Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway ...epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host–virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13–treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.
Cystic fibrosis (CF) is characterised by the accumulation of viscous adherent mucus in the lungs. While several hypotheses invoke a direct relationship with cystic fibrosis transmembrane conductance ...regulator (CFTR) dysfunction (
acidic airway surface liquid (ASL) pH, low bicarbonate (HCO
) concentration, airway dehydration), the dominant biochemical alteration of CF mucus remains unknown.
We characterised a novel cell line (CFTR-KO Calu3 cells) and the responses of human bronchial epithelial (HBE) cells from subjects with G551D or F508del mutations to ivacaftor and elexacaftor-tezacaftor-ivacaftor. A spectrum of assays such as short-circuit currents, quantitative PCR, ASL pH, Western blotting, light scattering/refractometry (size-exclusion chromatography with inline multi-angle light scattering), scanning electron microscopy, percentage solids and particle tracking were performed to determine the impact of CFTR function on mucus properties.
Loss of CFTR function in Calu3 cells resulted in ASL pH acidification and mucus hyperconcentration (dehydration). Modulation of CFTR in CF HBE cells did not affect ASL pH or mucin mRNA expression, but decreased mucus concentration, relaxed mucus network ultrastructure and improved mucus transport. In contrast with modulator-treated cells, a large fraction of airway mucins remained attached to naïve CF cells following short apical washes, as revealed by the use of reducing agents to remove residual mucus from the cell surfaces. Extended hydration, but not buffers alkalised with sodium hydroxide or HCO
, normalised mucus recovery to modulator-treated cell levels.
These results indicate that airway dehydration, not acidic pH and/or low HCO
, is responsible for abnormal mucus properties in CF airways and CFTR modulation predominantly restores normal mucin entanglement.
The severe acute respiratory syndrome coronavirus 2 pandemic is characterized by the emergence of novel variants of concern (VOCs) that replace ancestral strains. Here, we dissect the complex ...selective pressures by evaluating variant fitness and adaptation in human respiratory tissues. We evaluate viral properties and host responses to reconstruct forces behind D614G through Omicron (BA.1) emergence. We observe differential replication in airway epithelia, differences in cellular tropism, and virus-induced cytotoxicity. D614G accumulates the most mutations after infection, supporting zoonosis and adaptation to the human airway. We perform head-to-head competitions and observe the highest fitness for Gamma and Delta. Under these conditions, RNA recombination favors variants encoding the B.1.617.1 lineage 3′ end. Based on viral growth kinetics, Alpha, Gamma, and Delta exhibit increased fitness compared to D614G. In contrast, the global success of Omicron likely derives from increased transmission and antigenic variation. Our data provide molecular evidence to support epidemiological observations of VOC emergence.
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•The ancestral D614G is least adapted to the human airway compared to later variants•Gamma and Delta variants have the highest replicative fitness in human airway epithelia•Omicron displays poor replication and increased immune activation in the lower airway•Omicron success globally is likely due to increased transmission and antigenic variation
Meganck et al. recapitulate the drivers of evolution by evaluating SARS-CoV-2 variant fitness in primary human airway cells. Ancestral D614G displays pre-adaptation phenotypes, while Gamma and Delta variants have intrinsic high replicative fitness in the human airway. Omicron has poor replicative fitness and greater immune induction in the lower airway.
Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in ...the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV.
In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical ...properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G', G", G
, and tan δ) and gel point characteristics (γc and σc) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is <10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more "liquid-like" behavior overall (e.g., higher tan δ). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.
To determine the effect of suppression of vascular endothelial growth factor (VEGF) by monthly injection of ranibizumab on posterior retinal nonperfusion (RNP) in patients with diabetic macular edema ...(DME).
Unplanned retrospective analysis of prospectively collected data from 2 randomized, sham injection-controlled, double-masked, multicenter clinical trials.
Six hundred sixty-six patients with DME.
An independent reading center measured the area of RNP on fluorescein angiograms obtained in the phase 3 RISE and RIDE trials.
The percentage of patients with no posterior RNP.
The percentage of patients with no posterior RNP decreased in the sham group between baseline and month 24, but remained relatively stable in the 2 ranibizumab groups. After month 24, the sham group crossed over to receive monthly injections of ranibizumab 0.5 mg, and the differences between the sham and ranibizumab groups were reduced. The percentage of patients who showed an increase in posterior RNP from baseline increased over time in all 3 groups, but at a faster rate in the sham group, resulting in statistically significant differences at every time point between months 3 (9.6% vs. 18.5%; P = 0.016) and 24 (16.1% vs. 37.6%; P<0.0001) for ranibizumab 0.5 mg versus sham and from months 6 (12.3% vs. 23.0%; P = 0.013) through 24 (15.0% vs. 37.6%; P<0.0001) for ranibizumab 0.3 mg. Initiation of ranibizumab in the sham group at month 24 was followed by reduction in the percentage of patients with an increase in posterior RNP from baseline at months 30 and 36, whereas the 2 ranibizumab groups continued their gradual rise.
Just as high VEGF levels contribute to progression of retinal nonperfusion in retinal vein occlusion, the same is true in patients with DME, suggesting that regardless of the underlying disease process, high levels of VEGF can cause closure of retinal vessels. However, our data also suggest that VEGF-induced worsening of retinal perfusion in DME is superimposed on another cause of more gradually worsening perfusion, possibly glucotoxicity. Thus, monthly injections of ranibizumab can slow, but not completely prevent, retinal capillary closure in patients with DME.
To reduce treatment burden and optimise patient outcomes in diabetic macular oedema, we present 1-year results from two phase 3 trials of faricimab, a novel angiopoietin-2 and vascular endothelial ...growth factor-A bispecific antibody.
YOSEMITE and RHINE were randomised, double-masked, non-inferiority trials across 353 sites worldwide. Adults with vision loss due to centre-involving diabetic macular oedema were randomly assigned (1:1:1) to intravitreal faricimab 6·0 mg every 8 weeks, faricimab 6·0 mg per personalised treatment interval (PTI), or aflibercept 2·0 mg every 8 weeks up to week 100. PTI dosing intervals were extended, maintained, or reduced (every 4 weeks up to every 16 weeks) based on disease activity at active dosing visits. The primary endpoint was mean change in best-corrected visual acuity at 1 year, averaged over weeks 48, 52, and 56. Efficacy analyses included the intention-to-treat population (non-inferiority margin 4 Early Treatment Diabetic Retinopathy Study ETDRS letters); safety analyses included patients with at least one dose of study treatment. These trials are registered with ClinicalTrials.gov (YOSEMITE NCT03622580 and RHINE NCT03622593).
3247 patients were screened for eligibility in YOSEMITE (n=1532) and RHINE (n=1715). After exclusions, 940 patients were enrolled into YOSEMITE between Sept 5, 2018, and Sept 19, 2019, and 951 patients were enrolled into RHINE between Oct 9, 2018, and Sept 20, 2019. These 1891 patients were randomly assigned to faricimab every 8 weeks (YOSEMITE n=315, RHINE n=317), faricimab PTI (n=313, n=319), or aflibercept every 8 weeks (n=312, n=315). Non-inferiority for the primary endpoint was achieved with faricimab every 8 weeks (adjusted mean vs aflibercept every 8 weeks in YOSEMITE 10·7 ETDRS letters 97·52% CI 9·4 to 12·0 vs 10·9 ETDRS letters 9·6 to 12·2, difference −0·2 ETDRS letters −2·0 to 1·6; RHINE 11·8 ETDRS letters 10·6 to 13·0 vs 10·3 ETDRS letters 9·1 to 11·4 letters, difference 1·5 ETDRS letters −0·1 to 3·2) and faricimab PTI (YOSEMITE 11·6 ETDRS letters 10·3 to 12·9, difference 0·7 ETDRS letters −1·1 to 2·5; RHINE 10·8 ETDRS letters 9·6 to 11·9, difference 0·5 ETDRS letters −1·1 to 2·1). Incidence of ocular adverse events was comparable between faricimab every 8 weeks (YOSEMITE n=98 31%, RHINE n=137 43%), faricimab PTI (n=106 34%, n=119 37%), and aflibercept every 8 weeks (n=102 33%, n=113 36%).
Robust vision gains and anatomical improvements with faricimab were achieved with adjustable dosing up to every 16 weeks, demonstrating the potential for faricimab to extend the durability of treatment for patients with diabetic macular oedema.
F Hoffmann-La Roche.
To evaluate clinical characteristics of eyes in which investigator-determined new-onset exudative age-related macular degeneration (eAMD) developed during the FILLY trial.
Post hoc analysis of the ...phase 2 study of intravitreal pegcetacoplan in geographic atrophy (GA).
Patients with GA secondary to age-related macular degeneration (AMD), n = 246.
Either 15 mg intravitreal pegcetacoplan or sham given monthly or every other month for 12 months followed by a 6-month off-treatment period.
Time of new eAMD onset in the study eye, history of eAMD in the fellow eye, presence of double-layer sign (DLS) on structural OCT in the study eye, changes in retinal anatomic features by structural OCT and fluorescein angiography (FA), and changes in visual acuity.
Exudation was reported in 26 study eyes across treatment groups over 18 months. Mean time to eAMD diagnosis was 256 days (range, 31–555 days). Overall, a higher proportion of patients with a baseline history of eAMD in the fellow eye (P = 0.016) and a DLS in the study eye (P = 0.0001) demonstrated eAMD. Among study eyes in which eAMD developed, 18 of 26 (69%) had history of fellow-eye eAMD and 19 of 26 (73.1%) had DLS at baseline, compared with 76 of 217 study eyes (35%; P = 0.0007) and 70 of 215 study eyes (32.5%; P < 0.0001), respectively, in which eAMD did not develop. All 21 patients with structural OCT imaging at the time of eAMD diagnosis demonstrated subretinal fluid, intraretinal cysts, or both consistent with exudation. Among 17 patients who underwent FA at eAMD diagnosis, 10 showed detectable macular neovascularization (MNV), all occult lesions. Development of eAMD did not have an appreciable impact on visual acuity, and all patients responded to anti–vascular endothelial growth factor (VEGF) therapy.
Intravitreal pegcetacoplan slowed the rate of GA growth and was associated with an unexpected dose-dependent increased incidence of eAMD with no temporal clustering of onset. Exudative AMD seemed to be associated with baseline eAMD in the contralateral eye and a DLS, suggestive of nonexudative MNV, in the study eye. The safety profile of pegcetacoplan was acceptable to proceed to phase 3 studies without adjustments to enrollment criteria.
Neovascular (wet) age-related macular degeneration (nAMD) is driven by VEGFs A, C, and D, which promote angiogenesis and vascular permeability. Intravitreal injections of anti–VEGF-A drugs are the ...standard of care, but these do not inhibit VEGF-C and D, which may explain why many patients fail to respond fully. This trial aimed to test the safety and efficacy of OPT-302, a biologic inhibitor of VEGF-C and D, in combination with the anti–VEGF-A inhibitor ranibizumab.
Dose-ranging, phase 2b, randomized, double-masked, sham-controlled trial.
Participants with treatment-naive nAMD were enrolled from 109 sites across Europe, Israel, and the United States.
Participants were randomized to 6, 4-weekly, intravitreal injections of 0.5 mg OPT-302, 2.0 mg OPT-302, or sham, plus intravitreal 0.5 mg ranibizumab.
The primary outcome was mean change in ETDRS best-corrected visual acuity (BCVA) at 24 weeks. Secondary outcomes (comparing baseline with week 24) were the proportion of participants gaining or losing ≥ 15 ETDRS BCVA letters; area under the ETDRS BCVA over time curve; change in spectral-domain OCT (SD-OCT) central subfield thickness; and change in intraretinal fluid and subretinal fluid on SD-OCT.
Of 366 participants recruited from December 1, 2017, to November 30, 2018, 122, 123, and 121 were randomized to 0.5 mg OPT-302, 2.0 mg OPT-302, and sham, respectively. Mean (± standard deviation) visual acuity gain in the 2.0 mg OPT-302 group was significantly superior to sham (+14.2 ± 11.61 vs. +10.8 ± 11.52 letters; P = 0.01). The 0.5 mg OPT-302 group was not significantly different than the sham group (+9.44 ± 11.32 letters; P = 0.83). Compared with sham, the secondary BCVA outcomes favored the 2.0 mg OPT-302 group, with structural outcomes favoring both OPT-302 dosage groups. Adverse events (AEs) were similar across groups, with 16 (13.3%), 7 (5.6%), and 10 (8.3%) participants in the lower-dose, higher-dose, and sham groups, respectively, developing at least 1 serious AE. Two unrelated deaths both occurred in the sham arm.
Significantly superior vision gain was observed with OPT-302 2.0 mg combination therapy, versus standard of care, with favorable safety (ClinicalTrials.gov identifier: NCT03345082).
Proprietary or commercial disclosure may be found after the references.
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