(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .
Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice [1]
['Sandrine Belouzard', 'Univ Lille', 'Cnrs', 'Inserm', 'Chu Lille', 'Institut Pasteur De Lille', 'Center For Infection', 'Immunity Of Lille', 'Lille', 'Arnaud Machelart']
Date: 2022-07
In this study, we report the high-throughput screening of ~2,000 drugs, approved for human use, for their potential activity against SARS-CoV-2. Our data identify clofoctol as a promising antiviral candidate for the treatment of COVID-19 patients. This antibacterial drug was developed in the late 1970s. Its efficacy has been demonstrated for the treatment of Streptococcus pneumoniae—the leading cause of bacterial pneumonia worldwide—and Staphylococcus aureus [15,16]. The drug was marketed in France until 2005 under the trade name Octofène and is still prescribed in Italy under the trademark GramPlus. Mechanistically, clofoctol inhibits bacterial cell wall synthesis and induces membrane permeabilization [17,18]. Along with its bactericidal activity, clofoctol was recently shown to also inhibit protein translation and to impair tumor cell growth [19,20]. As such, clofoctol could be useful to treat some cancers and possibly other diseases [21].
Among the 2,000 drugs tested, clofoctol emerged as the most promising compound to inhibit SARS-CoV-2 replication in our experimental settings. Our data show that it can contribute to inhibition of SARS-CoV-2 propagation by blocking translation of viral RNA. However, we cannot exclude other effects of clofoctol on SARS-CoV-2 replication. The inhibition of translation by clofoctol could be due to the activation of the unfolded protein response (UPR) pathways. Clofoctol has indeed been reported to induce endoplasmic reticulum (ER) stress and to activate all three UPR pathways, i.e. the inositol requiring enzyme 1 (IRE1), the double stranded RNA-activated PK-like ER kinase (PERK), and the activating transcription factor 6 (ATF6) [22]. Although UPR activation is observed during SARS-CoV-2 infection [23], chemical activation of UPR by thapsigargin has been shown to inhibit coronavirus replication, including SARS-CoV-2 [24]. Furthermore, modulating the PERK-eIF2α pathway can inhibit the replication of the transmissible gastroenteritis porcine coronavirus [25]. Similarly, triggering the UPR with 2-deoxy-D-glucose inhibits the replication of another coronavirus, the porcine epidemic diarrhea virus [22]. Whether the clofoctol-induced inhibition of SARS-CoV-2 translation is linked to UPR activation will be the focus of further investigation.
Translation of viral RNA requires interaction of viral RNA with the host cell translational machinery. Several studies have uncovered RNA binding proteins (RBPs) important for infection. It is likely that clofoctol may act on one of these RBPs to inhibit viral translation. Comparison of translational factors recruited by SARS-CoV-2 or flavirirus (ZIKV, DENV) showed different preferences for elongation initiation factors. Indeed SARS-CoV-2 prefers EIF3B, 4H, 4B, 3F, and A3, whereas flaviviruses prefer EIF3A, 4G1, 3C, and 3D [26]. More specifically, CSDE1 has been identified as a proviral factor that binds viral RNAs [27]. This is particularly interesting as clofoctol has been shown to bind CSDE1 [19]. However, whether CSDE1 binds the UTR region or another region of the viral RNA will need further investigation.
Previous pharmacokinetic studies indicate that clofoctol is well absorbed by rectal administration, and can rapidly expose lung tissues [11,28]. Of interest, as early as 90 minutes after rectal administration, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC 50 measured in Vero-81 cells. In our experimental conditions, clofoctol was also detected in mouse lungs at a peak concentration reaching approximately tenfold its IC 50 . Notably, upon two days of treatment with doses allometrically similar to those approved for human treatment, its concentration in the lungs remained far above the IC 50 measured in vitro. Importantly, we demonstrate here that clofoctol treatment decreased the viral load in the lungs and drastically reduced pulmonary inflammation. These in vivo data, as well as the rapid onset of action expected in human pharmacokinetics, strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.
In our study, K18-hACE2 transgenic C57BL/6J mice were used. Although this model is useful to evaluate the efficacy of antivirals, it has some limitations. While it is much better tolerated in humans, clofoctol induces weight loss in mice, which is attributed to a decrease in gastric emptying. Moreover, SARS-CoV-2-infected mice die from encephalitis, a disease evolution not encountered in humans. It is noteworthy that, in our experimental conditions, clofoctol failed to significantly reduce mouse mortality upon SARS-CoV-2 infection. This lack of protection against mortality is likely due to the fact that mice were only treated for 2 days to limit weight loss to a maximum of 20% (Fig 5B), whereas treatment in human patients can last for up to 10 days without specific side effects. Maintaining clofoctol treatment is likely to be required to improve efficacy in this system, as exemplified by the reduced protective effect of clofoctol on the viral load at 4 dpi (S4A Fig). Attempts are currently in progress to reduce the effect of this compound on the weight loss observed in mice.
Together with its antiviral effects, clofoctol abrogated lung inflammation. To the best of our knowledge, the anti-inflammatory effect of clofoctol has never been reported before. Together with its effect on UPR pathways, clofoctol is known to interact with different targets including (i) the Cdc7/Dbf4 protein kinase complex, which regulates the initiation of DNA replication and (ii) the upstream-of-N-Ras protein (UNR), a highly conserved RNA-binding protein known to regulate gene expression. Of interest, by binding to UNR, clofoctol activates the transcription factor Kruppel-like factor 13 (KLF13) [20], known as a tumor suppressor gene and as a regulator of T cell differentiation [29,30]. Whether the UNR/KLF13 pathway triggered by clofoctol plays a role in decreasing inflammation during SARS-CoV-2 infection deserves further investigation. Additional functional studies are urgently needed to assess the global effect of clofoctol on COVID-19 pathology.
In conclusion, the antiviral and anti-inflammatory properties of clofoctol, associated with its safety profile and unique pharmacokinetics make a strong case for proposing clofoctol as an affordable therapeutic candidate for the treatment of COVID-19 patients. Finally, the relatively low cost of this drug suggests that it is a potential clinical option for treatment of COVID-19 patients in resource poor settings.
Methods
Ethics statement. All experiments involving SARS-CoV-2 were performed within the biosafety level 3 facility of the Institut Pasteur de Lille, after validation of the protocols by the local committee for the evaluation of the biological risks and complied with current national and institutional regulations and ethical guidelines (Institut Pasteur de Lille/B59-350009). The experimental protocols using animals were approved by the institutional ethical committee “Comité d’Ethique en Experimentation Animale (CEEA) 75, Nord Pas-de-Calais”. The animal study was authorized by the “Education, Research and Innovation Ministry” under registration number APAFIS#25517-2020052608325772v3.
Data reporting. No statistical methods were used to predetermine sample size. Compounds were spotted in a randomized order on the plates during the primary screen. All the other experiments were not randomized. Investigators were blinded to allocation during the primary screen and the corresponding validation, during both assay performance and outcome assessment. For all the other assays, the investigators were not blinded.
Cells and viruses. Vero-81 cells (ATCC, CCL-81), Vero-E6 cells (ATCC, CRL-1586), Huh-7 cells[31] and HEK293T/17 cells (ATCC, CRL-11268) were grown at 37°C with 5% CO 2 in Dulbecco’s modified eagle medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Eurobio). Calu-3 cells (Clinisciences, EP-CL-0054) were grown in minimum essential medium (Gibco, MEM) supplemented with glutamax (Gibco) and 10% heat-inactivated FBS. Lentiviral vectors expressing TMPRSS2 were produced by transfection of HEK293T cells with pTRIP-TMPRSS2, phCMV-VSVG and HIV gag-pol in the presence of Turbofect (Life Technologies) according to the manufacturer’s instruction. Supernatants were collected at 48h post-transfection and used to transduce Vero-E6 cells. The BetaCoV/France/IDF0372/2020 strain of SARS-CoV-2 was supplied by the French National Reference Center for Respiratory Viruses hosted by Institut Pasteur (Paris, France). The hCoV-19_IPL_France strain of SARS-CoV-2 (NCBI MW575140) was also used for in vivo experiments. All SARS-CoV-2 viruses, including the variants B.1.1.7, B.1.351 and B.1.617.2 were propagated in Vero-E6 cells expressing TMPRSS2 by inoculation at MOI 0.01. Cell supernatant medium was harvested at 72h post-infection and stored frozen at −80°C in small aliquots. All experiments were conducted in a biosafety level 3 (BSL3) laboratory. Recombinant HCoV-229E expressing the Renilla luciferase was a kind gift of Dr Volker Thiel (University of Bern, Switzerland) and was propagated onto Huh-7 cells.
Chemical libraries. The TEELibrary was built and supplied by the APTEEUS company. It was in its version n°4 and counted 1,942 small organic molecules approved for a use in human and selected within national and international drug repositories. It is mainly composed of active pharmaceutical ingredients (>90%) and it covers 85% of the Prestwick FDA approved collection. All molecules have been dissolved in an appropriate bio-compatible solvent (DMSO or water with adjusted pH), at a concentration compatible with the testing on living cells. The majority of them are prepared at a 10mM concentration in DMSO. CQ diphosphate was purchased from Sigma-Aldrich (Dorset, England and St. Louis, MO). CQ diphosphate was diluted to a final concentration of 10 mM in water. Clofoctol was purchased from Sigma-Aldrich (C2290) or provided directly by the manufacturer (Chiesi, Italy).
Drug screening assay. One day prior to infection, Vero-81 cells were seeded in black 384-well μClear plates (Greiner Bio-One), at a density of 3,000 cells per well in 30 μl DMEM, supplemented with 10% FBS and 1X Penicillin-Streptomycin solution (Gibco), using a MultiDrop Combi Reagent dispenser (ThermoFischer Scientific). The next day, compounds from the TEELibrary were first dispensed into the 384-well plates, using an Echo 550 Liquid Handler (Labcyte). To identify the compounds of interest, they were tested at a final compound concentration that usually does not induce cytotoxicity, most of them at 15 μM. On each plate, five 3-fold serial dilutions of CQ diphosphate ranging from 0.15 μM to 15 μM were added in six replicates, as a control compound of viral inhibition (positive controls). Eleven control virus wells devoid of compound and scattered over the plate, were supplemented with 0.15% DMSO or 0.15% H 2 O (negative controls), respectively. Cells were infected by adding 10 μL of SARS-CoV-2 per well at a MOI of 0.01 in 10% FBS-containing medium, using a Viafill Rapid Reagent Dispenser (Integra). The plates were then incubated at 37° with 5% CO 2 . At 3 days post-infection, cells were stained with 10 μg/mL Hoechst 33342 dye (Sigma-Aldrich) and 1 μg/mL PI (ThermoFischer Scientific) for 30 min at 37°C for CPE quantification by high-content imaging.
Dose response curves and hit validations. The selected hits were further validated in a 6-point dose-response confirmation assay. One day prior to infection, Vero-81 cells were seeded in 384-well plates, as previously described. The next day, six 3-fold serial dilutions of compounds (0.15 to 45 μM, in duplicate) were first added to the cells. Ten μL of virus diluted in medium was then added to the wells. On each plate, twenty-six virus control wells distributed over the plates were supplemented with 0.15% DMSO and H 2 O, respectively. CQ diphosphate was added as a control compound, at six 3-fold serial dilutions (0.15 μM to 45 μM, in duplicate). Plates were incubated for 3 days at 37°C prior to staining and CPE quantification by high-content imaging.
Image acquisition. Image acquisitions were performed on a high-resolution automated confocal microscope (Opera QEHS, PerkinElmer) using a 10x air objective (NA = 0.4) for cellular infection assays. Hoechst 33342-stained nuclei were detected using the 405 nm excitation laser (Ex) with a 450/50-nm emission filter (Em). Red signals, corresponding to PI-stained nuclei from dead cells, were detected using Ex at 561 nm and Em at 600 nm. A set of 3 fields was collected from each well.
Image-based analysis. For total cell and dead cell detection, images from the automated confocal microscope were analyzed using multi-parameter scripts developed using Columbus image analysis software (version 2.3.1; PerkinElmer) (S1 Table). A segmentation algorithm was applied to detect nuclei labeled by Hoechst 33342 (blue) and determine total nuclei number. Briefly, a mask was first determined from input image, using the intensity threshold of Hoechst dye signal to create a region of interest corresponding to Hoechst-stained population. The nuclei segmentation was then performed using the algorithm “Find Nuclei”, as described previously [32]. Morphology properties, as area and roundness, could be used to exclude smaller objects not corresponding to nuclei. The total number of cells was quantified as Hoechst-positive nuclei. Red fluorescence signal intensities in the previous selected nuclei were quantified and used for the selection of PI positive (PI+) and negative (PI-) nuclei. Subsequently, population of dead (PI+) and viable (PI-) cells were determined. The percentage of PI+ cells was calculated for each compound to select drugs having an effect on the decrease of cell death, corresponding to infection or viral replication inhibition.
Dose-response validation in different cell lines or with different variants. Vero-81, Vero-81-TMPRSS2 or Calu-3 cells were infected in duplicates at a MOI of 0.25 in the presence of increasing concentrations of clofoctol, ranging from 0 to 25 μM, and incubated either for 6h (Vero-81 cells) or 24h (Calu-3 cells). Then total RNA was extracted by using the Nucleospin RNA kit (Macherey Nagel) as recommended by the manufacturer. Genome quantification was performed as described [33].
Viral secretion. Vero-81 and Vero-81-TMPRSS2 cells were infected at a MOI of 0.25 for 1h, then the cells were rinsed 3 times with PBS and further incubated in the presence of increasing concentrations of clofoctol for 16h. Each condition was performed in duplicates. Cell supernatants were collected and viral titers were measured by the TCID 50 method.
Pseudoparticles infection. Retroviral Murine leukemia virus particle were pseudotyped with the SARS-CoV-2 Spike (BetaCoV/France/IDF0372/2020 strain) or the glycoprotein of the vesicular stomatitis virus (VSV-G). Briefly, HEK293T cells were co-transfected with a plasmid encoding Gag-Pol (pTG-Gag-Pol), a plasmid encoding the envelope glycoprotein and a plasmid containing a minigenome with a Firefly luciferase reporter gene. After 48h of incubation, cell supernatants were collected, filtered and used to transduce Huh-7 cells expressing human ACE2 in the presence of increasing concentrations of clofoctol or CQ. Transduced cells were lysed 48h later and luciferase activity was measured by using the luciferase assay system (Promega).
Time-of-addition experiment. Vero-81 cells were plated in 24-well plates and infected for 1h at a MOI of 0.5. Clofoctol, remdesivir or CQ were added to the cells at a concentration of 15 μM every hour starting one hour before inoculation. At 8h post-infection, the cells were lysed in non-reducing Laemmli loading buffer. Proteins were separated onto a 10% SDS-polyacrylamide gel electrophoresis and transferred on nitrocellulose membranes (Amersham). Membrane-bound N proteins were detected with a rabbit polyclonal antibody (Novus) and a horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch). Detection was carried out by chemoluminescence (Pierce) and signals were quantified by using the gel quantification function of ImageJ. The experiment was repeated 3 times in duplicates.
Immunofluorescence. Vero-81 cells were plated onto glass coverslips. The day after, the cells were infected for 1h with SARS-CoV-2 at a MOI of 0.25. Clofoctol, remdesivir or CQ were added at 15 μM at different steps of the infection. The cells were either incubated 1h before inoculation (pre-incubation) or during the inoculation and for 1h after virus removal (entry step) or starting 1h after the inoculation until cell fixation (post-entry). Additional conditions with the compounds present during the whole experiment were also included as well as controls with DMSO or H 2 0. Cells were incubated for 16h after infection and fixed with 4% paraformaldehyde. Then, cells were permeabilized for 5 min with 0.1% Triton X-100 in PBS and blocked for 30 min with 5% goat serum in PBS. Infected cells were detected by using an anti-dsRNA (J2 monoclonal antibody, Scicons) diluted in blocking buffer to detect the presence of replicating SARS-CoV-2 virus as previously determined [33]. After a 30-min incubation, cells were rinsed 3 times for 5 min in PBS and incubated for 30 min with a cyanine 3-conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch) and DAPI (4′,6-diamidino-2-phenylindole). The coverslips were rinsed with PBS 3 times for 5 min followed by a final water wash before mounting on microscope slides in Mowiol 4–88 containing medium. Images acquisitions were performed with an EVOS M5000 imaging system (Thermo Fischer Scientific) equipped with a 10X objective and light cubes for DAPI and RFP. The total number of cells was determined by counting the number of nuclei and the number of infected cells was determined by counting dsRNA-positive cells. The experiment was performed three times.
Viability assay. Vero cells, Huh-7 cells or Calu-3 cells were plated in 96-well plates and were then incubated the next day in 100 μl of culture medium containing increasing concentrations of clofoctol for 24h. An MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based viability assay (CellTiter 96 aqueous nonradioactive cell proliferation assay, Promega) was performed as recommended by the manufacturer. The absorbance of formazan at 490 nm is detected using an enzyme-linked immunosorbent assay (ELISA) plate reader (ELx808, BioTek Instruments, Inc.). Each measure was performed in triplicate.
Analysis of the effect of the drug on translation. A plasmid containing a synthetic gene encompassing the 5’-UTR (nucleotides 1–265) and the 3’UTR (nucleotides 29675–29903) of SARS-CoV-2 isolate Wuhan-Hu-1 (Genebank NC_045512.2) separated by two head-to-tail BbsI sites was produced by GeneCust. The coding sequence of Renilla luciferase amplified by PCR using primers containing BbsI sites was inserted between both UTRs by ligation of BbsI-restricted PCR and plasmid. In this way, the coding sequence of the luciferase was inserted between the UTRs without leaving an extra nucleotide in between. The plasmid was linearized by NsiI restriction, and the linearized DNA was then used as a template for in vitro transcription with the mMESSAGE mMACHINE T7 kit from Thermofischer Scientific, as recommended by the manufacturer. In vitro-transcribed capped RNA was delivered to Vero-81 and Huh-7 cells by electroporation. Cells were cultured for 8h in the presence of increasing concentrations of clofoctol. Renilla luciferase activities were measured with a Renilla luciferase assay from Promega. As a control, we used a bicistronic construct containing the Firefly luciferase sequence under the control of a cap structure, followed by the Renilla luciferase under the control of hepatitis C virus (HCV) IRES. Firefly and Renilla luciferase activities were measured with a dual-luciferase reporter assay system from Merck Millipore as previously reported [13]. To determine the effect of clofoctol on global protein synthesis, we used a puromycin-conjugation assay adapted from Schmidt et al.[34]. Briefly, confluent monolayers of Vero-81 or Huh-7 cells in P-24 wells were incubated with of 10 μg/ml puromycin in the presence of 0.05% DMSO, 25 μM clofoctol or 100 μM cycloheximide for 1h. Then, cells were rinsed 3 times with PBS, and lysed with 0.25 ml of SDS-PAGE loading buffer containing 2% SDS and 40 mM DTT. Lysates were incubated for 15 minutes at 70°C and 5 μl were spotted on nitrocellulose. The blot was incubated for 1 hour in blocking solution (20 mM TrisCl pH7.4, 137 mM NaCl, 0.1% NP40, 5% non-fat dry milk). Puromycin-conjugated polypeptides were detected using anti-puromycin mAb 12D10 (purchased from Sigma-Aldrich) diluted 1:20,000 in blocking solution, followed by horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody. Chemiluminescent signals were recorded with a LAS3000 apparatus and quantified with the imageJ software using gel analysis tool.
Pharmacokinetic study. Clofoctol diluted in 1.75% final Kolliphor RH40 (07076, Sigma) and 1.4% final ethanol in a sodium chloride solution (0.9%) was used for intraperitoneal (i.p.) injection. The concentration of clofoctol in plasma and lungs was measured at different time points post-clofoctol injection. Plasma samples and lung tissues were collected and treated with absolute ethanol, at a ratio of 1:10 (vol/vol) and 1:50 (vol/vol), respectively. Lung tissues were homogenized with a mechanical lysis system (Tissue Lyzer II). Supernatants were obtained by centrifugation before injection in LC-MS/MS. Samples were analysed using UPLC system Acquity I Class (Waters), combined with a triple quadrupole mass spectrometer Xevo TQD (Waters). The column, placed at 40°C, was an Acquity BEH C8 50*2.1mm, 1.7μm column (Waters) and the following mobile phases were used: 5mM ammonium formate pH 3.75 in water, as solvent (A) and 5 mM ammonium formate pH 3.75 in acetonitrile as solvent.
Experimental infection of K18-hACE2 transgenic mice. Eight week-old K18-human ACE2 expressing C57BL/6 mice (B6.Cg-Tg(K18-hACE2)2Prlmn/J) were purchased from the Jackson Laboratory. For infection, mice (both sexes) were anesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and then intranasally infected with 50 μl of DMEM containing (or not, in a mock sample) 5x102 TCID 50 of hCoV-19_IPL_France strain of SARS-CoV-2 (NCBI MW575140). Clofoctol (62.5 mg/kg in females and 10, 25, 50 and 62.5 mg/kg in males) was injected i.p. at 1h and 8h post-infection. The treatment was repeated the day after infection. Body weight was measured until day 2 post-infection. Mice were sacrificed at day 2 or day 4 post-infection.
Determination of viral loads in the lungs of mice. To determine the viral loads in lungs, half of right lobes were homogenized in Lysing Matrix D tubes (MP Bio) containing 1 mL of PBS using Mixer Mill MM 400 (Retsch) (15min– 15 Hz). After centrifugation at 11,000 rpm for 5 min, the clarified supernatant was harvested for virus titration. Dilutions of the supernatant were done in DMEM with 1% penicillin/streptomycin and dilutions were transferred to Vero-E6 cells in 96-well plates for TCID 50 assay. Quantitation of viral RNA in lung tissue was performed as follows. Briefly, half of the left lobe was homogenized in 1mL of RA1 buffer from the NucleoSpin RNA kit containing 20 mM of Tris(2-carboxyethyl)phosphine). Total RNAs in the tissue homogenate were extracted with NucleoSpin RNA from Macherey Nagel. RNA was eluted with 50μL of water.
Determination of the viral load and assessment of gene expression by RT-qPCR. Half of the right lobe was homogenized in 1 mL of RA1 buffer from the NucleoSpin RNA kit containing 20 mM of TCEP. Total RNAs in the tissue homogenate were extracted with NucleoSpin RNA from Macherey Nagel. RNAs were eluted with 60 μL of water. RNA was reverse-transcribed with the High-Capacity cDNA Archive Kit (Life Technologies, USA). The resulting cDNA was amplified using SYBR Green-based real-time PCR and the QuantStudio 12K Flex Real-Time PCR Systems (Applied Biosystems, USA) following manufacturers protocol. Relative quantifications were performed using the gene coding for RNA-dependent RNA polymerase (RdRp) and for glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Specific primers were designed using Primer Express software (Applied Biosystems, Villebon-sur-Yvette, France) and ordered to Eurofins Scientifics (Ebersberg, Germany). The list of primers is available in S3 Table. Relative mRNA levels (2-ΔΔCt) were determined by comparing (a) the PCR cycle thresholds (Ct) for the gene of interest and the house keeping gene (ΔCt) and (b) ΔCt values for treated and control groups (ΔΔCt). Data were normalized against expression of the gapdh gene and are expressed as a fold-increase over the mean gene expression level in mock-treated mice. Viral load is expressed as viral RNA normalized to Gapdh expression level (ΔCt).
Lung pathology scoring. Lung tissues were fixed in 4% PBS buffered formaldehyde for 7 days, rinsed in PBS, transferred in ethanol and then processed into paraffin-embedded tissues blocks. The subcontractor Sciempath Labo (Larçay, France) performed histological processing and analysis. The tissue sections in 3 μm were stained with haematoxylin and eosin (H&E) and whole mount tissues were scanned with a Nanozoomer (Hamatsu) and the morphological changes were assessed by a semi-quantitative score. For the scoring, a dual histopathology scoring system adapted from [35,36] was used to assess pulmonary changes in mice. Inflammation was scored as 0 = absent, 1 = 1–10% of lung section, 2 = 11–25% of lung section, 3 = 26–50% of lung section, and 4 = >50% of lung section affected.
[END]
---
[1] Url:
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010498
Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.
via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/
