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.

Mortality surrogates in combined pulmonary fibrosis and emphysema.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) with coexistent emphysema, termed combined pulmonary fibrosis and emphysema (CPFE) may associate with reduced forced vital capacity (FVC) declines compared to non-CPFE IPF patients. We examined associations between mortality and functional measures of disease progression in two IPF cohorts. METHODS: Visual emphysema presence (>0% emphysema) scored on computed tomography identified CPFE patients (CPFE/non-CPFE: derivation cohort n=317/n=183, replication cohort n=358/n=152), who were subgrouped using 10% or 15% visual emphysema thresholds, and an unsupervised machine-learning model considering emphysema and interstitial lung disease extents. Baseline characteristics, 1-year relative FVC and diffusing capacity of the lung for carbon monoxide (D LCO) decline (linear mixed-effects models), and their associations with mortality (multivariable Cox regression models) were compared across non-CPFE and CPFE subgroups. RESULTS: In both IPF cohorts, CPFE patients with ≥10% emphysema had a greater smoking history and lower baseline D LCO compared to CPFE patients with <10% emphysema. Using multivariable Cox regression analyses in patients with ≥10% emphysema, 1-year D LCO decline showed stronger mortality associations than 1-year FVC decline. Results were maintained in patients suitable for therapeutic IPF trials and in subjects subgrouped by ≥15% emphysema and using unsupervised machine learning. Importantly, the unsupervised machine-learning approach identified CPFE patients in whom FVC decline did not associate strongly with mortality. In non-CPFE IPF patients, 1-year FVC declines ≥5% and ≥10% showed strong mortality associations. CONCLUSION: When assessing disease progression in IPF, D LCO decline should be considered in patients with ≥10% emphysema and a ≥5% 1-year relative FVC decline threshold considered in non-CPFE IPF patients.

Individualised Exercise Training Enhances Antioxidant Buffering Capacity in Idiopathic Pulmonary Fibrosis.

Exercise training is recommended for patients with idiopathic pulmonary fibrosis (IPF); however, the mechanism(s) underlying its physiological benefits remain unclear. We investigated the effects of an individualised aerobic interval training programme on exercise capacity and redox status in IPF patients. IPF patients were recruited prospectively to an 8-week, twice-weekly cardiopulmonary exercise test (CPET)-derived structured responsive exercise training programme (SRETP). Systemic redox status was assessed pre- and post-CPET at baseline and following SRETP completion. An age- and sex-matched non-IPF control cohort was recruited for baseline comparison only. At baseline, IPF patients (n = 15) had evidence of increased oxidative stress compared with the controls as judged by; the plasma reduced/oxidised glutathione ratio (median, control 1856 vs. IPF 736 p = 0.046). Eleven IPF patients completed the SRETP (median adherence 88%). Following SRETP completion, there was a significant improvement in exercise capacity assessed via the constant work-rate endurance time (+82%, p = 0.003). This was accompanied by an improvement in post-exercise redox status (in favour of antioxidants) assessed via serum total free thiols (median increase, +0.26 µmol/g protein p = 0.005) and total glutathione concentration (+0.73 µM p = 0.03), as well as a decrease in post-exercise lipid peroxidation products (-1.20 µM p = 0.02). Following SRETP completion, post-exercise circulating nitrite concentrations were significantly lower compared with baseline (-0.39 µM p = 0.04), suggestive of exercise-induced nitrite utilisation. The SRETP increased both endurance time and systemic antioxidant capacity in IPF patients. The observed reduction in nitrite concentrations provides a mechanistic rationale to investigate nitrite/nitrate supplementation in IPF patients.

Predicting the risk of chest radiograph abnormality 12-weeks post hospitalisation with SARS CoV-2 PCR confirmed COVID-19.

BACKGROUND: Routine follow-up of patients hospitalised with COVID-19 is recommended, however due to the ongoing high number of infections this is not without significant health resource and economic burden. In a previous study we investigated the prevalence of, and risk factors for, persistent chest radiograph (CXR) abnormalities post-hospitalisation with COVID-19 and identified a 5-point composite score that strongly predicted risk of persistent CXR abnormality at 12-weeks. Here we sought to validate and refine our findings in an independent cohort of patients. METHODOLOGY: A single-centre prospective study of consecutive patients attending a virtual post-hospitalisation COVID-19 clinic and CXR as part of their standard clinical care between 2nd March - 22nd June 2021. Inpatient and follow-up CXRs were scored by the assessing clinician for extent of pulmonary infiltrates (0-4 in each lung) with complete resolution defined as a follow-up score of zero. RESULTS: 182 consecutive patients were identified of which 31% had persistent CXR abnormality at 12-weeks. Patients with persistent CXR abnormality were significantly older (p < 0.001), had a longer hospital length of stay (p = 0.005), and had a higher incidence of both level 2 or 3 facility admission (level 2/3 care) (p = 0.003) and ever-smoking history (p = 0.038). Testing our composite score in the present cohort we found it predicted persistent CXR abnormality with reasonable accuracy (area under the receiver operator curve [AUROC 0.64]). Refining this score replacing obesity with Age ≥ 50 years, we identify the SHADE-750 score (1-point each for; Smoking history, Higher-level care (level 2/3 admission), Age ≥ 50 years, Duration of admission ≥ 15 days and Enzyme-lactate dehydrogenase (LDH ≥ 750U/L), that accurately predicted risk of persistent CXR abnormality, both in the present cohort (AUROC 0.73) and when retrospectively applied to our 1st cohort (AUROC 0.79). Applied to both cohorts combined (n = 213) it again performed strongly (AUROC 0.75) with all patients with a score of zero (n = 18) having complete CXR resolution at 12-weeks. CONCLUSIONS: In two independent cohorts of patients hospitalised with COVID-19, we identify a 5-point score which accurately predicts patients at risk of persistent CXR abnormality at 12-weeks. This tool could be used by clinicians to identify patients in which radiological follow-up may not be required.

Efficacy testing of an immersion vaccine against Aeromonas salmonicida and immunocompetence in ballan wrasse (Labrus bergylta, Ascanius).

The development of effective vaccines is a critical step towards the domestication of emerging fish species for aquaculture. However, traditional vaccine delivery through intraperitoneal (i.p.) injection requires fish to reach a minimum size and age and therefore cannot provide protection at early developmental stages when infection may occur. This study investigated the effectiveness of immersion vaccination with respect to immunocompetence in a cleaner fish species (ballan wrasse, Labrus bergylta, Ascanius) used in Atlantic salmon farming as an alternative means to control sea lice. The species is susceptible to atypical strains of Aeromonas salmonicida (aAs) at early life stages (<15 g), when i.p. vaccination is not applicable. While immersion vaccination is currently used in commercial hatcheries, the optimal fish size for vaccination, and efficacy of the vaccine delivered by this route has not yet been established. Importantly, efficacy depends on the capability of the species immune system to recognise antigens and process antigens to trigger and produce an adaptive immune response, (process known as immunocompetence). In this study, the efficacy of a polyvalent autogenous vaccine administered by immersion in juvenile ballan wrasse and the subsequent immune response induced was investigated after prime and booster vaccination regimes. In addition, temporal expression (0-150 days post hatch) of adaptive immune genes including major histocompatibility complex (MHC II CD74 molecule) and immunoglobulin M (IgM) was assessed using quantitative PCR (qPCR). Prime and/or boost vaccination by immersion of juvenile ballan wrasse (0.5 g and 1.5 g corresponding to 80 and 170 days post hatch (dph), respectively) did not provide significant protection against aAs vapA V after bath challenge under experimental conditions. Despite no evident protection >80 dph, MHC II and IgM transcripts were first reported at 35 and 75 dph, respectively, suggesting a window of immunocompetence. The results provide important new information on the onset of adaptive immunity in ballan wrasse and highlight that immersion vaccination in the species for protection against aAs should be performed at later developmental stages (>1.5 g) in the hatchery.

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.

Comparative genomics of European avian pathogenic E. Coli (APEC).

BACKGROUND: Avian pathogenic Escherichia coli (APEC) causes colibacillosis, which results in significant economic losses to the poultry industry worldwide. However, the diversity between isolates remains poorly understood. Here, a total of 272 APEC isolates collected from the United Kingdom (UK), Italy and Germany were characterised using multiplex polymerase chain reactions (PCRs) targeting 22 equally weighted factors covering virulence genes, R-type and phylogroup. Following these analysis, 95 of the selected strains were further analysed using Whole Genome Sequencing (WGS). RESULTS: The most prevalent phylogroups were B2 (47%) and A1 (22%), although there were national differences with Germany presenting group B2 (35.3%), Italy presenting group A1 (53.3%) and UK presenting group B2 (56.1%) as the most prevalent. R-type R1 was the most frequent type (55%) among APEC, but multiple R-types were also frequent (26.8%). Following compilation of all the PCR data which covered a total of 15 virulence genes, it was possible to build a similarity tree using each PCR result unweighted to produce 9 distinct groups. The average number of virulence genes was 6-8 per isolate, but no positive association was found between phylogroup and number or type of virulence genes. A total of 95 isolates representing each of these 9 groupings were genome sequenced and analysed for in silico serotype, Multilocus Sequence Typing (MLST), and antimicrobial resistance (AMR). The UK isolates showed the greatest variability in terms of serotype and MLST compared with German and Italian isolates, whereas the lowest prevalence of AMR was found for German isolates. Similarity trees were compiled using sequencing data and notably single nucleotide polymorphism data generated ten distinct geno-groups. The frequency of geno-groups across Europe comprised 26.3% belonging to Group 8 representing serogroups O2, O4, O18 and MLST types ST95, ST140, ST141, ST428, ST1618 and others, 18.9% belonging to Group 1 (serogroups O78 and MLST types ST23, ST2230), 15.8% belonging to Group 10 (serogroups O8, O45, O91, O125ab and variable MLST types), 14.7% belonging to Group 7 (serogroups O4, O24, O35, O53, O161 and MLST type ST117) and 13.7% belonging to Group 9 (serogroups O1, O16, O181 and others and MLST types ST10, ST48 and others). The other groups (2, 3, 4, 5 and 6) each contained relatively few strains. However, for some of the genogroups (e.g. groups 6 and 7) partial overlap with SNPs grouping and PCR grouping (matching PCR groups 8 (13 isolates on 22) and 1 (14 isolates on 16) were observable). However, it was not possible to obtain a clear correlation between genogroups and unweighted PCR groupings. This may be due to the genome plasticity of E. coli that enables strains to carry the same virulence factors even if the overall genotype is substantially different. CONCLUSIONS: The conclusion to be drawn from the lack of correlations is that firstly, APEC are very diverse and secondly, it is not possible to rely on any one or more basic molecular or phenotypic tests to define APEC with clarity, reaffirming the need for whole genome analysis approaches which we describe here. This study highlights the presence of previously unreported serotypes and MLSTs for APEC in Europe. Moreover, it is a first step on a cautious reconsideration of the merits of classical identification criteria such as R typing, phylogrouping and serotyping.

Yersinia ruckeri Isolates Recovered from Diseased Atlantic Salmon (Salmo salar) in Scotland Are More Diverse than Those from Rainbow Trout (Oncorhynchus mykiss) and Represent Distinct Subpopulations.

UNLABELLED: Yersinia ruckeri is the etiological agent of enteric redmouth (ERM) disease of farmed salmonids. Enteric redmouth disease is traditionally associated with rainbow trout (Oncorhynchus mykiss, Walbaum), but its incidence in Atlantic salmon (Salmo salar) is increasing. Yersinia ruckeri isolates recovered from diseased Atlantic salmon have been poorly characterized, and very little is known about the relationship of the isolates associated with these two species. Phenotypic approaches were used to characterize 109 Y. ruckeri isolates recovered over a 14-year period from infected Atlantic salmon in Scotland; 26 isolates from infected rainbow trout were also characterized. Biotyping, serotyping, and comparison of outer membrane protein profiles identified 19 Y. ruckeri clones associated with Atlantic salmon but only five associated with rainbow trout; none of the Atlantic salmon clones occurred in rainbow trout and vice versa These findings suggest that distinct subpopulations of Y. ruckeri are associated with each species. A new O serotype (designated O8) was identified in 56 biotype 1 Atlantic salmon isolates and was the most common serotype identified from 2006 to 2011 and in 2014, suggesting an increased prevalence during the time period sampled. Rainbow trout isolates were represented almost exclusively by the same biotype 2, serotype O1 clone that has been responsible for the majority of ERM outbreaks in this species within the United Kingdom since the 1980s. However, the identification of two biotype 2, serotype O8 isolates in rainbow trout suggests that vaccines containing serotypes O1 and O8 should be evaluated in both rainbow trout and Atlantic salmon for application in Scotland. IMPORTANCE: Vaccination plays an important role in protecting Atlantic salmon against the bacterial pathogen Yersinia ruckeri, but, in recent years, there has been an increasing incidence of vaccine breakdown in salmon. This is largely because current vaccines are aimed at rainbow trout and are based on serotypes specific for this species. A wider range of serotypes is responsible for infection in Atlantic salmon, but very little is known about the diversity of these strains and their relationships to those recovered from rainbow trout. In the present study, we demonstrate that Y. ruckeri isolates recovered from diseased Atlantic salmon in Scotland are more diverse than those from rainbow trout; furthermore, isolates from the two species represent distinct subpopulations. In addition, a new O serotype was identified that is responsible for a significant proportion of the disease in Atlantic salmon. Our findings are likely to have important implications for the development of improved vaccines against Y. ruckeri.

Interaction of Salmonella choleraesuis, Salmonella dublin and Salmonella typhimurium with porcine and bovine terminal ileum in vivo.

Quantitative experiments on the interaction of Salmonella choleraesuis and Salmonella dublin with porcine and bovine intestinal epithelia yielded no evidence to suggest that host restriction of S. choleraesuis and S. dublin for pigs and calves respectively could be explained in terms of the patterns of intestinal invasion observed in ligated ileal loops in vivo, at 3 h after challenge. No evidence was found to support the idea that Peyer's patches, or specifically M cells, are the major route of entry for these serotypes in vivo. Three hours after loop inoculation, each serotype was recovered in comparable numbers from either absorptive or Peyer's patch mucosae present in the same ileal loop, indicating that both types of tissue are involved in the early stages of the enteropathogenic process induced by both serotypes. More detailed transmission electron microscopic (TEM) analyses of follicle-associated epithelia (FAE) challenged with S. choleraesuis showed that in the same region of FAE, organisms invaded both M cells and enterocytes directly; comparable detailed TEM studies with S. dublin could not be carried out because of the tissue-destructive properties of this serotype. S. dublin was clearly more histotoxic than S. choleraesuis as had previously been found in rabbits: this difference is almost certainly due to a tissue-damaging toxin which is neither host nor gut-tissue specific. The tissue-destructive potential of S. dublin has profound implications for the measurement of and the assignment of significance to the invasiveness of S. dublin. S. dublin was nearly always seen entering gut cells in micro-colonies whereas S. choleraesuis entered mainly as single organisms or small groups of two or three.

Invasiveness of Salmonella serotypes Typhimurium, Choleraesuis and Dublin for rabbit terminal ileum in vitro.

Ten recent clinical isolates of Salmonella serotype Typhimurium from man that were tested for their invasiveness in rabbit ileal explants in vitro, were compared with Typhimurium strain TML, a well-characterised invasive strain isolated from a case of human gastro-enteritis. Nine of the 10 strains showed invasiveness that was comparable to that of strain TML. One isolate (GM3) was apparently substantially less invasive; electron microscopy showed this strain to be histotoxic - the probable reason for its reduced recovery from ileal mucosa and thus apparent 'low' invasiveness. Salmonella serotype Choleraesuis strain A50, isolated from a case of systemic salmonellosis in pigs, and serotype Dublin strain 3246, isolated from a case of systemic salmonellosis in calves, were also examined. Dublin strain 3246, when grown at 37 degrees C and used immediately in the invasion assay, damaged the mucosa in a manner similar to that of Typhimurium strain GM3, whereas Dublin strain 3246 grown at 37 degrees C and stored overnight at 4 degrees C did not. This was reflected in an apparently lower invasiveness of freshly grown organisms compared with that of organisms stored at 4 degrees C. In contrast, the histotoxicity of Typhimurium strain GM3 was not affected by storage at 4 degrees C. When stored at 4 degrees C, the levels of invasiveness of Choleraesuis strain A50 and Dublin strain 3246 were not significantly different from each other or from Typhimurium strain TML.