Acriflavine, a clinically aproved drug, inhibits SARS-CoV-2 and other betacoronaviruses

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identified acriflavine as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

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Development and Optimization of A High-Throughput 3D Rat Purkinje Neuron Culture to Study Paraneoplastic Cerebellar Degeneration

10.21203/rs.3.rs-1166640/v1 ◽

2022 ◽

Author(s):

Ida Margrethe Uggerud ◽

Torbjorn Krakenes ◽

Hirokazu Hirai ◽

Christian Alexander Vedeler ◽

Manja Schubert

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Ex Vivo ◽

Purkinje Neuron ◽

Cerebellar Degeneration ◽

Paraneoplastic Cerebellar Degeneration ◽

Purkinje Neurons ◽

Branch Order ◽

Cellular Models

Abstract Improved understanding of the mechanisms involved in neurodegenerative disease has been hampered by the lack of robust cellular models that faithfully replicate in vivo features. Here, we present a refined protocol for generating age-dependent, well-developed and synaptically active rat Purkinje neurons in a 3D cell network culture which are responsive to a disease inducer. Using our model, we found that the application of autoantibody Yo, a paraneoplastic cerebellar degeneration (PCD) inducer, alters the structure of the dendritic arbour of cultured Purkinje neurons. The numbers of dendrites per branch-order, the branch-order in itself and the dendritic length were reduced by anti-Yo, proving a functional role for anti-Yo in the pathogenesis of PCD. Our new ex-vivo model is flexible and can be used to investigate disease mechanisms that disturb Purkinje neuron function and communication in 3D. Since it is possible to use the approach in a multi-well format, this method also has high-throughput screening potential.

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Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

Nature Communications ◽

10.1038/s41467-021-24730-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

David S. Fischer ◽

Meshal Ansari ◽

Karolin I. Wagner ◽

Sebastian Jarosch ◽

Yiqi Huang ◽

...

Keyword(s):

T Cells ◽

Single Cell ◽

Rna Sequencing ◽

Ex Vivo ◽

Severe Disease ◽

Human Leukocyte ◽

Cell Receptor ◽

Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

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Transcriptomic Analysis of Inbred Chicken Lines Reveals Infectious Bursal Disease Severity Is Associated with Greater Bursal Inflammation In Vivo and More Rapid Induction of Pro-Inflammatory Responses in Primary Bursal Cells Stimulated Ex Vivo

Viruses ◽

10.3390/v13050933 ◽

2021 ◽

Vol 13 (5) ◽

pp. 933

Author(s):

Amin S. Asfor ◽

Salik Nazki ◽

Vishwanatha R.A.P. Reddy ◽

Elle Campbell ◽

Katherine L. Dulwich ◽

...

Keyword(s):

Rho Gtpases ◽

Inflammatory Responses ◽

Ex Vivo ◽

Severe Disease ◽

Infectious Bursal Disease ◽

Rna Seq ◽

Rapid Induction ◽

Cytoskeletal Regulation ◽

Bursal Disease

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p < 0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

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Transcriptomic analysis reveals that severity of infectious bursal disease in White Leghorn inbred chicken lines is associated with greater bursal inflammation in vivo and more rapid induction of pro-inflammatory responses in primary bursal cells stimulated ex vivo.

10.1101/2021.03.18.436042 ◽

2021 ◽

Author(s):

Amin S Asfor ◽

Salik Nazki ◽

Vishwanatha RAP Reddy ◽

Elle Campbell ◽

Katherine L Dulwich ◽

...

Keyword(s):

Rho Gtpases ◽

Inflammatory Responses ◽

Ex Vivo ◽

Severe Disease ◽

Infectious Bursal Disease ◽

White Leghorn ◽

Rapid Induction ◽

Cytoskeletal Regulation ◽

Bursal Disease

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p<0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

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Inhibition of Chondroitin-4-Sulfate-Specific Adhesion of Plasmodium falciparum-Infected Erythrocytes by Sulfated Polysaccharides

Infection and Immunity ◽

10.1128/iai.73.7.4288-4294.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4288-4294 ◽

Cited By ~ 31

Author(s):

Katherine T. Andrews ◽

Nicole Klatt ◽

Yvonne Adams ◽

Petra Mischnick ◽

Reinhard Schwartz-Albiez

Keyword(s):

Plasmodium Falciparum ◽

Ex Vivo ◽

Severe Disease ◽

Placental Tissue ◽

Peripheral Circulation ◽

Sulfated Polysaccharides ◽

Mature Stage ◽

Dependent Manner ◽

Inhibitory Compounds

ABSTRACT Adhesion of Plasmodium falciparum-infected erythrocytes to placental chondroitin 4-sulfate (CSA) has been linked to the severe disease outcome of pregnancy-associated malaria. Soluble polysaccharides that release mature-stage parasitized erythrocytes into the peripheral circulation may help elucidate these interactions and have the potential to aid in developing therapeutic strategies. We have screened a panel of 11 sulfated polysaccharides for their capacities to inhibit adhesion of infected erythrocytes to CSA expressed on CHO-K1 cells and ex vivo human placental tissue. Two carrageenans and a cellulose sulfate (CS10) were able to inhibit adhesion to CSA and to cause already bound infected erythrocytes to de-adhere in a dose-dependent manner. CS10, like CSA and in contrast to all other compounds tested, remained bound to infected erythrocytes after washing and continued to inhibit binding. Both carrageenans and CS10 inhibited adhesion to placental tissue. Although highly sulfated dextran sulfate can inhibit CSA-mediated adhesion to CHO cells, this polysaccharide amplified adhesion to placental tissue severalfold, demonstrating the importance of evaluating inhibitory compounds in systems as close to in vivo as possible. Interestingly, and in contrast to all other compounds tested, which had a random distribution of sulfate groups, CS10 exhibited a clustered sulfate pattern along the polymer chain, similar to that of the undersulfated placental CSA preferred by placental-tissue-binding infected erythrocytes. Therefore, the specific antiadhesive capacity observed here seems to depend not only on the degree of charge and sulfation but also on a particular pattern of sulfation.

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Paracrine inhibition of GM-CSF signaling by human cytomegalovirus in monocytes differentiating to dendritic cells

Blood ◽

10.1182/blood-2011-02-337956 ◽

2011 ◽

Vol 118 (26) ◽

pp. 6783-6792 ◽

Cited By ~ 20

Author(s):

Jérome Carlier ◽

Hélène Martin ◽

Bernard Mariamé ◽

Benjamin Rauwel ◽

Catherine Mengelle ◽

...

Keyword(s):

Bystander Effect ◽

Ex Vivo ◽

Severe Disease ◽

Cytokine Signaling ◽

Virus Reactivation ◽

Therapeutic Approaches ◽

Dc Functions ◽

Innate And Adaptive Immunity ◽

Gm Csf

Abstract A primary HCMV infection or virus reactivation may cause severe disease in hosts with a deficient immune system. The virus can disturb both innate and adaptive immunity by targeting dendritic cell (DC) functions. Monocytes, the precursors of DCs in vivo (MoDCs), are the primary targets of HCMV; they can also harbor latent virus. The DCs generated from infected monocytes (CMV-MoDCs) have an altered phenotype and functional defects. We have shown that CMV-MoDCs do not secrete IL-12 in response to lipopolysaccharide stimulation, cannot ingest dead cells, induce TH1 differentiation, or the proliferation of naive allogeneic CD4+ T cells. We found that the GM-CSF signaling in an entire population of CMV-MoDCs was impaired, although only half of the cells were productively infected, and that IL-6 secretion and suppressors of cytokine signaling 3 induction contributed to this bystander effect. We also showed that MoDCs derived ex vivo from monocytes of viremic patients had the same altered phenotype as CMV-MoDCs, including decreased STAT5 phosphorylation, indicating defective GM-CSF signaling. We have thus described a new mechanism of HCMV-induced immunosupression, indicated how infection may disturb both GM-CSF–dependent physiologic processes and proposed GM-CSF–based therapeutic approaches.

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Development and optimization of a high-throughput 3D rat Purkinje neuron culture

10.1101/2020.05.20.105858 ◽

2020 ◽

Author(s):

Ida M. Uggerud ◽

Torbjorn Krakenes ◽

Hirokazu Hirai ◽

Christian A. Vedeler ◽

Manja Schubert

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Purkinje Neuron ◽

Paracrine Factors ◽

Neuron Culture ◽

Cell Network ◽

Cellular Models ◽

Age Dependent ◽

Neuron Function

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Functional properties of edible insects: a systematic review

Nutrition Research Reviews ◽

10.1017/s0954422421000366 ◽

2021 ◽

pp. 1-54

Author(s):

V. D’Antonio ◽

N. Battista ◽

G. Sacchetti ◽

C. Di Mattia ◽

M. Serafini

Keyword(s):

Glucose Metabolism ◽

Functional Role ◽

Dietary Intervention ◽

Ex Vivo ◽

Edible Insects ◽

Cellular Models ◽

Pubmed Database

Abstract Consumption of edible insects has been widely suggested as an environmentally sustainable substitute for meat to reduce GHG emissions. However, the novel research field for edible insects rely on the content of bioactive ingredients and on the ability to induce a functional effect in humans. The goal of this manuscript was to review the available body of evidence on the properties of edible insects in modulating oxidative and inflammatory stress, platelet aggregation, lipid and glucose metabolism and weight control. A search for literature investigating the functional role of edible insects was carried out in the PUBMED database using specific keywords. A total of 55 studies, meeting inclusion criteria after screening, were divided on the basis of the experimental approach: in vitro studies, cellular models/ex vivo studies or in vivo studies. In the majority of the studies, insects demonstrated the ability to reduce oxidative stress, modulate antioxidant status, restore the impaired activity of antioxidant enzymes and reduce markers of oxidative damage. Edible insects displayed anti-inflammatory activity reducing cytokines and modulating specific transcription factors. Results from animal studies suggest that edible insects can modulate lipid and glucose metabolism. The limited number of studies focused on the assessment of anticoagulation activity of edible insects make it difficult to draw conclusions. More evidence from dietary intervention studies in humans is needed to support the promising evidence from in vitro and animal models about the functional role of edible insects consumption.

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Identification of Celastrol as a novel HIV-1 Latency Reversal Agent by an Image-Based Screen

10.1101/2020.12.17.423211 ◽

2020 ◽

Author(s):

Hongbing Liu ◽

Pei-Wen Hu ◽

Julien Dubrulle ◽

Fabio Stossi ◽

Bryan C. Nikolai ◽

...

Keyword(s):

T Cells ◽

Ex Vivo ◽

Research Effort ◽

Viral Reservoir ◽

Reversal Agent ◽

Reversal Agents ◽

Healthy Donors ◽

Latent Hiv ◽

Hiv 1

AbstractAlthough current antiretroviral therapies (ART) are successful in controlling HIV-1 infection, a stable viral reservoir reactivates when ART is discontinued. Consequently, there is a major research effort to develop approaches to disrupt the latent viral reservoir and enhance the immune system’s ability to clear HIV-1. A number of small molecules, termed latency reversal agents (LRAs), have been identified which can reactivate latent HIV-1 in cell lines and patients’ cells ex vivo. However, clinical trials have suggested that combinations of LRAs will be required to efficiently reactivate HIV-1 in vivo, especially LRAs that act synergistically by functioning through distinct pathways. To identify novel LRAs, we used an image-based assay to screen a natural compound library for the ability to induce a low level of aggregation of resting primary CD4+ T cells from healthy donors. We identified celastrol as a novel LRA. Celastrol functions synergistically with other classes of LRA to reactivate latent HIV-1 in a Jurkat cell line, suggesting a novel mechanism in its LRA activity. Additionally, celastrol does not appear to activate resting CD4+ T cells at levels at which it can reactivate latent HIV-1. Celastrol appears to represent a novel class of LRAs and it therefore can serve as a lead compound for LRA development.

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Potential of phenolic compounds and their gut microbiota-derived metabolites to reduce microbial TMA formation: Application of an in vitro fermentation high throughput screening model

10.33774/chemrxiv-2021-q1q7z-v2 ◽

2022 ◽

Author(s):

Lisard Iglesias-Carres ◽

Emily Krueger ◽

Jacob Herring ◽

Jeffery Tessem ◽

Andrew Neilson

Keyword(s):

Gut Microbiota ◽

Caffeic Acid ◽

High Throughput Screening ◽

Ex Vivo ◽

Inhibitory Effects ◽

In Vitro Fermentation ◽

Choline Metabolism ◽

Fermentation Model

Trimethylamine N-oxide (TMAO) is a pro-atherosclerotic product of dietary choline metabolism generated by a microbiome-host axis. The first step in this pathway is enzymatic metabolism of choline to trimethylamine (TMA) by the gut microbiota. This reaction could be targeted to reduce atherosclerosis risk. We aimed to evaluate potential inhibitory effects of select dietary phenolics and their relevant gut microbial metabolites on TMA production via a human ex vivo-in vitro fermentation model. Various phenolics inhibited choline use and TMA production. The most bioactive compounds tested (caffeic acid, catechin and epicatechin) reduced TMA-d9 formation (compared to control) by 57.5 ± 1.3% to 72.5 ± 0.4% at 8 h and preserved remaining choline-d9 concentrations by 194.1 ± 6.4% to 256.1 ± 6.3% compared to control conditions at 8 h. These inhibitory effects were achieved without altering cell respiration or cell growth. However, inhibitory effects decreased at late fermentation times, which suggest that these compounds delay choline metabolism rather than completely inhibiting TMA formation. Overall, caffeic acid, catechin and epicatechin were the most effective non-cytotoxic inhibitors of choline use and TMA production. Thus, these compounds are proposed as lead bioactives to test in vivo.

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