LKB1 depletion-mediated epithelial-mesenchymal transition induces fibroblast activation in lung fibrosis.

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.

Genetic and geographic influence on phenotypic variation in European sarcoidosis patients.

Introduction: Sarcoidosis is a highly variable disease in terms of organ involvement, type of onset and course. Associations of genetic polymorphisms with sarcoidosis phenotypes have been observed and suggest genetic signatures. Methods: After obtaining a positive vote of the competent ethics committee we genotyped 1909 patients of the deeply phenotyped Genetic-Phenotype Relationship in Sarcoidosis (GenPhenReSa) cohort of 31 European centers in 12 countries with 116 potentially disease-relevant single-nucleotide polymorphisms (SNPs). Using a meta-analysis, we investigated the association of relevant phenotypes (acute vs. sub-acute onset, phenotypes of organ involvement, specific organ involvements, and specific symptoms) with genetic markers. Subgroups were built on the basis of geographical, clinical and hospital provision considerations. Results: In the meta-analysis of the full cohort, there was no significant genetic association with any considered phenotype after correcting for multiple testing. In the largest sub-cohort (Serbia), we confirmed the known association of acute onset with TNF and reported a new association of acute onset an HLA polymorphism. Multi-locus models with sets of three SNPs in different genes showed strong associations with the acute onset phenotype in Serbia and Lublin (Poland) demonstrating potential region-specific genetic links with clinical features, including recently described phenotypes of organ involvement. Discussion: The observed associations between genetic variants and sarcoidosis phenotypes in subgroups suggest that gene-environment-interactions may influence the clinical phenotype. In addition, we show that two different sets of genetic variants are permissive for the same phenotype of acute disease only in two geographic subcohorts pointing to interactions of genetic signatures with different local environmental factors. Our results represent an important step towards understanding the genetic architecture of sarcoidosis.

A Qualitative Study of Medical Students' Perspectives on Distance Learning During COVID-19.

Introduction: With the Coronavirus disease 2019 [COVID-19] pandemic, changes were undertaken out of necessity to allow medical students to continue their education. The aim of this study is to create key themes for educators to consider when implementing distance learning strategies into the curriculum based on 2nd year graduate entry medical students experience of learning and engagement with the use of distance learning during the COVID-19 pandemic. Materials and Methods: A qualitative study with a phenomenological methodology was set within a constructivist paradigm. A volunteer sampling strategy was used to recruit participants. Nine semi-structured, audio-recorded interviews were undertaken and transcribed verbatim. A thematic analysis was undertaken of the transcripts using the Braun and Clarke framework with an open-coded approach. Results: Exploration of the student experience generated an understanding of the learning process. The concept of adaptability emerged based on the themes of technology, environment, study skills and human interaction. Discussion: Necessary changes to the formal curriculum affected medical students learning and experience that demanded adaptability. The 'new normal' generated a context within which students were communicating and interacting in ways creating individual challenges for students and educators. Conclusion: With the advancements in information, communication and technology, distance learning is likely to be further incorporated in undergraduate training long term. Its position should be one that is harmonious within the wider educational realm that engages and meets the needs of the students. The rich understanding exposes adaptations and considerations for educators to improve the student experience.

Spatially resolved deconvolution of the fibrotic niche in lung fibrosis.

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.

Bidirectional epithelial-mesenchymal crosstalk provides self-sustaining profibrotic signals in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1-mediated epithelial-mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-ß-induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial-mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1-tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-ß-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor-like repeats. Together, these data identify that aberrant bidirectional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.

"You have to change your whole life": A qualitative study of the dynamics of treatment adherence among adults with tuberculosis in the United Kingdom.

Maintaining adherence to treatment for tuberculosis (TB) is essential if the disease is to be eliminated. As part of formative research to develop an intervention to improve adherence, we documented the lived experiences of adults receiving anti-TB treatment (ATT) in three UK cities and examined how personal, social, and structural circumstances interacted to impact on individuals' adherence to treatment. Using a topic guide that explored social circumstances and experiences of TB care, we conducted in-depth interviews with 18 adults (six women) who were being or had been treated for TB (patients) and four adults (all women) who were caring for a friend, relative, or partner being treated for TB (caregivers). We analysed transcripts using an adapted framework method that classified factors affecting adherence as personal, social, structural, health systems, or treatment-related. Eleven of 18 patients were born outside the UK (in South, Central, and East Asia, and Eastern and Southern Africa); among the seven who were UK-born, four were Black, Asian, or Minority Ethnic and three were White British. TB and its treatment were often disruptive: in addition to debilitating symptoms and side effects of ATT, participants faced job insecurity, unstable housing, stigma, social isolation, worsening mental health, and damaged relationships. Those who had a strong support network, stable employment, a routine that could easily be adapted, a trusting relationship with their TB team, and clear understanding of the need for treatment reported finding it easier to adhere to ATT. Changes in circumstances sometimes had dramatic effects on an individual's ability to take ATT; participants described how the impact of certain acute events (e.g., the onset of side effects or fatigue, episodes of stigmatisation, loss of income) were amplified by their timing or through their interaction with other elements of the individual's life. We suggest that the dynamic and fluctuating nature of these factors necessitates comprehensive and regular review of needs and potential problems, conducted before and during ATT; this, coupled with supportive measures that consider (and seek to mitigate) the influence of social and structural factors, may help improve adherence.

Breathlessness and cough in the acute setting.

The symptom of breathlessness is well recognized as part of the presentation of a wide range of medical conditions. It can be a manifestation of a life-threatening emergency. In acute medical settings, the priority is to quickly recognize patients who are critically unwell and require emergency treatment. For these patients, rapid initial assessment and immediate treatment are essential. However, once symptoms have stabilized or in less acute settings, a more thorough assessment is required. Cough is a common respiratory symptom, often part of a symptom complex, which is troublesome for the patient. It is important to recognize worrying associated features to prompt further investigation. In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection emerged from a zoonotic source, resulting in many cases of infection, hospitalizations and deaths, and has since spread in a pandemic across every continent. A substantial percentage of patients with COVID-19 develop an acute respiratory illness requiring hospital care. Cough and acute breathlessness are two of the most prevalent symptoms in this infection; any patient presenting to an acute setting should currently be assumed to have this infection and immediately tested with a viral swab from the upper airway to guide management.

Comprehensive plasma proteomic profiling reveals biomarkers for active tuberculosis.

BACKGROUNDTuberculosis (TB) kills more people than any other infection, and new diagnostic tests to identify active cases are required. We aimed to discover and verify novel markers for TB in nondepleted plasma.METHODSWe applied an optimized quantitative proteomics discovery methodology based on multidimensional and orthogonal liquid chromatographic separation combined with high-resolution mass spectrometry to study nondepleted plasma of 11 patients with active TB compared with 10 healthy controls. Prioritized candidates were verified in independent UK (n = 118) and South African cohorts (n = 203).RESULTSWe generated the most comprehensive TB plasma proteome to date, profiling 5022 proteins spanning 11 orders-of-magnitude concentration range with diverse biochemical and molecular properties. We analyzed the predominantly low-molecular weight subproteome, identifying 46 proteins with significantly increased and 90 with decreased abundance (peptide FDR ≤ 1%, q ≤ 0.05). Verification was performed for novel candidate biomarkers (CFHR5, ILF2) in 2 independent cohorts. Receiver operating characteristics analyses using a 5-protein panel (CFHR5, LRG1, CRP, LBP, and SAA1) exhibited discriminatory power in distinguishing TB from other respiratory diseases (AUC = 0.81).CONCLUSIONWe report the most comprehensive TB plasma proteome to date, identifying novel markers with verification in 2 independent cohorts, leading to a 5-protein biosignature with potential to improve TB diagnosis. With further development, these biomarkers have potential as a diagnostic triage test.FUNDINGColciencias, Medical Research Council, Innovate UK, NIHR, Academy of Medical Sciences, Program for Advanced Research Capacities for AIDS, Wellcome Centre for Infectious Diseases Research.

Paracrine SPARC signaling dysregulates alveolar epithelial barrier integrity and function in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic scarring disease in which aging, environmental exposure(s) and genetic susceptibility have been implicated in disease pathogenesis, however, the causes and mechanisms of the progressive fibrotic cascade are still poorly understood. As epithelial-mesenchymal interactions are essential for normal wound healing, through human 2D and 3D in vitro studies, we tested the hypothesis that IPF fibroblasts (IPFFs) dysregulate alveolar epithelial homeostasis. Conditioned media from IPFFs exaggerated the wound-healing response of primary human Type II alveolar epithelial cells (AECs). Furthermore, AECs co-cultured with IPFFs exhibited irregular epithelialization compared with those co-cultured with control fibroblasts (NHLFs) or AECs alone, suggesting that epithelial homeostasis is dysregulated in IPF as a consequence of the abnormal secretory phenotype of IPFFs. Secretome analysis of IPFF conditioned media and functional studies identified the matricellular protein, SPARC, as a key mediator in the epithelial-mesenchymal paracrine signaling, with increased secretion of SPARC by IPFFs promoting persistent activation of alveolar epithelium via an integrin/focal adhesion/cellular-junction axis resulting in disruption of epithelial barrier integrity and increased macromolecular permeability. These findings suggest that in IPF fibroblast paracrine signaling promotes persistent alveolar epithelial activation, so preventing normal epithelial repair responses and restoration of tissue homeostasis. Furthermore, they identify SPARC-mediated paracrine signaling as a potential therapeutic target to promote the restoration of lung epithelial homoestasis in IPF patients.

Autophagy inhibition-mediated epithelial-mesenchymal transition augments local myofibroblast differentiation in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF), the prototypic progressive fibrotic interstitial lung disease, is thought to be a consequence of repetitive micro-injuries to an ageing, susceptible alveolar epithelium. Ageing is a risk factor for IPF and incidence has been demonstrated to increase with age. Decreased (macro)autophagy with age has been reported extensively in a variety of systems and diseases, including IPF. However, it is undetermined whether the role of faulty autophagy is causal or coincidental in the context of IPF. Here, we report that in alveolar epithelial cells inhibition of autophagy promotes epithelial-mesenchymal transition (EMT), a process implicated in embryonic development, wound healing, cancer metastasis and fibrosis. We further demonstrate that this is attained, at least in part, by increased p62/SQSTM1 expression that promotes p65/RELA mediated-transactivation of an EMT transcription factor, Snail2 (SNAI2), which not only controls EMT but also regulates the production of locally acting profibrogenic mediators. Our data suggest that reduced autophagy induces EMT of alveolar epithelial cells and can contribute to fibrosis via aberrant epithelial-fibroblast crosstalk.

Paracrine signalling during ZEB1-mediated epithelial-mesenchymal transition augments local myofibroblast differentiation in lung fibrosis.

The contribution of epithelial-mesenchymal transition (EMT) to human lung fibrogenesis is controversial. Here we provide evidence that ZEB1-mediated EMT in human alveolar epithelial type II (ATII) cells contributes to the development of lung fibrosis by paracrine signalling to underlying fibroblasts. Activation of EGFR-RAS-ERK signalling in ATII cells induced EMT via ZEB1. ATII cells had extremely low extracellular matrix gene expression even after induction of EMT, however conditioned media from ATII cells undergoing RAS-induced EMT augmented TGFß-induced profibrogenic responses in lung fibroblasts. This epithelial-mesenchymal crosstalk was controlled by ZEB1 via the expression of tissue plasminogen activator (tPA). In human fibrotic lung tissue, nuclear ZEB1 expression was detected in alveolar epithelium adjacent to sites of extracellular matrix (ECM) deposition, suggesting that ZEB1-mediated paracrine signalling has the potential to contribute to early fibrotic changes in the lung interstitium. Targeting this novel ZEB1 regulatory axis may be a viable strategy for the treatment of pulmonary fibrosis.

Nanoscale dysregulation of collagen structure-function disrupts mechano-homeostasis and mediates pulmonary fibrosis.

Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.

Tracking and responding to an outbreak of tuberculosis using MIRU-VNTR genotyping and whole genome sequencing as epidemiological tools.

Background: We describe an outbreak that contributed to a near doubling of the incidence of tuberculosis in Southampton, UK. We examine the importance of 24 locus mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) genotyping in its identification and management and the role of whole genome sequencing (WGS) in tracing the spread of the strain. Methods: Outbreak cases were defined as those diagnosed between January and December 2011 with indistinguishable 24 locus-MIRU-VNTR genotypes or, cases linked epidemiologically. A cluster questionnaire was administered by TB nurses to identify contacts and social settings. Results: Overall, 25 patients fulfilled the case definition. No cases with this MIRU-VNTR genotype had been detected in the UK previously. Connections were found between all cases through household contacts or social venues including a football club, Internet cafe and barber's shop. Public health actions included extended contact tracing, venue screening and TB awareness-raising. The outbreak resulted in a high rate of transmission and high incidence of clinical disease among contacts. Conclusions: This outbreak illustrates the value of combining active case-finding with prospective MIRU-VNTR genotyping to identify settings to undertake public health action. In addition WGS revealed that the VNTR-defined cluster was a single outbreak and that active TB transmission not reactivation was responsible for this outbreak in non-UK born individuals.

Dissection of the host-pathogen interaction in human tuberculosis using a bioengineered 3-dimensional model.

Cell biology differs between traditional cell culture and 3-dimensional (3-D) systems, and is modulated by the extracellular matrix. Experimentation in 3-D presents challenges, especially with virulent pathogens. Mycobacterium tuberculosis (Mtb) kills more humans than any other infection and is characterised by a spatially organised immune response and extracellular matrix remodelling. We developed a 3-D system incorporating virulent mycobacteria, primary human blood mononuclear cells and collagen-alginate matrix to dissect the host-pathogen interaction. Infection in 3-D led to greater cellular survival and permitted longitudinal analysis over 21 days. Key features of human tuberculosis develop, and extracellular matrix integrity favours the host over the pathogen. We optimised multiparameter readouts to study emerging therapeutic interventions: cytokine supplementation, host-directed therapy and immunoaugmentation. Each intervention modulates the host-pathogen interaction, but has both beneficial and harmful effects. This methodology has wide applicability to investigate infectious, inflammatory and neoplastic diseases and develop novel drug regimes and vaccination approaches.

The safety of new drug treatments for idiopathic pulmonary fibrosis.

INTRODUCTION: The management of idiopathic pulmonary fibrosis (IPF) has been transformed by the recent approval of two anti-fibrotic drugs, nintedanib and pirfenidone. An increasing number of patients with IPF are receiving treatment with these novel therapies, and the risk of adverse events that may be associated with their use must be carefully evaluated. Areas covered: Safety data about nintedanib and pirfenidone is critically evaluated, including data from randomized clinical trials and post-marketing reports. Management strategies to minimize the occurrence of side effects are summarized. Expert opinion: The safety profile of the two anti-fibrotic drugs approved for clinical use in IPF patients appears to be comparable. Data from clinical trials and initial post-marketing surveillance indicate that most of the observed side effects are mild and easily manageable. However, approximately 1/5 of patients may discontinue treatment as a consequence of side effects. Careful patient counselling, and regular follow-up during therapy could reduce the rate of discontinuations. Ongoing post-marketing surveillance may further inform our understanding of the safety profile of these therapies.