Regenerative Metaplastic Clones in COPD Lung Drive Inflammation and Fibrosis.

Chronic obstructive pulmonary disease (COPD) is a progressive condition of chronic bronchitis, small airway obstruction, and emphysema that represents a leading cause of death worldwide. While inflammation, fibrosis, mucus hypersecretion, and metaplastic epithelial lesions are hallmarks of this disease, their origins and dependent relationships remain unclear. Here we apply single-cell cloning technologies to lung tissue of patients with and without COPD. Unlike control lungs, which were dominated by normal distal airway progenitor cells, COPD lungs were inundated by three variant progenitors epigenetically committed to distinct metaplastic lesions. When transplanted to immunodeficient mice, these variant clones induced pathology akin to the mucous and squamous metaplasia, neutrophilic inflammation, and fibrosis seen in COPD. Remarkably, similar variants pre-exist as minor constituents of control and fetal lung and conceivably act in normal processes of immune surveillance. However, these same variants likely catalyze the pathologic and progressive features of COPD when expanded to high numbers.

Dietary restriction impacts health and lifespan of genetically diverse mice.

Caloric restriction extends healthy lifespan in multiple species1. Intermittent fasting, an alternative form of dietary restriction, is potentially more sustainable in humans, but its effectiveness remains largely unexplored2-8. Identifying the most efficacious forms of dietary restriction is key for developing interventions to improve human health and longevity9. Here we performed an extensive assessment of graded levels of caloric restriction (20% and 40%) and intermittent fasting (1 and 2 days fasting per week) on the health and survival of 960 genetically diverse female mice. We show that caloric restriction and intermittent fasting both resulted in lifespan extension in proportion to the degree of restriction. Lifespan was heritable and genetics had a larger influence on lifespan than dietary restriction. The strongest trait associations with lifespan included retention of body weight through periods of handling-an indicator of stress resilience, high lymphocyte proportion, low red blood cell distribution width and high adiposity in late life. Health effects differed between interventions and exhibited inconsistent relationships with lifespan extension. 40% caloric restriction had the strongest lifespan extension effect but led to a loss of lean mass and changes in the immune repertoire that could confer susceptibility to infections. Intermittent fasting did not extend the lifespan of mice with high pre-intervention body weight, and two-day intermittent fasting was associated with disruption of erythroid cell populations. Metabolic responses to dietary restriction, including reduced adiposity and lower fasting glucose, were not associated with increased lifespan, suggesting that dietary restriction does more than just counteract the negative effects of obesity. Our findings indicate that improving health and extending lifespan are not synonymous and raise questions about which end points are the most relevant for evaluating aging interventions in preclinical models and clinical trials.

Residual embryonic cells as precursors of a Barrett's-like metaplasia.

Barrett's esophagus is an intestine-like metaplasia and precursor of esophageal adenocarcinoma. Triggered by gastroesophageal reflux disease, the origin of this metaplasia remains unknown. p63-deficient mice, which lack squamous epithelia, may model acid-reflux damage. We show here that p63 null embryos rapidly develop intestine-like metaplasia with gene expression profiles similar to Barrett's metaplasia. We track its source to a unique embryonic epithelium that is normally undermined and replaced by p63-expressing cells. Significantly, we show that a discrete population of these embryonic cells persists in adult mice and humans at the squamocolumnar junction, the source of Barrett's metaplasia. We show that upon programmed damage to the squamous epithelium, these embryonic cells migrate toward adjacent, specialized squamous cells in a process that may recapitulate early Barrett's. Our findings suggest that certain precancerous lesions, such as Barrett's, initiate not from genetic alterations but from competitive interactions between cell lineages driven by opportunity.

Inflammatory Activity of Epithelial Stem Cell Variants from Cystic Fibrosis Lungs Is Not Resolved by CFTR Modulators.

Rationale: CFTR (cystic fibrosis transmembrane conductance regulator) modulator drugs restore function to mutant channels in patients with cystic fibrosis (CF) and lead to improvements in body mass index and lung function. Although it is anticipated that early childhood treatment with CFTR modulators will significantly delay or even prevent the onset of advanced lung disease, lung neutrophils and inflammatory cytokines remain high in patients with CF with established lung disease despite modulator therapy, underscoring the need to identify and ultimately target the sources of this inflammation in CF lungs. Objectives: To determine whether CF lungs, like chronic obstructive pulmonary disease (COPD) lungs, harbor potentially pathogenic stem cell "variants" distinct from the normal p63/Krt5 lung stem cells devoted to alveolar fates, to identify specific variants that might contribute to the inflammatory state of CF lungs, and to assess the impact of CFTR genetic complementation or CFTR modulators on the inflammatory variants identified herein. Methods: Stem cell cloning technology developed to resolve pathogenic stem cell heterogeneity in COPD and idiopathic pulmonary fibrosis lungs was applied to end-stage lungs of patients with CF (three homozygous CFTR:F508D, one CFTR F508D/L1254X; FEV1, 14-30%) undergoing therapeutic lung transplantation. Single-cell-derived clones corresponding to the six stem cell clusters resolved by single-cell RNA sequencing of these libraries were assessed by RNA sequencing and xenografting to monitor inflammation, fibrosis, and mucin secretion. The impact of CFTR activity on these variants after CFTR gene complementation or exposure to CFTR modulators was assessed by molecular and functional studies. Measurements and Main Results: End-stage CF lungs display a stem cell heterogeneity marked by five predominant variants in addition to the normal lung stem cell, of which three are proinflammatory both at the level of gene expression and their ability to drive neutrophilic inflammation in xenografts in immunodeficient mice. The proinflammatory functions of these three variants were unallayed by genetic or pharmacological restoration of CFTR activity. Conclusions: The emergence of three proinflammatory stem cell variants in CF lungs may contribute to the persistence of lung inflammation in patients with CF with advanced disease undergoing CFTR modulator therapy.

Prognostic factors associated with unhealed perineal wounds post-proctectomy for perianal Crohn's disease: a two-centre study.

AIM: The aim of this work was to determine the factors associated with poor wound healing in patients with perianal Crohn's disease (pCD) who had undergone proctectomy in the era of biologic therapies. METHOD: Case record review was performed on 103 patients with pCD who underwent proctectomy at St Mark's Hospital, Harrow and the Western General Hospital, Edinburgh between 2005 and 2017. Healing rates at 6 and 12 months post-proctectomy were considered; univariate analysis was performed. RESULTS: Sixty out of 103 patients (58.3%) had failure of wound healing at 6 months and 41/103 (39.8%) at 12 months. In total, 63.1% (65/103) patients received biologic therapies prior to proctectomy; however, exposure to biologics was not a significant factor in predicting failure of wound healing at 12 months (infliximab p = 0.255; adalimumab p = 0.889; vedolizumab p = 0.153). Male gender was the only variable associated with poor wound healing at 12 months on univariate analysis (p = 0.017). A lower pre-operative C-reactive protein was associated with early wound healing at 6 months compared with at 12 months (p = 0.041) on univariate analysis. Other parameters not associated with rates of wound healing included smoking status, corticosteroid exposure, thiopurine exposure, number of previous biologics, perianal sepsis on MRI within the last 12 months, duration of CD prior to proctectomy and pre-operative albumin. CONCLUSION: More than a third of patients had unhealed wounds 12 months after proctectomy. We report that unhealed wounds are more common in male patients. Importantly, our results also suggest that exposure to biologics does not affect rates of wound healing.

p63(+)Krt5(+) distal airway stem cells are essential for lung regeneration.

Lung diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis involve the progressive and inexorable destruction of oxygen exchange surfaces and airways, and have emerged as a leading cause of death worldwide. Mitigating therapies, aside from impractical organ transplantation, remain limited and the possibility of regenerative medicine has lacked empirical support. However, it is clinically known that patients who survive sudden, massive loss of lung tissue from necrotizing pneumonia or acute respiratory distress syndrome often recover full pulmonary function within six months. Correspondingly, we recently demonstrated lung regeneration in mice following H1N1 influenza virus infection, and linked distal airway stem cells expressing Trp63 (p63) and keratin 5, called DASC(p63/Krt5), to this process. Here we show that pre-existing, intrinsically committed DASC(p63/Krt5) undergo a proliferative expansion in response to influenza-induced lung damage, and assemble into nascent alveoli at sites of interstitial lung inflammation. We also show that the selective ablation of DASC(p63/Krt5) in vivo prevents this regeneration, leading to pre-fibrotic lesions and deficient oxygen exchange. Finally, we demonstrate that single DASC(p63/Krt5)-derived pedigrees differentiate to type I and type II pneumocytes as well as bronchiolar secretory cells following transplantation to infected lung and also minimize the structural consequences of endogenous stem cell loss on this process. The ability to propagate these cells in culture while maintaining their intrinsic lineage commitment suggests their potential in stem cell-based therapies for acute and chronic lung diseases.

Hierarchical analysis of RNA-seq reads improves the accuracy of allele-specific expression.

Motivation: Allele-specific expression (ASE) refers to the differential abundance of the allelic copies of a transcript. RNA sequencing (RNA-seq) can provide quantitative estimates of ASE for genes with transcribed polymorphisms. When short-read sequences are aligned to a diploid transcriptome, read-mapping ambiguities confound our ability to directly count reads. Multi-mapping reads aligning equally well to multiple genomic locations, isoforms or alleles can comprise the majority (>85%) of reads. Discarding them can result in biases and substantial loss of information. Methods have been developed that use weighted allocation of read counts but these methods treat the different types of multi-reads equivalently. We propose a hierarchical approach to allocation of read counts that first resolves ambiguities among genes, then among isoforms, and lastly between alleles. We have implemented our model in EMASE software (Expectation-Maximization for Allele Specific Expression) to estimate total gene expression, isoform usage and ASE based on this hierarchical allocation. Results: Methods that align RNA-seq reads to a diploid transcriptome incorporating known genetic variants improve estimates of ASE and total gene expression compared to methods that use reference genome alignments. Weighted allocation methods outperform methods that discard multi-reads. Hierarchical allocation of reads improves estimation of ASE even when data are simulated from a non-hierarchical model. Analysis of RNA-seq data from F1 hybrid mice using EMASE reveals widespread ASE associated with cis-acting polymorphisms and a small number of parent-of-origin effects. Availability and implementation: EMASE software is available at https://github.com/churchill-lab/emase. Supplementary information: Supplementary data are available at Bioinformatics online.

Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies.

BACKGROUND: Since they were first derived more than three decades ago, embryonic stem cells have been proposed as a source of replacement cells in regenerative medicine, but their plasticity and unlimited capacity for self-renewal raises concerns about their safety, including tumour formation ability, potential immune rejection, and the risk of differentiating into unwanted cell types. We report the medium-term to long-term safety of cells derived from human embryonic stem cells (hESC) transplanted into patients. METHODS: In the USA, two prospective phase 1/2 studies were done to assess the primary endpoints safety and tolerability of subretinal transplantation of hESC-derived retinal pigment epithelium in nine patients with Stargardt's macular dystrophy (age >18 years) and nine with atrophic age-related macular degeneration (age >55 years). Three dose cohorts (50,000, 100,000, and 150,000 cells) were treated for each eye disorder. Transplanted patients were followed up for a median of 22 months by use of serial systemic, ophthalmic, and imaging examinations. The studies are registered with ClinicalTrials.gov, numbers NCT01345006 (Stargardt's macular dystrophy) and NCT01344993 (age-related macular degeneration). FINDINGS: There was no evidence of adverse proliferation, rejection, or serious ocular or systemic safety issues related to the transplanted tissue. Adverse events were associated with vitreoretinal surgery and immunosuppression. 13 (72%) of 18 patients had patches of increasing subretinal pigmentation consistent with transplanted retinal pigment epithelium. Best-corrected visual acuity, monitored as part of the safety protocol, improved in ten eyes, improved or remained the same in seven eyes, and decreased by more than ten letters in one eye, whereas the untreated fellow eyes did not show similar improvements in visual acuity. Vision-related quality-of-life measures increased for general and peripheral vision, and near and distance activities, improving by 16-25 points 3-12 months after transplantation in patients with atrophic age-related macular degeneration and 8-20 points in patients with Stargardt's macular dystrophy. INTERPRETATION: The results of this study provide the first evidence of the medium-term to long-term safety, graft survival, and possible biological activity of pluripotent stem cell progeny in individuals with any disease. Our results suggest that hESC-derived cells could provide a potentially safe new source of cells for the treatment of various unmet medical disorders requiring tissue repair or replacement. FUNDING: Advanced Cell Technology.

Delayed Acute Pancreatitis After Transpapillary Gallbladder Drainage With Plastic Stents.

Endoscopic retrograde cholangiopancreatography-guided transpapillary gallbladder drainage has emerged as an effective alternative for management of acute cholecystitis in nonoperable candidates. Delayed acute pancreatitis has not been previously described as an adverse event with this procedure. In this article, we describe 3 patients who developed acute pancreatitis between 2 and 6 weeks after stent insertion with no alternative inciting cause. Delayed acute pancreatitis may represent a rare and previously uncharacterized adverse event related to transpapillary gallbladder drainage.

Ferroptosis regulates hemolysis in stored murine and human red blood cells.

Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. Here, we leveraged a diversity outbred mouse population to map the genetic drivers of fresh/stored RBC metabolism and extravascular hemolysis upon storage and transfusion in 350 mice. We identify the ferrireductase Steap3 as a critical regulator of a ferroptosis-like process of lipid peroxidation. Steap3 polymorphisms were associated with RBC iron content, in vitro hemolysis, and in vivo extravascular hemolysis both in mice and 13,091 blood donors from the Recipient Epidemiology and Donor evaluation Study. Using metabolite Quantitative Trait Loci analyses, we identified a network of gene products (FADS1/2, EPHX2 and LPCAT3) - enriched in donors of African descent - associated with oxylipin metabolism in stored human RBCs and related to Steap3 or its transcriptional regulator, the tumor protein TP53. Genetic variants were associated with lower in vivo hemolysis in thousands of single-unit transfusion recipients. Highlights: Steap3 regulates lipid peroxidation and extravascular hemolysis in 350 diversity outbred miceSteap3 SNPs are linked to RBC iron, hemolysis, vesiculation in 13,091 blood donorsmQTL analyses of oxylipins identified ferroptosis-related gene products FADS1/2, EPHX2, LPCAT3Ferroptosis markers are linked to hemoglobin increments in transfusion recipients.

SEESAW: detecting isoform-level allelic imbalance accounting for inferential uncertainty.

Detecting allelic imbalance at the isoform level requires accounting for inferential uncertainty, caused by multi-mapping of RNA-seq reads. Our proposed method, SEESAW, uses Salmon and Swish to offer analysis at various levels of resolution, including gene, isoform, and aggregating isoforms to groups by transcription start site. The aggregation strategies strengthen the signal for transcripts with high uncertainty. The SEESAW suite of methods is shown to have higher power than other allelic imbalance methods when there is isoform-level allelic imbalance. We also introduce a new test for detecting imbalance that varies across a covariate, such as time.

Multi-omics analysis identifies drivers of protein phosphorylation.

BACKGROUND: Phosphorylation of proteins is a key step in the regulation of many cellular processes including activation of enzymes and signaling cascades. The abundance of a phosphorylated peptide (phosphopeptide) is determined by the abundance of its parent protein and the proportion of target sites that are phosphorylated. RESULTS: We quantified phosphopeptides, proteins, and transcripts in heart, liver, and kidney tissue samples of mice from 58 strains of the Collaborative Cross strain panel. We mapped ~700 phosphorylation quantitative trait loci (phQTL) across the three tissues and applied genetic mediation analysis to identify causal drivers of phosphorylation. We identified kinases, phosphatases, cytokines, and other factors, including both known and potentially novel interactions between target proteins and genes that regulate site-specific phosphorylation. Our analysis highlights multiple targets of pyruvate dehydrogenase kinase 1 (PDK1), a regulator of mitochondrial function that shows reduced activity in the NZO/HILtJ mouse, a polygenic model of obesity and type 2 diabetes. CONCLUSIONS: Together, this integrative multi-omics analysis in genetically diverse CC strains provides a powerful tool to identify regulators of protein phosphorylation. The data generated in this study provides a resource for further exploration.

Cloning a profibrotic stem cell variant in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible, and rapidly fatal interstitial lung disease marked by the replacement of lung alveoli with dense fibrotic matrices. Although the mechanisms initiating IPF remain unclear, rare and common alleles of genes expressed in lung epithelia, combined with aging, contribute to the risk for this condition. Consistently, single-cell RNA sequencing (scRNA-seq) studies have identified lung basal cell heterogeneity in IPF that might be pathogenic. We used single-cell cloning technologies to generate "libraries" of basal stem cells from the distal lungs of 16 patients with IPF and 10 controls. We identified a major stem cell variant that was distinguished from normal stem cells by its ability to transform normal lung fibroblasts into pathogenic myofibroblasts in vitro and to activate and recruit myofibroblasts in clonal xenografts. This profibrotic stem cell variant, which was shown to preexist in low quantities in normal and even fetal lungs, expressed a broad network of genes implicated in organ fibrosis and showed overlap in gene expression with abnormal epithelial signatures identified in previously published scRNA-seq studies of IPF. Drug screens highlighted specific vulnerabilities of this profibrotic variant to inhibitors of epidermal growth factor and mammalian target of rapamycin signaling as prospective therapeutic targets. This profibrotic stem cell variant in IPF was distinct from recently identified profibrotic stem cell variants in chronic obstructive pulmonary disease and may extend the notion that inappropriate accrual of minor and preexisting stem cell variants contributes to chronic lung conditions.

Post-operative follow-up care after acute spinal trauma: What is the reality?

•Only 23.7% of acute spinal trauma patients who underwent instrumentation met or surpassed one-year of clinical follow-up care.•Factors associated with lower rates of completed follow-up are ISS, presence of non-ambulatory spinal cord injury, history of IVDA, and insurance.•Increased rates of completed follow-up were seen in patients with a Workers'.

Long-term cardiovascular outcomes after orlistat therapy in patients with obesity: a nationwide, propensity-score matched cohort study.

AIMS: The rising prevalence of obesity and its associated comorbidities represent a growing public health issue; in particular, obesity is known to be a major risk factor for cardiovascular disease. Despite the evidence behind the efficacy of orlistat in achieving weight loss in patients with obesity, no study thus far has quantified its long-term effect on cardiovascular outcomes. The purpose of this study is to explore long-term cardiovascular outcomes after orlistat therapy. METHODS AND RESULTS: A propensity-score matched cohort study was conducted on the nation-wide electronic primary and integrated secondary healthcare records of the Clinical Practice Research Datalink (CPRD). The 36 876 patients with obesity in the CPRD database who had completed a course of orlistat during follow-up were matched on a 1:1 basis with equal numbers of controls who had not taken orlistat. Patients were followed up for a median of 6 years for the occurrence of the primary composite endpoint of major adverse cardiovascular events (fatal or non-fatal myocardial infarction or ischaemic stroke), and a number of secondary endpoints including primary endpoint components individually, the occurrence of new-onset heart failure, coronary revascularization, new chronic kidney disease stage III+ (CKD3+), and all-cause mortality. During the median study follow-up of 6 years, the occurrence of major adverse cardiovascular events was lower in the orlistat cohort [hazard ratio (HR) 0.74; 95% confidence interval (CI) 0.66-0.83, P < 0.001]. Patients who took orlistat experienced lower rates of myocardial infarction (HR 0.77; 95% CI 0.66-0.88, P < 0.001) and ischaemic stroke (HR 0.68; 95% CI 0.56 to -0.84, P < 0.001) as well as new-onset heart failure (HR 0.79; 95% CI 0.67-0.94, P = 0.007). There was no differences in revascularization rates (HR 1.12; 95% CI 0.91-1.38, P = 0.27), but a lower rate of both CKD3+ development (HR 0.78; 95% CI 0.73-0.83, P < 0.001) and mortality (HR 0.39, 95% CI 0.36 to -0.41, P < 0.001) was observed. CONCLUSION: In this nation-wide, propensity-score matched study, orlistat was associated with lower rates of overall major adverse cardiovascular events, new-onset heart failure, renal failure, and mortality. This study adds to current evidence on the known improvements in cardiovascular risk factor profiles of orlistat treatment by suggesting a potential role in primary prevention.

QTLViewer: an interactive webtool for genetic analysis in the Collaborative Cross and Diversity Outbred mouse populations.

The Collaborative Cross and the Diversity Outbred mouse populations are related multiparental populations, derived from the same 8 isogenic founder strains. They carry >50 M known genetic variants, which makes them ideal tools for mapping genetic loci that regulate phenotypes, including physiological and molecular traits. Mapping quantitative trait loci requires statistical and computational training, which can present a barrier to access for some researchers. The QTLViewer software allows users to graphically explore Collaborative Cross and Diversity Outbred quantitative trait locus mapping and related analyses performed through the R/qtl2 package. Additionally, the QTLViewer website serves as a repository for published Collaborative Cross and Diversity Outbred studies, increasing the accessibility of these genetic resources to the broader scientific community.

Hypoaminoacidemia underpins glucagon-mediated energy expenditure and weight loss.

Glucagon analogs show promise as components of next-generation, multi-target, anti-obesity therapeutics. The biology of chronic glucagon treatment, in particular, its ability to induce energy expenditure and weight loss, remains poorly understood. Using a long-acting glucagon analog, G108, we demonstrate that glucagon-mediated body weight loss is intrinsically linked to the hypoaminoacidemia associated with its known amino acid catabolic action. Mechanistic studies reveal an energy-consuming response to low plasma amino acids in G108-treated mice, prevented by dietary amino acid supplementation and mimicked by a rationally designed low amino acid diet. Therefore, low plasma amino acids are a pre-requisite for G108-mediated energy expenditure and weight loss. However, preventing hypoaminoacidemia with additional dietary protein does not affect the ability of G108 to improve glycemia or hepatic steatosis in obese mice. These studies provide a mechanism for glucagon-mediated weight loss and confirm the hepatic glucagon receptor as an attractive molecular target for metabolic disease therapeutics.

Striking the Balance: GLP-1/Glucagon Co-Agonism as a Treatment Strategy for Obesity.

Obesity and Type 2 diabetes represent global health challenges, and there is an unmet need for long-lasting and effective pharmacotherapies. Although long-acting glucagon-like peptide-1 (GLP-1) analogues are now in routine use for diabetes and are now being utilised for obesity per se, the need for ever better treatments has driven the development of co-agonists, with the theoretical advantages of improved efficacy by targeting multiple pathways and reduced adverse effects. In this review, we highlight the past and present progress in our understanding and development of treatments based on GLP-1/glucagon co-agonism. We also reflect on the divergent effects of varying the GLP-1:glucagon activity and ratio in the context of pre-clinical and human clinical trial findings. In particular, the multiple metabolic actions of glucagon highlight the importance of understanding the contributions of individual hormone action to inform the safe, effective and tailored use of GLP-1/glucagon co-agonists to target weight loss and metabolic disease in the future.