Defining predictors of responsiveness to advanced therapies in Crohn's disease and ulcerative colitis: protocol for the IBD-RESPONSE and nested CD-metaRESPONSE prospective, multicentre, observational cohort study in precision medicine.

INTRODUCTION: Characterised by chronic inflammation of the gastrointestinal tract, inflammatory bowel disease (IBD) symptoms including diarrhoea, abdominal pain and fatigue can significantly impact patient's quality of life. Therapeutic developments in the last 20 years have revolutionised treatment. However, clinical trials and real-world data show primary non-response rates up to 40%. A significant challenge is an inability to predict which treatment will benefit individual patients.Current understanding of IBD pathogenesis implicates complex interactions between host genetics and the gut microbiome. Most cohorts studying the gut microbiota to date have been underpowered, examined single treatments and produced heterogeneous results. Lack of cross-treatment comparisons and well-powered independent replication cohorts hampers the ability to infer real-world utility of predictive signatures.IBD-RESPONSE will use multi-omic data to create a predictive tool for treatment response. Future patient benefit may include development of biomarker-based treatment stratification or manipulation of intestinal microbial targets. IBD-RESPONSE and downstream studies have the potential to improve quality of life, reduce patient risk and reduce expenditure on ineffective treatments. METHODS AND ANALYSIS: This prospective, multicentre, observational study will identify and validate a predictive model for response to advanced IBD therapies, incorporating gut microbiome, metabolome, single-cell transcriptome, human genome, dietary and clinical data. 1325 participants commencing advanced therapies will be recruited from ~40 UK sites. Data will be collected at baseline, week 14 and week 54. The primary outcome is week 14 clinical response. Secondary outcomes include clinical remission, loss of response in week 14 responders, corticosteroid-free response/remission, time to treatment escalation and change in patient-reported outcome measures. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Wales Research Ethics Committee 5 (ref: 21/WA/0228). Recruitment is ongoing. Following study completion, results will be submitted for publication in peer-reviewed journals and presented at scientific meetings. Publications will be summarised at www.ibd-response.co.uk. TRIAL REGISTRATION NUMBER: ISRCTN96296121.

Dual bronchodilators in Bronchiectasis study (DIBS): protocol for a pragmatic, multicentre, placebo-controlled, three-arm, double-blinded, randomised controlled trial studying bronchodilators in preventing exacerbations of bronchiectasis.

INTRODUCTION: Bronchiectasis is a long-term lung condition, with dilated bronchi, chronic inflammation, chronic infection and acute exacerbations. Recurrent exacerbations are associated with poorer clinical outcomes such as increased severity of lung disease, further exacerbations, hospitalisations, reduced quality of life and increased risk of death. Despite an increasing prevalence of bronchiectasis, there is a critical lack of high-quality studies into the disease and no treatments specifically approved for its treatment. This trial aims to establish whether inhaled dual bronchodilators (long acting beta agonist (LABA) and long acting muscarinic antagonist (LAMA)) taken as either a stand-alone therapy or in combination with inhaled corticosteroid (ICS) reduce the number of exacerbations of bronchiectasis requiring treatment with antibiotics during a 12 month treatment period. METHODS: This is a multicentre, pragmatic, double-blind, randomised controlled trial, incorporating an internal pilot and embedded economic evaluation. 600 adult patients (≥18 years) with CT confirmed bronchiectasis will be recruited and randomised to either inhaled dual therapy (LABA+LAMA), triple therapy (LABA+LAMA+ICS) or matched placebo, in a 2:2:1 ratio (respectively). The primary outcome is the number of protocol defined exacerbations requiring treatment with antibiotics during the 12 month treatment period. ETHICS AND DISSEMINATION: Favourable ethical opinion was received from the North East-Newcastle and North Tyneside 2 Research Ethics Committee (reference: 21/NE/0020). Results will be disseminated in peer-reviewed publications, at national and international conferences, in the NIHR Health Technology Assessments journal and to participants and the public (using lay language). TRIAL REGISTRATION NUMBER: ISRCTN15988757.

Left cardiac isomerism in the Sonic hedgehog null mouse.

Sonic hedgehog (Shh) is a secreted morphogen necessary for the production of sidedness in the developing embryo. In this study, we describe the morphology of the atrial chambers and atrioventricular junctions of the Shh null mouse heart. We demonstrate that the essential phenotypic feature is isomerism of the left atrial appendages, in combination with an atrioventricular septal defect and a common atrioventricular junction. These malformations are known to be frequent in humans with left isomerism. To confirm the presence of left isomerism, we show that Pitx2c, a recognized determinant of morphological leftness, is expressed in the Shh null mutants on both the right and left sides of the inflow region, and on both sides of the solitary arterial trunk exiting from the heart. It has been established that derivatives of the second heart field expressing Isl1 are asymmetrically distributed in the developing normal heart. We now show that this population is reduced in the hearts from the Shh null mutants, likely contributing to the defects. To distinguish the consequences of reduced contributions from the second heart field from those of left-right patterning disturbance, we disrupted the movement of second heart field cells into the heart by expressing dominant-negative Rho kinase in the population of cells expressing Isl1. This resulted in absence of the vestibular spine, and presence of atrioventricular septal defects closely resembling those seen in the hearts from the Shh null mutants. The primary atrial septum, however, was well formed, and there was no evidence of isomerism of the atrial appendages, suggesting that these features do not relate to disruption of the contributions made by the second heart field. We demonstrate, therefore, that the Shh null mouse is a model of isomerism of the left atrial appendages, and show that the recognized associated malformations found at the venous pole of the heart in the setting of left isomerism are likely to arise from the loss of the effects of Shh in the establishment of laterality, combined with a reduced contribution made by cells derived from the second heart field.

Dual role for neural crest cells during outflow tract septation in the neural crest-deficient mutant Splotch(2H).

Splotch(2H) (Sp(2H)) is a well-recognized mouse model of neural crest cell (NCC) deficiency that develops a spectrum of cardiac outflow tract malformations including common arterial trunk, double outlet right ventricle, ventricular septal defects and pharyngeal arch artery patterning defects, as well as defects in other neural-crest derived organ systems. These defects have been ascribed to reduced NCC in the pharyngeal and outflow regions. Here we provide a detailed map of NCC within the pharyngeal arches and outflow tract of Sp(2H)/Sp(2H) embryos and fetuses, relating this to the development of the abnormal anatomy of these structures. In the majority of Sp(2H)/Sp(2H) embryos we show that deficiency of NCC in the pharyngeal region results in a failure to stabilize, and early loss of, posterior pharyngeal arch arteries. Furthermore, marked reduction in the NCC-derived mesenchyme in the dorsal wall of the aortic sac disrupts fusion with the distal outflow tract cushions, preventing the initiation of outflow tract septation and resulting in common arterial trunk. In around 25% of Sp(2H)/Sp(2H) embryos, posterior arch arteries are stabilized and fusion occurs between the dorsal wall of the aortic sac and the outflow cushions, initiating outflow tract septation; these embryos develop double outlet right ventricle. Thus, NCC are required in the pharyngeal region both for stabilization of posterior arch arteries and initiation of outflow tract septation. Loss of NCC also disrupts the distribution of second heart field cells in the pharyngeal and outflow regions. These secondary effects of NCC deficiency likely contribute to the overall outflow phenotype, suggesting that disrupted interactions between these two cell types may underlie many common outflow defects.

A late role for bmp2b in the morphogenesis of semicircular canal ducts in the zebrafish inner ear.

BACKGROUND: The Bone Morphogenetic Protein (BMP) genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear. METHODOLOGY/PRINCIPAL FINDINGS: We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b(-/-)) embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.

Autonomic innervation of the developing heart: origins and function.

Maintenance of homeostatic circulation in mammals and birds is reliant upon autonomic innervation of the heart. Neural branches of mixed cellular origin and function innervate the heart at the arterial and venous poles as it matures, eventually coupling autonomic output to the cardiac components, including the conduction system. The development of neural identity is controlled by specific networks of genes and growth factors, whereas functional properties are governed by the use of different neurotransmitters. In this review, we summarize briefly the anatomic arrangement of the vertebrate autonomic nervous system and describe, in detail, the innervation of the heart. We discuss the timing of cardiac innervation in the chick and mouse, emphasizing the relationship of the cardiac neural networks to the anatomical structures within the heart. We also discuss the variable contribution of the neural crest to vagal cardiac nerves, and summarize the main neurotransmitters secreted by the developing sympathetic and parasympathetic autonomic divisions. We provide an overview of the main growth factor and gene families involved in neural development, discussing how these factors may impact upon the development of cardiac abnormalities in congenital syndromes associated with autonomic dysfunction.

Non-cell-autonomous roles for the planar cell polarity gene Vangl2 in development of the coronary circulation.

Establishment of cellular polarity is essential for the development of many tissues. In this study, we describe defects in the formation of the coronary vasculature in the loop-tail (Lp) mutant in which the planar cell polarity (PCP) gene, Vangl2, is disrupted. Although Vangl2 is expressed exclusively in the myocardial cells of the developing heart, the coronary vessels do not develop an intact smooth muscle layer, and there are enlarged, ectopic vessels on the surface of the heart. Reduced fibronectin deposition in the subepicardial space is associated with limited migration of epicardially derived cells (EPDCs) into the ventricular myocardium and likely contributes to these defects. Analysis of cardiomyocytes shows that the actin cytoskeleton is disrupted and the cytoarchitecture of the ventricular myocardium is abnormal in Lp/Lp hearts. Moreover, activation of RhoA/Rho kinase signaling is disrupted in these cells. Conditional inhibition of myocardial Rho kinase activity disrupts the organization of the cardiomyocytes and formation of the coronary vessels to produce the same spectrum of defects as seen in Lp. These data suggest that Vangl2 and Rho kinase act cell autonomously in the myocardium to regulate the organization of cardiomyocytes but also have non-cell-autonomous effects on the formation of the coronary vasculature.

Cells migrating from the neural crest contribute to the innervation of the venous pole of the heart.

Cells migrating from the neural crest are known to septate the outflow tract of the developing heart, and to contribute to the formation of the arterial valves, their supporting sinuses, the coronary arteries and cardiac neural ganglia. Neural crest cells have also been suggested to contribute to development of the venous pole of the heart, but the extent and fate of such cells remains unclear. In this study, in the mouse, it is shown that cells from the neural crest contribute to the parasympathetic and, to a lesser extent, the sympathetic innervation of the venous pole of the heart. Nerves within the venous pole of the heart are shown to be of mixed origin, with some being derived from the neural crest, while others have an alternative origin, presumably placodal. The neurons innervating the nodal tissue, which can exert chronotropic effects on cardiac conduction, are shown not to be derived from the neural crest. In particular, no evidence was found to support previous suggestions that cells from the neural crest make a direct contribution to the myocardial atrioventricular conduction axis, although a small subset of these cells do co-localize with the developing left bundle branch. We have therefore confirmed that cells from the neural crest migrate to the venous pole of the heart, and that their major role is in the development of the parasympathetic innervation. In addition, in some embryos, a population of cells derived from the neural crest persist in the leaflets of the atrioventricular valves, but their role in subsequent development remains unknown.

Disruption of planar cell polarity signaling results in congenital heart defects and cardiomyopathy attributable to early cardiomyocyte disorganization.

The Drosophila scribble gene regulates apical-basal polarity and is implicated in control of cellular architecture and cell growth control. Mutations in mammalian Scrib (circletail; Crc mutant) also result in abnormalities suggestive of roles in planar cell polarity regulation. We show that Crc mutants develop heart malformations and cardiomyopathy attributable to abnormalities in cardiomyocyte organization within the early heart tube. N-Cadherin is lost from the cardiomyocyte cell membrane and cell-cell adhesion is disrupted. This results in abnormalities in heart looping and formation of both the trabeculae and compact myocardium, which ultimately results in cardiac misalignment defects and ventricular noncompaction. Thus, these late abnormalities arise from defects occurring at the earliest stages of heart development. Mislocalization of Vangl2 in Crc/Crc cardiomyocytes suggests Scrib is acting in the planar cell polarity pathway in this tissue. Moreover, double heterozygosity for mutations in both Scrib and Vangl2 can cause cardiac defects similar to those found in homozygous mutants for each gene but without other major defects. We propose that heterozygosity for mutations in different genes in the planar cell polarity pathway may be an important mechanism for congenital heart defects and cardiomyopathy in humans.

An aneuploid mouse strain carrying human chromosome 21 with Down syndrome phenotypes.

Aneuploidies are common chromosomal defects that result in growth and developmental deficits and high levels of lethality in humans. To gain insight into the biology of aneuploidies, we manipulated mouse embryonic stem cells and generated a trans-species aneuploid mouse line that stably transmits a freely segregating, almost complete human chromosome 21 (Hsa21). This "transchromosomic" mouse line, Tc1, is a model of trisomy 21, which manifests as Down syndrome (DS) in humans, and has phenotypic alterations in behavior, synaptic plasticity, cerebellar neuronal number, heart development, and mandible size that relate to human DS. Transchromosomic mouse lines such as Tc1 may represent useful genetic tools for dissecting other human aneuploidies.