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The mutation profile of the SARS-CoV-2 Omicron (lineage BA.1) variant posed a concern for naturally acquired and vaccine-induced immunity. We investigated the ability of prior infection with an early SARS-CoV-2 ancestral isolate (Australia/VIC01/2020, VIC01) to protect against disease caused by BA.1. We established that BA.1 infection in naïve Syrian hamsters resulted in a less severe disease than a comparable dose of the ancestral virus, with fewer clinical signs including less weight loss. We present data to show that these clinical observations were almost absent in convalescent hamsters challenged with the same dose of BA.1 50 days after an initial infection with ancestral virus. These data provide evidence that convalescent immunity against ancestral SARS-CoV-2 is protective against BA.1 in the Syrian hamster model of infection. Comparison with published pre-clinical and clinical data supports consistency of the model and its predictive value for the outcome in humans. Further, the ability to detect protection against the less severe disease caused by BA.1 demonstrates continued value of the Syrian hamster model for evaluation of BA.1-specific countermeasures.
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COVID-19 , Animales , Cricetinae , Humanos , Convalecencia , Mesocricetus , SARS-CoV-2RESUMEN
Respiratory diseases account for over 5 million deaths yearly and are a huge burden to healthcare systems worldwide. Murine models have been of paramount importance to decode human lung biology in vivo, but their genetic, anatomical, physiological and immunological differences with humans significantly hamper successful translation of research into clinical practice. Thus, to clearly understand human lung physiology, development, homeostasis and mechanistic dysregulation that may lead to disease, it is essential to develop models that accurately recreate the extraordinary complexity of the human pulmonary architecture and biology. Recent advances in micro-engineering technology and tissue engineering have allowed the development of more sophisticated models intending to bridge the gap between the native lung and its replicates in vitro Alongside advanced culture techniques, remarkable technological growth in downstream analyses has significantly increased the predictive power of human biology-based in vitro models by allowing capture and quantification of complex signals. Refined integrated multi-omics readouts could lead to an acceleration of the translational pipeline from in vitro experimental settings to drug development and clinical testing in the future. This review highlights the range and complexity of state-of-the-art lung models for different areas of the respiratory system, from nasal to large airways, small airways and alveoli, with consideration of various aspects of disease states and their potential applications, including pre-clinical drug testing. We explore how development of optimised physiologically relevant in vitro human lung models could accelerate the identification of novel therapeutics with increased potential to translate successfully from the bench to the patient's bedside.
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Pulmón , Enfermedades Respiratorias , Humanos , Animales , Ratones , Pulmón/fisiología , Ingeniería de Tejidos/métodosRESUMEN
BACKGROUND: Effective treatment for severe asthma is a significant unmet need. While eosinophilic inflammation caused by type 2 cytokines is responsive to corticosteroid and biologic therapies, many severe asthmatics exhibit corticosteroid-unresponsive mixed granulocytic inflammation. OBJECTIVE: Here, we tested the hypothesis that the pro-allergic cytokine, IL-13, can drive both corticosteroid-sensitive and corticosteroid-resistant responses. RESULTS: By integration of in vivo and in vitro models of IL-13-driven inflammation, we identify a role for the epidermal growth factor receptor (EGFR/ERBB1) as a mediator of corticosteroid-unresponsive inflammation and bronchial hyperresponsiveness driven by IL-13. Topological data analysis using human epithelial transcriptomic data from the U-BIOPRED cohort identified severe asthma groups with features consistent with the presence of IL-13 and EGFR/ERBB activation, with involvement of distinct EGFR ligands. Our data suggest that IL-13 may play a dual role in severe asthma: on the one hand driving pathologic corticosteroid-refractory mixed granulocytic inflammation, but on the other hand underpinning beneficial epithelial repair responses, which may confound responses in clinical trials. CONCLUSION AND CLINICAL RELEVANCE: Detailed dissection of those molecular pathways that are downstream of IL-13 and utilize the ERBB receptor and ligand family to drive corticosteroid-refractory inflammation should enhance the development of new treatments that target this sub-phenotype(s) of severe asthma, where there is an unmet need.
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Corticoesteroides/farmacología , Asma/inmunología , Bronquios/inmunología , Resistencia a Medicamentos/inmunología , Receptores ErbB/inmunología , Interleucina-13/inmunología , Mucosa Respiratoria/inmunología , Animales , Asma/tratamiento farmacológico , Asma/genética , Asma/patología , Bronquios/patología , Resistencia a Medicamentos/genética , Receptores ErbB/genética , Interleucina-13/genética , Ratones , Ratones Transgénicos , Mucosa Respiratoria/patologíaAsunto(s)
Proteínas ADAM/inmunología , Asma/inmunología , Células Epiteliales/inmunología , Factor de Crecimiento Transformador beta/inmunología , Proteínas ADAM/genética , Alérgenos/inmunología , Animales , Células COS , Chlorocebus aethiops , Pulmón/inmunología , Ratones Transgénicos , Dominios Proteicos , Pyroglyphidae/inmunología , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The factors that determine fibrosis progression or normal tissue repair are largely unknown. We previously demonstrated that autophagy inhibition-mediated epithelial-mesenchymal transition (EMT) in human alveolar epithelial type II (ATII) cells augments local myofibroblast differentiation in pulmonary fibrosis by paracrine signalling. Here, we report that liver kinase B1 (LKB1) inactivation in ATII cells inhibits autophagy and induces EMT as a consequence. In IPF lungs, this is caused by downregulation of CAB39L, a key subunit within the LKB1 complex. 3D co-cultures of ATII cells and MRC5 lung fibroblasts coupled with RNA sequencing (RNA-seq) confirmed that paracrine signalling between LKB1-depleted ATII cells and fibroblasts augmented myofibroblast differentiation. Together these data suggest that reduced autophagy caused by LKB1 inhibition can induce EMT in ATII cells and contribute to fibrosis via aberrant epithelial-fibroblast crosstalk.
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Matrix stiffening by lysyl oxidase-like 2 (LOXL2)-mediated collagen cross-linking is proposed as a core feedforward mechanism that promotes fibrogenesis. Failure in clinical trials of simtuzumab (the humanized version of AB0023, a monoclonal antibody against human LOXL2) suggested that targeting LOXL2 may not have disease relevance; however, target engagement was not directly evaluated. We compare the spatial transcriptome of active human lung fibrogenesis sites with different human cell culture models to identify a disease-relevant model. Within the selected model, we then evaluate AB0023, identifying that it does not inhibit collagen cross-linking or reduce tissue stiffness, nor does it inhibit LOXL2 catalytic activity. In contrast, it does potently inhibit angiogenesis consistent with an alternative, non-enzymatic mechanism of action. Thus, AB0023 is anti-angiogenic but does not inhibit LOXL2 catalytic activity, collagen cross-linking, or tissue stiffening. These findings have implications for the interpretation of the lack of efficacy of simtuzumab in clinical trials of fibrotic diseases.
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The ongoing emergence of SARS-CoV-2 virus variants remains a source of concern because it is accompanied by the potential for increased virulence as well as evasion of immunity. Here we show that, although having an almost identical spike gene sequence as another Omicron variant (BA.5.2.1), a BA.4 isolate lacked all the typical disease characteristics of other isolates seen in the Golden Syrian hamster model despite replicating almost as effectively. Animals infected with BA.4 had similar viral shedding profiles to those seen with BA.5.2.1 (up to day 6 post-infection), but they all failed to lose weight or present with any other significant clinical signs. We hypothesize that this lack of detectable signs of disease during infection with BA.4 was due to a small (nine nucleotide) deletion (∆686-694) in the viral genome (ORF1ab) responsible for the production of non-structural protein 1, which resulted in the loss of three amino acids (aa 141-143).
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COVID-19 , Animales , Cricetinae , SARS-CoV-2/genética , Mesocricetus , Aminoácidos , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
The golden Syrian hamster (Mesocricetus auratus) is now commonly used in preclinical research for the study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the assessment of vaccines, drugs and therapeutics. Here, we show that hamsters inoculated via the intranasal route with the same infectious virus dose of prototypical SARS-CoV-2 administered in a different volume present with different clinical signs, weight loss and viral shedding, with a reduced volume resulting in reduced severity of disease similar to that obtained by a 500-fold reduction in the challenge dose. The tissue burden of the virus and the severity of pulmonary pathology were also significantly affected by different challenge inoculum volumes. These findings suggest that a direct comparison between the severity of SARS-CoV-2 variants or studies assessing the efficacy of treatments determined by hamster studies cannot be made unless both the challenge dose and inoculation volume are matched when using the intranasal route. Additionally, analysis of sub-genomic and total genomic RNA PCR data demonstrated no link between sub-genomic and live viral titres and that sub-genomic analyses do not provide any information beyond that provided by more sensitive total genomic PCR.
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COVID-19 , Cricetinae , Animales , Humanos , Mesocricetus , COVID-19/patología , SARS-CoV-2 , Pulmón , Gravedad del Paciente , Modelos Animales de EnfermedadRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disorder characterized by the proliferation of interstitial fibroblasts and the deposition of extracellular matrix causing impaired gas exchange. Spiruchostatin A (SpA) is a histone deacetylase inhibitor (HDI) with selectivity toward Class I enzymes, which distinguishes it from other nonspecific HDIs that are reported to inhibit (myo)fibroblast proliferation and differentiation. Because the selectivity of HDIs may be important clinically, we postulated that SpA inhibits the proliferation and differentiation of IPF fibroblasts. Primary fibroblasts were grown from lung biopsy explants obtained from patients with IPF or from normal control subjects, using two-dimensional or three-dimensional culture models. The effect of SpA on fibroproliferation in serum-containing medium ± transforming growth factor (TGF)-ß(1) was quantified by methylene blue binding. The acetylation of histone H3, the expression of the cell-cycle inhibitor p21(waf1), and the myofibroblast markers α-smooth muscle actin (α-SMA) and collagens I and III were determined by Western blotting, quantitative RT-PCR, immunofluorescent staining, or colorimetry. SpA inhibited the proliferation of IPF or normal fibroblasts in a time-dependent and concentration-dependent manner (concentration required to achieve 50% inhibition = 3.8 ± 0.4 nM versus 7.8 ± 0.2 nM, respectively; P < 0.05), with little cytotoxicity. Western blot analyses revealed that SpA caused a concentration-dependent increase in histone H3 acetylation, paralleling its antiproliferative effect. SpA also increased p21(waf1) expression, suggesting that direct cell-cycle regulation was the mechanism of inhibiting proliferation. Although treatment with TGF-ß(1) induced myofibroblast differentiation associated with increased expression of α-SMA, collagen I and collagen III and soluble collagen release, these responses were potently inhibited by SpA. These data support the concept that bicyclic tetrapeptide HDIs merit further investigation as potential treatments for IPF.
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Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Péptidos Cíclicos/farmacología , Fibrosis Pulmonar/patología , Células Cultivadas , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
A defining pathological feature of human lung fibrosis is localized tissue heterogeneity, which challenges the interpretation of transcriptomic studies that typically lose spatial information. Here we investigate spatial gene expression in diagnostic tissue using digital profiling technology. We identify distinct, region-specific gene expression signatures as well as shared gene signatures. By integration with single-cell data, we spatially map the cellular composition within and distant from the fibrotic niche, demonstrating discrete changes in homeostatic and pathologic cell populations even in morphologically preserved lung, while through ligand-receptor analysis, we investigate cellular cross-talk within the fibrotic niche. We confirm findings through bioinformatic, tissue, and in vitro analyses, identifying that loss of NFKB inhibitor zeta in alveolar epithelial cells dysregulates the TGFß/IL-6 signaling axis, which may impair homeostatic responses to environmental stress. Thus, spatially resolved deconvolution advances understanding of cell composition and microenvironment in human lung fibrogenesis.
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Fibrosis Pulmonar , Células Epiteliales Alveolares/metabolismo , Fibrosis , Humanos , Pulmón/patología , Fibrosis Pulmonar/metabolismo , Transducción de SeñalRESUMEN
Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFß increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.
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Colágeno/fisiología , Fibrosis Pulmonar/metabolismo , Biomarcadores , Células Cultivadas , Colágeno/química , Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Factor 1 Inducible por Hipoxia , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Estrés Oxidativo/fisiología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
OBJECTIVE: To investigate the effect of kinesiotape on hop distance, pain, and motoneuronal excitability in healthy people and people with Achilles tendinopathy (AT). DESIGN: Within-subject design. SETTING: An academic health science center, which is an acute London National Health Service trust. PARTICIPANTS: With ethical approval and informed consent, a convenience sample of 26 healthy people and 29 people with AT were recruited. Seven participants were lost after functional testing, leaving 24 participants in each group. INTERVENTIONS: Kinesiotape applied over the Achilles tendon. MAIN OUTCOME MEASURES: The single-leg hop test and visual analog scale were measured with and without the tape. Using the Hoffman (H) reflex, change in motoneuronal excitability of calf muscles was measured before tape application, with the tape on and after its removal. RESULTS: There were no changes to hop distance when tape was applied (P = 0.55). Additionally, there were no changes to pain (P = 0.74). The H reflex amplitude of soleus and gastrocnemius increased in the healthy group after its removal (P = 0.01 and P = 0.03, respectively), whereas the H reflex remained unchanged in people with AT (P = 0.43 and 0.16, respectively). CONCLUSIONS: Calf muscles were facilitated by kinesiotape in healthy participants. Despite this, there was no change to hop distance. Kinesiotape had no effect on hop distance, pain, or motoneuronal excitability in people with AT. These results do not support the use of kinesiotape applied in this way for this condition.
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Tendón Calcáneo/fisiopatología , Cinta Atlética , Salud , Neuronas Motoras/fisiología , Manejo del Dolor , Tendinopatía/terapia , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fuerza Muscular/fisiología , Músculo Esquelético/fisiopatología , Dimensión del Dolor , Reflejo Anormal/fisiología , Adulto JovenRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive disease that usually affects elderly people. It has a poor prognosis and there are limited therapies. Since epigenetic alterations are associated with IPF, histone deacetylase (HDAC) inhibitors offer a novel therapeutic strategy to address the unmet medical need. This study investigated the potential of romidepsin, an FDA-approved HDAC inhibitor, as an anti-fibrotic treatment and evaluated biomarkers of target engagement that may have utility in future clinical trials. The anti-fibrotic effects of romidepsin were evaluated both in vitro and in vivo together with any harmful effect on alveolar type II cells (ATII). Bronchoalveolar lavage fluid (BALF) from IPF or control donors was analyzed for the presence of lysyl oxidase (LOX). In parallel with an increase in histone acetylation, romidepsin potently inhibited fibroblast proliferation, myofibroblast differentiation and LOX expression. ATII cell numbers and their lamellar bodies were unaffected. In vivo, romidepsin inhibited bleomycin-induced pulmonary fibrosis in association with suppression of LOX expression. LOX was significantly elevated in BALF of IPF patients compared to controls. These data show the anti-fibrotic effects of romidepsin, supporting its potential use as novel treatment for IPF with LOX as a companion biomarker for evaluation of early on-target effects.
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Depsipéptidos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Acetilación , Biomarcadores , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Depsipéptidos/uso terapéutico , Epigénesis Genética , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/patología , MasculinoRESUMEN
Asthma is a chronic inflammatory airways disease that usually begins in early life and involves gene-environment interactions. Although most asthma exhibits allergic inflammation, many allergic individuals do not have asthma. Here, we report how the asthma gene a disintegrin and metalloprotease 33 (ADAM33) acts as local tissue susceptibility gene that promotes allergic asthma. We show that enzymatically active soluble ADAM33 (sADAM33) is increased in asthmatic airways and plays a role in airway remodeling, independent of inflammation. Furthermore, remodeling and inflammation are both suppressed in Adam33-null mice after allergen challenge. When induced in utero or added ex vivo, sADAM33 causes structural remodeling of the airways, which enhances postnatal airway eosinophilia and bronchial hyperresponsiveness following subthreshold challenge with an aeroallergen. This substantial gene-environment interaction helps to explain the end-organ expression of allergic asthma in genetically susceptible individuals. Finally, we show that sADAM33-induced airway remodeling is reversible, highlighting the therapeutic potential of targeting ADAM33 in asthma.