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1.
Nature ; 2024 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-38898278

RESUMEN

The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the dynamics of early cellular responses to this disease remains limited1. Here in our SARS-CoV-2 human challenge study, we used single-cell multi-omics profiling of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in seronegative individuals challenged with pre-Alpha SARS-CoV-2. Our analyses revealed rapid changes in cell-type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific time points and infection status. We observed that the interferon response in blood preceded the nasopharyngeal response. Moreover, nasopharyngeal immune infiltration occurred early in samples from individuals with only transient infection and later in samples from individuals with sustained infection. High expression of HLA-DQA2 before inoculation was associated with preventing sustained infection. Ciliated cells showed multiple immune responses and were most permissive for viral replication, whereas nasopharyngeal T cells and macrophages were infected non-productively. We resolved 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our new computational pipeline Cell2TCR identifies activated antigen-responding T cells based on a gene expression signature and clusters these into clonotype groups and motifs. Overall, our detailed time series data can serve as a Rosetta stone for epithelial and immune cell responses and reveals early dynamic responses associated with protection against infection.

2.
Mol Ther ; 32(5): 1497-1509, 2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38429928

RESUMEN

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.


Asunto(s)
Adhesión Celular , Epidermólisis Ampollosa , Laminina , Lentivirus , Humanos , Laminina/metabolismo , Laminina/genética , Epidermólisis Ampollosa/genética , Epidermólisis Ampollosa/metabolismo , Epidermólisis Ampollosa/terapia , Epidermólisis Ampollosa/patología , Niño , Lentivirus/genética , Masculino , Femenino , Preescolar , Terapia Genética/métodos , Vectores Genéticos/genética , Células Epiteliales/metabolismo , Células Cultivadas , Expresión Génica , Adolescente , Lactante
3.
Thorax ; 2024 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-38050163

RESUMEN

INTRODUCTION: Respiratory syncytial virus (RSV) causes a severe respiratory condition, bronchiolitis, in infants but not in adults. Bronchiolitis is characterised by neutrophilic infiltration in the airways, but whether neutrophils enhance recovery from infection or contribute to its pathology remains unknown. METHODS: We used a novel in-vitro model to compare term umbilical cord blood (infant) (n=17 donors) and adult neutrophils (n=15 donors) during migration across RSV-infected differentiated human nasal airway epithelial cells (AECs) in a basolateral to apical direction. RESULTS: Greater numbers of infant neutrophils (mean (95% CI)) (336 684 (242 352 to 431 015)) migrated across RSV-infected AECs to the apical compartment (equivalent to the airway lumen) compared with adult neutrophils (56 586 (24 954 to 88 218)) (p<0.0001). Having reached the apical compartment of infected AECs, much greater numbers of infant neutrophils (140 787 (103 117 to 178 456)) became apoptotic compared with adult (5853 (444 to 11 261)) (p=0.002). Infant neutrophils displayed much greater expression of CD11b, CD64, neutrophil elastase (NE) and myeloperoxidase (MPO) than adult neutrophils at baseline and at all points of migration. However, as adult neutrophils migrated, expression of CD11b, CD64, NE and MPO became greater than at baseline. DISCUSSION: The high proportion of infant neutrophils migrating across RSV-infected AECs correlates with the neutrophilic infiltrate seen in infants with severe RSV bronchiolitis, with large numbers undergoing apoptosis, which may represent a protective mechanism during infection. Compared with adult neutrophils, infant neutrophils already have high expression of surface markers before contact with AECs or migration, with less capacity to increase further in response to RSV infection or migration.

4.
Exp Brain Res ; 242(5): 1115-1126, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38483567

RESUMEN

The use of functional near-infrared spectroscopy (fNIRS) for brain imaging during human movement continues to increase. This technology measures brain activity non-invasively using near-infrared light, is highly portable, and robust to motion artifact. However, the spatial resolution of fNIRS is lower than that of other imaging modalities. It is unclear whether fNIRS has sufficient spatial resolution to differentiate nearby areas of the cortex, such as the leg areas of the motor cortex. Therefore, the purpose of this study was to determine fNIRS' ability to discern laterality of lower body contractions. Activity in the primary motor cortex was recorded in forty participants (mean = 23.4 years, SD = 4.5, female = 23, male = 17) while performing unilateral lower body contractions. Contractions were performed at 30% of maximal force against a handheld dynamometer. These contractions included knee extension, knee flexion, dorsiflexion, and plantar flexion of the left and right legs. fNIRS signals were recorded and stored for offline processing and analysis. Channels of fNIRS data were grouped into regions of interest, with five tolerance conditions ranging from strict to lenient. Four of five tolerance conditions resulted in significant differences in cortical activation between hemispheres. During right leg contractions, the left hemisphere was more active than the right hemisphere. Similarly, during left leg contractions, the right hemisphere was more active than the left hemisphere. These results suggest that fNIRS has sufficient spatial resolution to distinguish laterality of lower body contractions. This makes fNIRS an attractive technology in research and clinical applications in which laterality of brain activity is required during lower body activity.


Asunto(s)
Lateralidad Funcional , Corteza Motora , Espectroscopía Infrarroja Corta , Humanos , Espectroscopía Infrarroja Corta/métodos , Masculino , Femenino , Adulto Joven , Lateralidad Funcional/fisiología , Adulto , Corteza Motora/fisiología , Contracción Muscular/fisiología , Mapeo Encefálico/métodos
5.
Anal Chem ; 94(16): 6089-6096, 2022 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-35417141

RESUMEN

Precisely controlling the size of engineered biomolecules and pharmaceutical compounds is often critical to their function. Standard methods for size characterization, such as dynamic light scattering or size exclusion chromatography, can be sample intensive and may not provide the sensitivity needed for mass- or concentration-limited biological systems. Taylor dispersion analysis (TDA) is a proven analytical method for direct, calibration-free size determination which utilizes only nL-pL sample volumes. In TDA, diffusion coefficients, which are mathematically transformed to hydrodynamic radii, are determined by characterizing band broadening of an analyte under well-controlled laminar flow conditions. Here, we describe the design and development of a 3D printed instrument for TDA, which is the first such instrument to offer dual-point laser-induced fluorescence (LIF) detection. The instrument utilized a fully 3D printed eductor as a vacuum source for precise and stable pressure-driven flow within a capillary, evidenced by a linear response in generated static pressure to applied gas pressure (R2 = 0.997) and a 30-fold improvement in stability of static pressure (0.05% RSD) as compared to a standard mechanical pump (1.53%). Design aspects of the LIF detection system were optimized to maximize S/N for excitation and emission optical axes, and high sensitivity was achieved as evidenced by an 80 pM limit of detection for the protein R-Phycoerythrin and low nM limits of detection for three additional fluorophores. The utility of the instrument was demonstrated via sizing of R-Phycoerythrin at pM concentrations.


Asunto(s)
Hidrodinámica , Ficoeritrina , Dispersión Dinámica de Luz , Rayos Láser , Impresión Tridimensional
6.
J Antimicrob Chemother ; 78(1): 8-20, 2022 12 23.
Artículo en Inglés | MEDLINE | ID: mdl-36411255

RESUMEN

Conventional cell culture systems involve growing cells in stationary cultures in the presence of growth medium containing various types of supplements. At confluency, the cells are divided and further expanded in new culture dishes. This passage from confluent monolayer to sparse cultures does not reflect normal physiological conditions and represents quite a drastic physiological change that may affect the natural cell physiobiology. Hollow-fibre bioreactors were in part developed to overcome these limitations and since their inception, they have widely been used in production of monoclonal antibodies and recombinant proteins. These bioreactors are increasingly used to study antibacterial drug effects via simulation of in vivo pharmacokinetic profiles. The use of the hollow-fibre infection model (HFIM) in viral infection studies is less well developed and in this review we have analysed and summarized the current available literature on the use of these bioreactors, with an emphasis on viruses. Our work has demonstrated that this system can be applied for viral expansion, studies of drug resistance mechanisms, and studies of pharmacokinetic/pharmacodynamic (PK/PD) of antiviral compounds. These platforms could therefore have great applications in large-scale vaccine development, and in studies of mechanisms driving antiviral resistance, since the HFIM could recapitulate the same resistance mechanisms and mutations observed in vivo in clinic. Furthermore, some dosage and spacing regimens evaluated in the HFIM system, as allowing maximal viral suppression, are in line with clinical practice and highlight this 'in vivo-like' system as a powerful tool for experimental validation of in vitro-predicted antiviral activities.


Asunto(s)
Antibacterianos , Virosis , Humanos , Antibacterianos/farmacología , Antivirales/farmacocinética , Modelos Biológicos , Virosis/tratamiento farmacológico
7.
J Physiol ; 599(18): 4255-4267, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34287894

RESUMEN

Currently, therapeutics for COVID-19 are limited. To overcome this, it is important that we use physiologically relevant models to reproduce the pathology of infection and evaluate the efficacy of antiviral drugs. Models of airway infection, including the use of a human infection challenge model or well-defined, disease relevant in vitro systems can help determine the key components that perpetuate the severity of the disease. Here, we briefly review the human models that are currently being used in COVID-19 research and drug development.


Asunto(s)
COVID-19 , Antivirales/uso terapéutico , Humanos , SARS-CoV-2
8.
Eur Respir J ; 58(4)2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-33795320

RESUMEN

BACKGROUND: Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies followed by ciliated differentiation at the air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that can be expanded from such biopsies. METHODS: We describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. RESULTS: Initial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells in vitro. CONCLUSION: Our study provides a platform for higher throughput analyses of airway epithelia that is applicable in a range of settings and suggests novel avenues for drug evaluation and development in PCD caused by nonsense mutations.


Asunto(s)
Trastornos de la Motilidad Ciliar , Síndrome de Kartagener , Cilios , Trastornos de la Motilidad Ciliar/diagnóstico , Trastornos de la Motilidad Ciliar/tratamiento farmacológico , Trastornos de la Motilidad Ciliar/genética , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/tratamiento farmacológico , Síndrome de Kartagener/genética , Depuración Mucociliar
9.
J Virol ; 94(13)2020 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-32295918

RESUMEN

Respiratory syncytial virus (RSV) is a major cause of pediatric respiratory disease. Large numbers of neutrophils are recruited into the airways of children with severe RSV disease. It is not clear whether or how neutrophils enhance recovery from disease or contribute to its pathology. Using an in vitro model of the differentiated airway epithelium, we found that the addition of physiological concentrations of neutrophils to RSV-infected nasal cultures was associated with greater epithelial damage with lower ciliary activity, cilium loss, less tight junction expression (ZO-1), and more detachment of epithelial cells than is seen with RSV infection alone. This was also associated with a decrease in infectious virus and fewer RSV-positive cells in cultures after neutrophil exposure than in preexposure cultures. Epithelial damage in response to RSV infection was associated with neutrophil activation (within 1 h) and neutrophil degranulation, with significantly greater cellular expression of CD11b and myeloperoxidase and higher levels of neutrophil elastase and myeloperoxidase activity in apical surface media than in media with mock-infected airway epithelial cells (AECs). We also recovered more apoptotic neutrophils from RSV-infected cultures (>40%) than from mock-infected cultures (<5%) after 4 h. The results of this study could provide important insights into the role of neutrophils in host response in the airway.IMPORTANCE This study shows that the RSV-infected human airway drives changes in the behavior of human neutrophils, including increasing activation markers and delaying apoptosis, that result in greater airway damage and viral clearance.


Asunto(s)
Neutrófilos/inmunología , Mucosa Respiratoria/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Adulto , Células Epiteliales/virología , Humanos , Neutrófilos/virología , Cultivo Primario de Células , Mucosa Respiratoria/virología , Infecciones por Virus Sincitial Respiratorio/fisiopatología , Virus Sincitial Respiratorio Humano/fisiología , Virus Sincitiales Respiratorios/metabolismo , Virus Sincitiales Respiratorios/patogenicidad , Virus Sincitiales Respiratorios/fisiología , Virosis/metabolismo
10.
Thorax ; 73(9): 847-856, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29748250

RESUMEN

INTRODUCTION: Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. METHODS: We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air-liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. RESULTS: Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and ßENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. CONCLUSION: Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.


Asunto(s)
Fibrosis Quística/genética , Fibrosis Quística/patología , Canales Epiteliales de Sodio/genética , Silenciador del Gen , ARN Interferente Pequeño , Transfección/métodos , Animales , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Humanos , Ratones , Nanopartículas
11.
Am J Respir Crit Care Med ; 194(2): 156-68, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-26840431

RESUMEN

RATIONALE: Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. OBJECTIVES: To define a scalable cell culture system to deliver airway epithelium to clinical grafts. METHODS: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. MEASUREMENTS AND MAIN RESULTS: 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. CONCLUSIONS: Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.


Asunto(s)
Células Epiteliales/metabolismo , Enfermedades Respiratorias/terapia , Células Madre/metabolismo , Ingeniería de Tejidos/métodos , Diferenciación Celular/fisiología , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Depuración Mucociliar/fisiología , Reacción en Cadena de la Polimerasa , Mucosa Respiratoria/fisiología
12.
Am J Respir Crit Care Med ; 190(2): 196-207, 2014 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-24941423

RESUMEN

RATIONALE: Respiratory syncytial virus (RSV) and Streptococcus pneumoniae are major respiratory pathogens. Coinfection with RSV and S. pneumoniae is associated with severe and often fatal pneumonia but the molecular basis for this remains unclear. OBJECTIVES: To determine if interaction between RSV and pneumococci enhances pneumococcal virulence. METHODS: We used confocal microscopy and Western blot to identify the receptors involved in direct binding of RSV and pneumococci, the effects of which were studied in both in vivo and in vitro models of infection. Human ciliated respiratory epithelial cell cultures were infected with RSV for 72 hours and then challenged with pneumococci. Pneumococci were collected after 2 hours exposure and changes in gene expression determined using quantitative real-time polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: Following incubation with RSV or purified G protein, pneumococci demonstrated a significant increase in the inflammatory response and bacterial adherence to human ciliated epithelial cultures and markedly increased virulence in a pneumonia model in mice. This was associated with extensive changes in the pneumococcal transcriptome and significant up-regulation in the expression of key pneumococcal virulence genes, including the gene for the pneumococcal toxin, pneumolysin. We show that mechanistically this is caused by RSV G glycoprotein binding penicillin binding protein 1a. CONCLUSIONS: The direct interaction between a respiratory virus protein and the pneumococcus resulting in increased bacterial virulence and worsening disease outcome is a new paradigm in respiratory infection.


Asunto(s)
Coinfección/microbiología , Proteínas de Unión a las Penicilinas/metabolismo , Neumonía Neumocócica/microbiología , Infecciones por Virus Sincitial Respiratorio/microbiología , Virus Sincitiales Respiratorios/metabolismo , Streptococcus pneumoniae/patogenicidad , Proteínas Virales de Fusión/metabolismo , Animales , Adhesión Bacteriana , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Coinfección/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Femenino , Regulación Bacteriana de la Expresión Génica , Humanos , Ratones , Microscopía Confocal , Neumonía Neumocócica/metabolismo , Neumonía Neumocócica/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/microbiología , Infecciones por Virus Sincitial Respiratorio/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/virología , Transcriptoma , Regulación hacia Arriba , Virulencia
13.
Eur Respir J ; 43(2): 485-96, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23520320

RESUMEN

Respiratory syncytial virus is a major cause of respiratory disease. There are conflicting accounts of the response of human epithelial cells to respiratory syncytial virus and a lack of data on its effect on ciliary function. Our aim was to study the early stages of respiratory syncytial virus infection of primary human basal and ciliated cultures. Using high speed videomicroscopy, we found that ciliary beat frequency was unaffected by respiratory syncytial virus infection over 72 h; however, ciliary dyskinesia significantly increased within 24 h of infection (p<0.05). Transmission electron microscopy revealed that ultrastructural abnormalities were confined to ciliated cells, including increased cilia loss and mitochondrial damage. Confocal immunofluorescence microscopy showed that respiratory syncytial virus antigen gradually spread from the cell surface to the ciliary tip of infected cells over 3 days. Interestingly, ciliated cultures secreted fewer viruses than basal (progenitor) cell cultures and produced a chemokine response focused on recruitment of neutrophils. This study highlights differences in infection models and underscores the need to explore further the role of ciliated cells in the establishment of respiratory syncytial virus infection. Increased ciliary dyskinesia combined with ciliary loss and epithelial damage is likely to result in reduced mucociliary clearance early in the infective process.


Asunto(s)
Trastornos de la Motilidad Ciliar/diagnóstico , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Adulto , Antígenos Virales/metabolismo , Células Cultivadas , Cilios/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Humanos , Microscopía por Video , Persona de Mediana Edad , Depuración Mucociliar , Virus Sincitiales Respiratorios , Células TH1/citología , Adulto Joven
14.
Mucosal Immunol ; 17(1): 124-136, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38007005

RESUMEN

SARS-CoV-2 initially infects cells in the nasopharynx and oral cavity. The immune system at these mucosal sites plays a crucial role in minimizing viral transmission and infection. To develop new strategies for preventing SARS-CoV-2 infection, this study aimed to identify proteins that protect against viral infection in saliva. We collected 551 saliva samples from 290 healthcare workers who had tested positive for COVID-19, before vaccination, between June and December 2020. The samples were categorized based on their ability to block or enhance infection using in vitro assays. Mass spectrometry and enzyme-linked immunosorbent assay experiments were used to identify and measure the abundance of proteins that specifically bind to SARS-CoV-2 antigens. Immunoglobulin (Ig)A specific to SARS-CoV-2 antigens was detectable in over 83% of the convalescent saliva samples. We found that concentrations of anti-receptor-binding domain IgA >500 pg/µg total protein in saliva correlate with reduced viral infectivity in vitro. However, there is a dissociation between the salivary IgA response to SARS-CoV-2, and systemic IgG titers in convalescent COVID-19 patients. Then, using an innovative technique known as spike-baited mass spectrometry, we identified novel spike-binding proteins in saliva, most notably vimentin, which correlated with increased viral infectivity in vitro and could serve as a therapeutic target against COVID-19.


Asunto(s)
COVID-19 , Humanos , Anticuerpos Antivirales , Antígenos Virales , Inmunoglobulina A , SARS-CoV-2 , Vimentina
15.
Nat Microbiol ; 9(5): 1293-1311, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38622380

RESUMEN

Children infected with SARS-CoV-2 rarely progress to respiratory failure. However, the risk of mortality in infected people over 85 years of age remains high. Here we investigate differences in the cellular landscape and function of paediatric (<12 years), adult (30-50 years) and older adult (>70 years) ex vivo cultured nasal epithelial cells in response to infection with SARS-CoV-2. We show that cell tropism of SARS-CoV-2, and expression of ACE2 and TMPRSS2 in nasal epithelial cell subtypes, differ between age groups. While ciliated cells are viral replication centres across all age groups, a distinct goblet inflammatory subtype emerges in infected paediatric cultures and shows high expression of interferon-stimulated genes and incomplete viral replication. In contrast, older adult cultures infected with SARS-CoV-2 show a proportional increase in basaloid-like cells, which facilitate viral spread and are associated with altered epithelial repair pathways. We confirm age-specific induction of these cell types by integrating data from in vivo COVID-19 studies and validate that our in vitro model recapitulates early epithelial responses to SARS-CoV-2 infection.


Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Células Epiteliales , Mucosa Nasal , SARS-CoV-2 , Serina Endopeptidasas , Humanos , COVID-19/virología , SARS-CoV-2/fisiología , SARS-CoV-2/patogenicidad , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Adulto , Persona de Mediana Edad , Anciano , Células Epiteliales/virología , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Mucosa Nasal/virología , Niño , Factores de Edad , Replicación Viral , Preescolar , Tropismo Viral , Masculino , Femenino , Anciano de 80 o más Años , Células Cultivadas , Adolescente , Lactante
16.
Nat Commun ; 15(1): 1652, 2024 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-38396069

RESUMEN

Viral clearance, antibody response and the mutagenic effect of molnupiravir has not been elucidated in at-risk populations. Non-hospitalised participants within 5 days of SARS-CoV-2 symptoms randomised to receive molnupiravir (n = 253) or Usual Care (n = 324) were recruited to study viral and antibody dynamics and the effect of molnupiravir on viral whole genome sequence from 1437 viral genomes. Molnupiravir accelerates viral load decline, but virus is detectable by Day 5 in most cases. At Day 14 (9 days post-treatment), molnupiravir is associated with significantly higher viral persistence and significantly lower anti-SARS-CoV-2 spike antibody titres compared to Usual Care. Serial sequencing reveals increased mutagenesis with molnupiravir treatment. Persistence of detectable viral RNA at Day 14 in the molnupiravir group is associated with higher transition mutations following treatment cessation. Viral viability at Day 14 is similar in both groups with post-molnupiravir treated samples cultured up to 9 days post cessation of treatment. The current 5-day molnupiravir course is too short. Longer courses should be tested to reduce the risk of potentially transmissible molnupiravir-mutated variants being generated. Trial registration: ISRCTN30448031.


Asunto(s)
COVID-19 , Citidina/análogos & derivados , Hidroxilaminas , SARS-CoV-2 , Adulto , Humanos , SARS-CoV-2/genética , Pacientes Ambulatorios , Formación de Anticuerpos , Anticuerpos Antivirales , Antivirales/uso terapéutico
17.
Biomaterials ; 301: 122203, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37515903

RESUMEN

Lung infections are one of the leading causes of death worldwide, and this situation has been exacerbated by the emergence of COVID-19. Pre-clinical modelling of viral infections has relied on cell cultures that lack 3D structure and the context of lung extracellular matrices. Here, we propose a bioreactor-based, whole-organ lung model of viral infection. The bioreactor takes advantage of an automated system to achieve efficient decellularization of a whole rat lung, and recellularization of the scaffold using primary human bronchial cells. Automatization allowed for the dynamic culture of airway epithelial cells in a breathing-mimicking setup that led to an even distribution of lung epithelial cells throughout the distal regions. In the sealed bioreactor system, we demonstrate proof-of-concept for viral infection within the epithelialized lung by infecting primary human airway epithelial cells and subsequently injecting neutrophils. Moreover, to assess the possibility of drug screening in this model, we demonstrate the efficacy of the broad-spectrum antiviral remdesivir. This whole-organ scale lung infection model represents a step towards modelling viral infection of human cells in a 3D context, providing a powerful tool to investigate the mechanisms of the early stages of pathogenic infections and the development of effective treatment strategies for respiratory diseases.


Asunto(s)
COVID-19 , Neumonía , Virosis , Ratas , Humanos , Animales , Pulmón , Células Epiteliales , Andamios del Tejido/química
18.
iScience ; 25(11): 105409, 2022 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-36388965

RESUMEN

The airway epithelium is a protective barrier that is maintained by the self-renewal and differentiation of basal stem cells. Increasing age is a principle risk factor for chronic lung diseases, but few studies have explored age-related molecular or functional changes in the airway epithelium. We retrieved epithelial biopsies from histologically normal tracheobronchial sites from pediatric and adult donors and compared their cellular composition and gene expression profile (in laser capture-microdissected whole epithelium, fluorescence-activated cell-sorted basal cells, and basal cells in cell culture). Histologically, pediatric and adult tracheobronchial epithelium was similar in composition. We observed age-associated changes in RNA sequencing studies, including higher interferon-associated gene expression in pediatric epithelium. In cell culture, pediatric cells had higher colony formation ability, sustained in vitro growth, and outcompeted adult cells in a direct competitive proliferation assay. Our results demonstrate cell-intrinsic differences between airway epithelial cells from children and adults in both homeostatic and proliferative states.

19.
J Aerosol Med Pulm Drug Deliv ; 34(3): 181-188, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-32960118

RESUMEN

Background: Inhaled corticosteroids (ICSs) are the main prophylactic treatment for asthma and are used in other diseases, including chronic pulmonary obstructive disease, yet the interaction of ICS particles with the ciliated epithelium remains unclear. The aim of this study was to investigate the earliest interaction of aerosolized fluticasone propionate (FP) particles with human ciliated respiratory epithelium. Methods: A bespoke system was developed to allow aerosolized FP particles to be delivered to ciliated epithelial cultures by nebulization and from a pressurized metered-dose inhaler (pMDI) through a spacer with interactions observed in real time using high-speed video microscopy. Interaction with nonrespiratory cilia was investigated using steroids on brain ependymal ciliary cultures. The dissolution rate of steroid particles was determined. Results: FP particles delivered by aerosol attached to the tips of rapidly beating cilia. Within 2 hours, 8.7% ± 1.8% (nebulization) and 12.1% ± 2.1% (pMDI through spacer) of ciliated cells had one or more particles attached to motile cilia. These levels decreased to 5.8% ± 1.6% (p = 0.59; nebulization) and 5.3% ± 2.2% (p = 0.14; pMDI through spacer) at 24 hours. Particle attachment did not affect ciliary beat frequency (p > 0.05) but significantly (p < 0.001) reduced ciliary beat amplitude. Steroid particles also attached to the tips of motile ependymal brain cilia and also reduced beat amplitude (24 hours: >2 particles bound p < 0.001). Dissolution of FP particles was slow with only 22.8% ± 1.3% of nebulized and 12.8% ± 0.5% of pMDI-delivered drug dissolving by 24 hours. Conclusions: FP particles adhere to the tips of rapidly moving cilia with significant numbers remaining bound at 24 hours, resisting the shear stress generated by ciliary beating. In vivo, this mechanism may predispose to high local drug concentrations and enhance respiratory and systemic corticosteroid exposure.


Asunto(s)
Cilios , Inhaladores de Dosis Medida , Administración por Inhalación , Fluticasona , Humanos , Pulmón
20.
Nat Med ; 27(5): 904-916, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33879890

RESUMEN

Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy.


Asunto(s)
COVID-19/inmunología , Proteoma , SARS-CoV-2/inmunología , Análisis de la Célula Individual/métodos , Transcriptoma , Estudios Transversales , Humanos , Monocitos/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología
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