (ii) decreased Th1 cell cytokine secretion, and (iii) resulted in decreased cytolytic activity towards adipocytes. Moreover, (iv) the implication of PD-L1 in obese ASC-mediated T cell dysfunction was demonstrated through PD-L1 blockade. Finally, (v) conditioned media gathered from these cocultures enhanced PD-L1 expression in freshly differentiated adipocytes, depending on IFNγ. Altogether, our results suggest that PD-L1 is overexpressed in the WAT of obese individuals during IFNγ secretion, leading to T cell dysfunction and notably reduced cytolytic activity. Such a mechanism could shed light on why adipose-tissue-infiltrating viruses, such as SARS-CoV-2, can worsen disease in obese individuals.


Subject(s)
Adipose Tissue, White/metabolism , B7-H1 Antigen/biosynthesis , Gene Expression Regulation , Mesenchymal Stem Cells/cytology , Obesity/metabolism , T-Lymphocytes/immunology , Animals , COVID-19/immunology , Cell Differentiation , Coculture Techniques , Humans , Immunohistochemistry , Inflammation , Interferon-gamma/metabolism , Leukocytes, Mononuclear/cytology , Male , Mice , Mice, Inbred C57BL , Obesity/immunology , SARS-CoV-2 , T-Lymphocytes/cytology
12.
Front Immunol ; 12: 692729, 2021.
Article in English | MEDLINE | ID: covidwho-1369667

ABSTRACT

Epidemiological studies and clinical trials suggest Bacillus Calmette-Guérin (BCG) vaccine has protective effects against coronavirus disease 2019 (COVID-19). There are now over 30 clinical trials evaluating if BCG vaccination can prevent or reduce the severity of COVID-19. However, the mechanism by which BCG vaccination can induce severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses is unknown. Here, we identify 8 novel BCG-derived peptides with significant sequence homology to either SARS-CoV-2 NSP3 or NSP13-derived peptides. Using an in vitro co-culture system, we show that human CD4+ and CD8+ T cells primed with a BCG-derived peptide developed enhanced reactivity to its corresponding homologous SARS-CoV-2-derived peptide. As expected, HLA differences between individuals meant that not all persons developed immunogenic responses to all 8 BCG-derived peptides. Nevertheless, all of the 20 individuals that were primed with BCG-derived peptides developed enhanced T cell reactivity to at least 7 of 8 SARS-CoV-2-derived peptides. These findings provide an in vitro mechanism that may account, in part, for the epidemiologic observation that BCG vaccination confers some protection from COVID-19.


Subject(s)
BCG Vaccine/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cross Reactions , SARS-CoV-2/immunology , Adult , COVID-19/immunology , COVID-19/prevention & control , Cells, Cultured , Coculture Techniques , Female , Flow Cytometry , Humans , Male , Sequence Analysis, Protein , Sequence Homology , Vaccines, Subunit/immunology , Young Adult
13.
J Hazard Mater ; 421: 126679, 2022 01 05.
Article in English | MEDLINE | ID: covidwho-1313241

ABSTRACT

Intensive disinfection of wastewater during the COVID-19 pandemic might elevate the generation of toxic disinfection byproducts (DBPs), which has triggered global concerns about their ecological risks to natural aquatic ecosystems. In this study, the toxicity of 17 DBPs typically present in wastewater effluents on three representative microalgae, including Scenedesmus sp. (Chlorophyta), Microcystis aeruginosa (Cyanophyta), and Cyclotella sp. (Bacillariophyta) was investigated. The sensitivities of the three microalgae to DBPs varied greatly from species to species, indicating that DBPs may change the structure of phytoplankton communities. Later, co-cultures of these phytoplankton groups as a proxy of ecological freshwater scenario were conducted to explore the impacts of DBPs on phytoplankton community succession. M. aeruginosa became surprisingly dominant in co-cultures, representing over 50% after dosing with monochloroacetic acid (MCAA, 0.1-10 mg/L). The highest proportion of M. aeruginosa was 70.3% when exposed to 2 mg/L MCAA. Although Scenedesmus sp. dominated in monochloroacetonitrile (MCAN) exposure, M. aeruginosa accounted for no less than 30% even at 40 mg/L MCAN. In this study, DBPs disrupted the original inter-algal relationship in favor of M. aeruginosa, suggesting that DBPs may contribute to the outbreak of cyanobacterial blooms in aquatic ecosystems.


Subject(s)
Disinfectants/toxicity , Phytoplankton/drug effects , Scenedesmus , Coculture Techniques , Disinfection , Ecosystem , Fresh Water , Scenedesmus/drug effects
14.
Adv Healthc Mater ; 10(15): e2100879, 2021 08.
Article in English | MEDLINE | ID: covidwho-1283191

ABSTRACT

High-throughput tissue barrier models can yield critical insights on how barrier function responds to therapeutics, pathogens, and toxins. However, such models often emphasize multiplexing capability at the expense of physiologic relevance. Particularly, the distal lung's air-blood barrier is typically modeled with epithelial cell monoculture, neglecting the substantial contribution of endothelial cell feedback in the coordination of barrier function. An obstacle to establishing high-throughput coculture models relevant to the epithelium/endothelium interface is the requirement for underside cell seeding, which is difficult to miniaturize and automate. Therefore, this paper describes a scalable, low-cost seeding method that eliminates inversion by optimizing medium density to float cells so they attach under the membrane. This method generates a 96-well model of the distal lung epithelium-endothelium barrier with serum-free, glucocorticoid-free air-liquid differentiation. The polarized epithelial-endothelial coculture exhibits mature barrier function, appropriate intercellular junction staining, and epithelial-to-endothelial transmission of inflammatory stimuli such as polyinosine:polycytidylic acid (poly(I:C)). Further, exposure to influenza A virus PR8 and human beta-coronavirus OC43 initiates a dose-dependent inflammatory response that propagates from the epithelium to endothelium. While this model focuses on the air-blood barrier, the underside seeding method is generalizable to various coculture tissue models for scalable, physiologic screening.


Subject(s)
Blood-Air Barrier , Lung , Coculture Techniques , Endothelial Cells , Epithelium , Humans
15.
Microb Biotechnol ; 13(4): 997-1011, 2020 07.
Article in English | MEDLINE | ID: covidwho-1280255

ABSTRACT

In contrast to the current paradigm of using microbial mono-cultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed-species consortia to perform functions that are difficult to programme into individual strains. In this work, we developed a synthetic microbial consortium composed of two genetically engineered microbes, a cyanobacterium (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). These microbial species specialize in the co-culture: cyanobacteria fix CO2 through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P. putida, which has been engineered to consume sucrose and to degrade the environmental pollutant 2,4-dinitrotoluene (2,4-DNT). By encapsulating S. elongatus within a barium-alginate hydrogel, cyanobacterial cells were protected from the toxic effects of 2,4-DNT, enhancing the performance of the co-culture. The synthetic consortium was able to convert 2,4-DNT with light and CO2 as key inputs, and its catalytic performance was stable over time. Furthermore, cycling this synthetic consortium through low nitrogen medium promoted the sucrose-dependent accumulation of polyhydroxyalkanoate, an added-value biopolymer, in the engineered P. putida strain. Altogether, the synthetic consortium displayed the capacity to remediate the industrial pollutant 2,4-DNT while simultaneously synthesizing biopolymers using light and CO2 as the primary inputs.


Subject(s)
Pseudomonas putida , Biotransformation , Coculture Techniques , Dinitrobenzenes , Pseudomonas putida/genetics , Synechococcus
16.
Stem Cells Dev ; 30(15): 758-772, 2021 08 01.
Article in English | MEDLINE | ID: covidwho-1254367

ABSTRACT

Cytokine storm is recognized as one of the factors contributing to organ failures and mortality in patients with COVID-19. Due to chronic inflammation, COVID-19 patients with diabetes mellitus (DM) or renal disease (RD) have more severe symptoms and higher mortality. However, the factors that contribute to severe outcomes of COVID-19 patients with DM and RD have received little attention. In an effort to investigate potential treatments for COVID-19, recent research has focused on the immunomodulation functions of mesenchymal stem cells (MSCs). In this study, the correlation between DM and RD and the severity of COVID-19 was examined by a combined approach with a meta-analysis and experimental research. The results of a systematic review and meta-analysis suggested that the odd of mortality in patients with both DM and RD was increased in comparison to those with a single comorbidity. In addition, in the experimental research, the data showed that high glucose and uremic toxins contributed to the induction of cytokine storm in human lung adenocarcinoma epithelial cells (Calu-3 cells) in response to SARS-CoV Peptide Pools. Of note, the incorporation of Wharton's jelly MSC-derived extracellular vesicles (WJ-EVs) into SARS-CoV peptide-induced Calu-3 resulted in a significant decrease in nuclear NF-κB p65 and the downregulation of the cytokine storm under high concentrations of glucose and uremic toxins. This clearly suggests the potential for WJ-EVs to reduce cytokine storm reactions in patients with both chronic inflammation diseases and viral infection.


Subject(s)
Cytokine Release Syndrome/prevention & control , Extracellular Vesicles/physiology , Mesenchymal Stem Cells/cytology , SARS-CoV-2/physiology , Wharton Jelly/cytology , Adult , Aged , COVID-19/blood , COVID-19/complications , COVID-19/metabolism , COVID-19/therapy , Cells, Cultured , Coculture Techniques , Cytokine Release Syndrome/genetics , Cytokine Release Syndrome/metabolism , Cytokine Release Syndrome/virology , Cytokines/genetics , Cytokines/metabolism , Diabetes Complications/blood , Diabetes Complications/metabolism , Diabetes Complications/therapy , Diabetes Complications/virology , Diabetes Mellitus/blood , Diabetes Mellitus/metabolism , Diabetes Mellitus/therapy , Diabetes Mellitus/virology , Dose-Response Relationship, Drug , Female , Gene Expression Regulation/drug effects , Glucose/metabolism , Glucose/pharmacology , Humans , Inflammation Mediators/metabolism , Male , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/physiology , Pregnancy , Toxins, Biological/metabolism , Toxins, Biological/pharmacology , Umbilical Cord/cytology , Uremia/blood , Uremia/complications , Uremia/metabolism , Uremia/therapy
17.
Elife ; 102021 04 23.
Article in English | MEDLINE | ID: covidwho-1200330

ABSTRACT

Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.


Subject(s)
COVID-19/pathology , Giant Cells/pathology , Host-Pathogen Interactions , SARS-CoV-2/physiology , Virus Internalization , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Cholesterol , Coculture Techniques , Humans , Lung/pathology , Membrane Fusion , Membrane Lipids/metabolism
19.
Arterioscler Thromb Vasc Biol ; 41(1): 415-429, 2021 01.
Article in English | MEDLINE | ID: covidwho-1027162

ABSTRACT

OBJECTIVE: The study's aim was to analyze the capacity of human valve interstitial cells (VICs) to participate in aortic valve angiogenesis. Approach and Results: VICs were isolated from human aortic valves obtained after surgery for calcific aortic valve disease and from normal aortic valves unsuitable for grafting (control VICs). We examined VIC in vitro and in vivo potential to differentiate in endothelial and perivascular lineages. VIC paracrine effect was also examined on human endothelial colony-forming cells. A pathological VIC (VICp) mesenchymal-like phenotype was confirmed by CD90+/CD73+/CD44+ expression and multipotent-like differentiation ability. When VICp were cocultured with endothelial colony-forming cells, they formed microvessels by differentiating into perivascular cells both in vivo and in vitro. VICp and control VIC conditioned media were compared using serial ELISA regarding quantification of endothelial and angiogenic factors. Higher expression of VEGF (vascular endothelial growth factor)-A was observed at the protein level in VICp-conditioned media and confirmed at the mRNA level in VICp compared with control VIC. Conditioned media from VICp induced in vitro a significant increase in endothelial colony-forming cell proliferation, migration, and sprouting compared with conditioned media from control VIC. These effects were inhibited by blocking VEGF-A with blocking antibody or siRNA approach, confirming VICp involvement in angiogenesis by a VEGF-A dependent mechanism. CONCLUSIONS: We provide here the first proof of an angiogenic potential of human VICs isolated from patients with calcific aortic valve disease. These results point to a novel function of VICp in valve vascularization during calcific aortic valve disease, with a perivascular differentiation ability and a VEGF-A paracrine effect. Targeting perivascular differentiation and VEGF-A to slow calcific aortic valve disease progression warrants further investigation.


Subject(s)
Aortic Valve Stenosis/metabolism , Aortic Valve/metabolism , Aortic Valve/pathology , Calcinosis/metabolism , Cell Differentiation , Cell Lineage , Endothelial Progenitor Cells/metabolism , Neovascularization, Pathologic , Vascular Endothelial Growth Factor A/metabolism , Adult , Aged , Aged, 80 and over , Animals , Aortic Valve Stenosis/pathology , Calcinosis/pathology , Case-Control Studies , Cells, Cultured , Coculture Techniques , Endothelial Progenitor Cells/pathology , Endothelial Progenitor Cells/transplantation , Female , Humans , Male , Mice, Nude , Middle Aged , Osteogenesis , Paracrine Communication , Phenotype , Signal Transduction , Vascular Endothelial Growth Factor A/genetics
20.
Mol Cell Biochem ; 476(1): 93-107, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-737128

ABSTRACT

Mesenchymal stem cells (MSCs) can alleviate acute respiratory distress syndrome (ARDS), but the mechanisms involved are unclear, especially about their specific effects on cellular mitochondrial respiratory function. Thirty mice were allocated into the Control, LPS, and LPS + Bone marrow mesenchymal stem cell (BMSC) group (n = 10/group). Mouse alveolar epithelial cells (MLE-12) and macrophage cells (RAW264.7) were divided into the same groups. Pathological variation, inflammation-related factors, reactive oxygen species (ROS), ATP levels, and oxygen consumption rate (OCR) were analyzed. Pathologic features of ARDS were observed in the LPS group and were significantly alleviated by BMSCs. The trend in inflammation-related factors among the three groups was the LPS group > LPS + BMSC group > Control group. In the MLE-12 co-culture system, IL-6 was increased in the LPS group but not significantly reduced in the LPS + BMSC group. In the RAW264.7 co-culture system, IL-1ß, TNF-α, and IL-10 levels were all increased in the LPS group, IL-1ß and TNF-α levels were reduced by BMSCs, while IL-10 level kept increasing. ROS and ATP levels were increased and decreased respectively in both MLE-12 and RAW264.7 cells in the LPS groups but reversed by BMSCs. Basal OCR, ATP-linked OCR, and maximal OCR were lower in the LPS groups. Impaired basal OCR and ATP-linked OCR in MLE-12 cells were partially restored by BMSCs, while impaired basal OCR and maximal OCR in RAW264.7 cells were restored by BMSCs. BMSCs improved the mitochondrial respiration dysfunction of macrophages and alveolar epithelial cells induced by LPS, alleviated lung tissue injury, and inflammatory response in a mouse model of ARDS.


Subject(s)
Epithelium/metabolism , Mesenchymal Stem Cells/cytology , Mitochondria/metabolism , Pulmonary Alveoli/metabolism , Respiratory Distress Syndrome/metabolism , Adenosine Triphosphate/metabolism , Animals , Bone Marrow Cells/cytology , Coculture Techniques , Inflammation , Interleukin-10/metabolism , Interleukin-6/metabolism , Lipopolysaccharides/metabolism , Lung Injury/metabolism , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Oxygen Consumption , RAW 264.7 Cells
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