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The murine bone marrow has a central role in immune function and health as the primary source of leukocytes in adult mice. Laboratory mice provide a human-homologous, genetically manipulable and reproducible model that has enabled an immeasurable volume of high-quality immunological research. However, recent research has questioned the translatability of laboratory mouse research into humans and proposed that the exposure of mice to their wild and natural environment may hold the key to further immunological breakthroughs. To date, there have been no studies providing an in-depth cellular analysis of the wild mouse bone marrow. This study utilized wild mice from an isolated island population (Isle of May, Scotland, UK) and performed flow cytometric and histological analysis to characterize the myeloid, lymphoid, hematopoietic progenitor, and adipocyte compartments within the wild mouse bone marrow. We find that, compared to laboratory mouse bone marrow, the wild mouse bone marrow differs in every cell type assessed. Some of the major distinctions include; a smaller B cell compartment with an enriched presence of plasma cells, increased proportions of KLRG1+ CD8+ T cells, diminished CD11b expression in the myeloid lineage and a five-fold enlargement of the eosinophil compartment. We conclude that the wild mouse bone marrow is dramatically distinct from its laboratory counterparts, with multiple phenotypes that to our knowledge have never been observed in laboratory models. Further research into these unique features may uncover novel immunological mechanisms and grant a greater understanding of the role of the immune system in a natural setting.

The gut microbiota is known to play an important role in maintaining gut health through a symbiotic relationship with the host. Altered gut microbiota is a common feature of several diseases of the gastrointestinal tract; however, the causal relationship between microbiota and disease pathogenesis is poorly understood. Necrotising enterocolitis (NEC) and inflammatory bowel disease (IBD) are both severe inflammatory diseases affecting the gastrointestinal tract. Although they affect very different patient populations, with NEC primarily being a disease of prematurity and IBD predominantly affecting adults although children can be affected, they both demonstrate common features of gut microbial dysbiosis and a dysregulated host immune response. By comparing and contrasting the changes in gut microbiota, host immune response and function, we aim to highlight common features in diseases that may seem clinically unrelated. Key areas of interest are the role of pattern recognition receptors in altered recognition and responses to the gut microbiota by the host immune system and the associated dysfunctional gut epithelial barrier. The challenge of identifying causal relationships between microbiota and disease is ever-present; however, considering a disease-agnostic approach may help to identify mechanistic pathways shared across several clinical diseases.

Digital transformation of the healthcare workforce is a priority if we are to leverage the potential of digital technologies, artificial intelligence in clinical decision support and the potential of data captured within electronic health records. Educational programmes need to be diverse and support the digital novices through to the champions whom will be responsible for procuring and implementing digital solutions. In order to professionalise the workforce in this area, digital competencies need to be built into training from early on and be underpinned by frameworks that help to guide regulators and professional bodies and support educational providers to deliver them. Here we describe Manchester's involvement in the development of digital competency frameworks and our digital transformation education programmes that we have created, including a Massive Online Open Course and a professional development course for England's Topol Digital Fellows.

Digital transformation of the healthcare workforce is a priority if we are to leverage the potential of digital technologies, artificial intelligence in clinical decision support and the potential of data captured within electronic health records. Educational programmes need to be diverse and support the digital novices through to the champions whom will be responsible for procuring and implementing digital solutions. In order to professionalise the workforce in this area, digital competencies need to be built into training from early on and be underpinned by frameworks that help to guide regulators and professional bodies and support educational providers to deliver them. Here we describe Manchester’s involvement in the development of digital competency frameworks and our digital transformation education programmes that we have created, including a Massive Online Open Course and a professional development course for England’s Topol Digital Fellows.

With large-scale population sequencing projects gathering pace, there is a need for strategies that advance disease gene prioritization1,2. Metrics that provide information about a gene and its ability to tolerate protein-altering variation can aid in clinical interpretation of human genomes and can advance disease gene discovery1,2,3,4. Previous reported methods analyzed the total variant load in a gene1,2,3,4, but did not analyze the distribution pattern of variants within a gene. Using data from 138,632 exome and genome sequences2, we developed gene variation intolerance rank (GeVIR), a continuous gene-level metric for 19,361 genes that is able to prioritize both dominant and recessive Mendelian disease genes5, that outperforms missense constraint metrics3 and that is comparable—but complementary—to loss-of-function (LOF) constraint metrics2. GeVIR is also able to prioritize short genes, for which LOF constraint cannot be estimated with confidence2. The majority of the most intolerant genes identified here have no defined phenotype and are candidates for severe dominant disorders.

Background Identifying the factors that contribute to chronicity in inflamed colitic tissue is not trivial. However, in mouse models of colitis, we can investigate at preclinical timepoints. We sought to validate murine Trichuris muris infection as a model for identification of factors that promote development of chronic colitis. Methods We compared preclinical changes in mice with a resolving immune response to T. muris (resistant) vs mice that fail to expel the worms and develop chronic colitis (susceptible). Findings were then validated in healthy controls and patients with suspected or confirmed IBD. Results The receptor for advanced glycation end products (RAGE) was highly dysregulated between resistant and susceptible mice before the onset of any pathological signs. Increased soluble RAGE (sRAGE) in the serum and feces of resistant mice correlated with reduced colitis scores. Mouse model findings were validated in a preliminary clinical study: fecal sRAGE was differentially expressed in patients with active IBD compared with IBD in remission, patients with IBD excluded, or healthy controls. Conclusions Preclinical changes in mouse models can identify early pathways in the development of chronic inflammation that human studies cannot. We identified the decoy receptor sRAGE as a potential mechanism for protection against chronic inflammation in colitis in mice and humans. We propose that the RAGE pathway is clinically relevant in the onset of chronic colitis and that further study of sRAGE in IBD may provide a novel diagnostic and therapeutic target.

Findings from gut microbiome studies are strongly influenced by both experimental and analytical factors that can unintentionally bias their interpretation. Environment is also critical. Both co-housing and maternal effects are expected to affect microbiomes and have the potential to confound other manipulated factors, such as genetics. We therefore analysed microbiome data from a mouse experiment using littermate controls and tested differences among genotypes (wildtype versus colitis prone-mdr1a−/−), gut niches (stool versus mucus), host ages (6 versus 18 weeks), social groups (co-housed siblings of different genotypes) and maternal influence. We constructed a 16S phylogenetic tree from bacterial communities, fitting random forest models using all 428,234 clades identified. Models discriminated all criteria except host genotype, where no community differences were found. Host social groups differed in abundant, low-level, taxa whereas intermediate phylogenetic and abundance scales distinguished ages and niches. Thus, a carefully controlled experiment treating evolutionary clades of microbes equivalently without reference to taxonomy, clearly identifies whether and how gut microbial communities are distinct across ecologically important factors (niche and host age) and other experimental factors, notably cage effects and maternal influence. These findings highlight the importance of considering such environmental factors in future microbiome studies.

Background Dendritic cells (DCs) play a key role in shaping T cell responses. To do this, DCs must be able to migrate to the site of the infection and the lymph nodes to prime T cells and initiate the appropriate immune response. Integrins such as β2 integrin play a key role in leukocyte adhesion, migration, and cell activation. However, the role of β2 integrin in DC migration and function in the context of infection-induced inflammation in the gut is not well understood. This study looked at the role of β2 integrin in DC migration and function during infection with the nematode worm Trichuris muris. Itgb2tm1Bay mice lacking functional β2 integrin and WT littermate controls were infected with T. muris and the response to infection and kinetics of the DC response was assessed. Results In infection, the lack of functional β2 integrin significantly reduced DC migration to the site of infection but not the lymph nodes. The lack of functional β2 integrin did not negatively impact T cell activation in response to T. muris infection. Conclusions This data suggests that β2 integrins are important in DC recruitment to the infection site potentially impacting the initiation of innate immunity but is dispensible for DC migration to lymph nodes and T cell priming in the context of T. muris infection.

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture. This article has an associated First Person interview with the first author of the paper., Summary: A novel inhibitory mechanism of glucocorticoid (GC) action on cell migration is dependent on the α-tubulin deacetylase HDAC6, which has implications for the design of novel GCs with improved therapeutic benefit.

Trichuris trichiura is a parasite that infects 500 million people worldwide, leading to colitis, growth retardation and Trichuris dysentery syndrome. There are no licensed vaccines available to prevent Trichuris infection and current treatments are of limited efficacy. Trichuris infections are linked to poverty, reducing children’s educational performance and the economic productivity of adults. We employed a systematic, multi-stage process to identify a candidate vaccine against trichuriasis based on the incorporation of selected T-cell epitopes into virus-like particles. We conducted a systematic review to identify the most appropriate in silico prediction tools to predict histocompatibility complex class II (MHC-II) molecule T-cell epitopes. These tools were used to identify candidate MHC-II epitopes from predicted ORFs in the Trichuris genome, selected using inclusion and exclusion criteria. Selected epitopes were incorporated into Hepatitis B core antigen virus-like particles (VLPs). Bone marrow-derived dendritic cells and bone marrow-derived macrophages responded in vitro to VLPs irrespective of whether the VLP also included T-cell epitopes. The VLPs were internalized and co-localized in the antigen presenting cell lysosomes. Upon challenge infection, mice vaccinated with the VLPs+T-cell epitopes showed a significantly reduced worm burden, and mounted Trichuris-specific IgM and IgG2c antibody responses. The protection of mice by VLPs+T-cell epitopes was characterised by the production of mesenteric lymph node (MLN)-derived Th2 cytokines and goblet cell hyperplasia. Collectively our data establishes that a combination of in silico genome-based CD4+ T-cell epitope prediction, combined with VLP delivery, offers a promising pipeline for the development of an effective, safe and affordable helminth vaccine., Author summary The soil transmitted helminth Trichuris trichiura is a major parasite in developing countries; development of a comprehensive vaccine has been elusive. Here we used a systematic approach based on in silico identification of MHC-II T-cell epitopes from genome sequences, their incorporation into a virus-like particle (VLP), characterization of the assemblies and testing in an in vivo murine infection model. Animals vaccinated with a preparation of four different VLP-antigen fusions showed significant reductions in intestinal worm burdens and associated antibody responses consistent with protection. The results suggest that a pipeline based on in silico prediction of potent MHC-II T-cell epitopes, followed by incorporation into VLPs, could be a strategy which enables rapid translation into a vaccine against Trichuris trichiura.

Managers in complex organisations often have to make decisions on whether new software developments are worth undertaking or not. Such decisions are hard to make, especially at an enterprise level. Both costs and risks are regularly underestimated, despite the existence of a plethora of software and systems engineering methodologies aimed at predicting and controlling them. Our objective is to help managers and stakeholders of large, complex organisations (like the National Health Service in the UK) make better informed decisions on the costs and risks of planned new software systems that will reuse or extend their existing information infrastructure.We analysed case studies describing new software developments undertaken by providers of health care services in the UK, looking for common points of risk and high cost. The results highlighted the movement of data within and between organisations as a key factor. Data movement can be hindered by numerous technical barriers, but also by other challenges arising from social aspects of the organisation. These latter aspects are often harder to predict, and are ignored by many of the more common software engineering methodologies.In this paper, we propose data journey modelling, a new method aiming to predict places of high cost and risk when existing data needs to move to a new development. The method is lightweight and combines technical and social aspects, but relies only on information that is likely to be already known to key stakeholders, or will be cheap to acquire.To assess the effectiveness of our method, we conducted a retrospective evaluation in an NHS Foundation Trust hospital. Using the method, we were able to predict most of the points of high cost/risk that the hospital staff had identified, along with several other possible directions that the staff did not identify for themselves, but agreed could be promising.

Gut microbiome analysis using 16S rRNA frequently focuses on summary statistics (e.g. diversity) or single taxonomic scales (e.g. Operational Taxonomic units, OTUs). This approach risks misinterpreting the phylogenetic or abundance scales of community differences (e.g. over-emphasising the role of single strains). We therefore constructed a 16S phylogenetic tree from mouse stool and colonic mucus communities. Random forest models, of all 428,234 clades, tested community differences among niches (stool versus mucus), host ages (6 versus 18 weeks), genotypes (wildtype versus colitis prone-mdr1a-/-) and social groups (co-housed siblings). Models discriminated all criteria except host genotype, where no community differences were found. Host social groups differed in abundant, low-level, taxa whereas intermediate phylogenetic and abundance scales distinguished ages and niches. Thus, treating evolutionary clades of microbes equivalently without reference to OTUs or taxonomy, clearly identifies whether and how gut microbial communities are distinct and provides a novel way to define functionally important bacteria.

AimsIdentifying the factors that contribute to chronicity in inflamed colitic tissue is not trivial. However, in mouse models of colitis, we can investigate at preclinical timepoints. We sought to validate murine Trichuris muris infection as a model for identification of factors that promote development of chronic colitis.MethodsWe compared preclinical changes in mice with a resolving immune response to T. muris (resistant) versus mice that fail to expel the worms and develop chronic colitis (susceptible). Findings were then validated in healthy controls and patients with suspected or confirmed IBD.ResultsThe Receptor for Advanced Glycation End Products (Rage) was highly dysregulated between resistant and susceptible mice prior to the onset of any pathological signs. Increased soluble RAGE (sRAGE) in the serum and faeces of resistant mice correlated with reduced colitis scores. Mouse model findings were validated in a preliminary clinical study: faecal sRAGE was differentially expressed in patients with active IBD compared with IBD in remission, patients with IBD excluded or healthy controls.ConclusionPre-clinical changes in mouse models can identify early pathways in the development of chronic inflammation that human studies cannot. We identified the decoy receptor sRAGE as a potential mechanism for protection against chronic inflammation in colitis in mice and humans. We propose that the RAGE pathway is clinically relevant in the onset of chronic colitis, and that further study of sRAGE in IBD may provide a novel diagnostic and therapeutic target.

Summary The gut has the largest commensal bacterial population in the body and its composition can be impacted by host factors such as production of immunoglobulin A (IgA). Eosinophils in the gut have been implicated in the production of antibacterial factors and maintenance of IgA‐secreting plasma cells. We used an eosinophil‐deficient mouse (∆dblGATA‐1 −/−) and littermate controls to investigate the role of eosinophils in the regulation of the microbiota, with particular emphasis on mucus‐resident species in the small and large intestine. We found no differences in IgA production or IgA‐expressing plasma cells between naive littermates in the small or large intestine. However, denaturing gel gradient electrophoresis revealed differences in the bacterial communities of the mucus and stools between wild‐type mice and ∆dblGATA‐1 −/− mice, with the greatest separation between the mucus microbial communities. Mucus‐resident bacteria in ∆dblGATA‐1 −/− mice had reduced diversity in the mucus compared with the stools. A quantitative PCR panel of selected bacteria showed that the most significant differences in the microbiota were between mucus‐resident bacteria and those in stool, such as the abundance of Clostridiales and Bacteroides. Our data implicate eosinophils in the regulation of the microbiota, especially the bacteria most hyperlocal to the gut barrier. Although we see differences between host genotypes in the overall microbial communities, further work is required to establish specifically which bacteria are different between these groups. Most importantly, the data revealed that the mucus and stool microbiota are discrete communities. Stool analysis alone may be insufficient to comprehensively explore and define the role of the gut microbiota in health and disease.

Part 1: Regular Papers; International audience; Information sharing within complex organisations is often source of considerable cost and risk. In previous work, we showed that points of highest IT cost/risk within an organisation are often located at the points where data moves from one context of use to another. We proposed a lightweight method of modelling the journeys data make within an organisation, and showed how to identify risky or costly boundaries. In this paper, we build on this previous work by evaluating the stability and completeness of the three core boundaries of our proposed method with staff from different clinical genomics hospital departments in the UK. Assessing our boundaries in the four new studies we found that although our core boundaries are stable in the new area of Clinical Genomics, domain-specific requirements of organisations can drive the need for additional boundaries. Finally, we discuss the feasibility of a general, low-cost process for identifying further boundaries of interest when applying the method to a new domain.

Background: Inflammatory bowel disease (IBD) is associated with an inappropriate immune response to the gut microbiota. Notably, patients with IBD reportedly have alterations in fecal microbiota. However, the colonic microbiota occupies both the gut lumen and the mucus covering the epithelium. Thus, information about mucus-resident microbiota fails to be conveyed in the routine microbiota analyses of stool samples. Further, studies analyzing microbiota in IBD have mainly focused on stool samples taken after onset of inflammation. Our objective was to investigate both temporal and spatial changes in colonic microbiota communities preceding the onset of colitis. Methods: We studied mucus and stool microbiota using a spontaneous model of colitis, the mdr1a-/- mouse, and their respective wild-type littermate controls in a time series mode. Results: Using this approach we have shown that microbial dysbiosis was evident in the mucus but not stools, with reduced abundance of Clostridiales was evident in the mucus but not stools, of colitis-prone mice mdr1a-/- mice 12 weeks before the onset of detectable inflammation. This altered microbial composition was coupled with a significantly thinner mucus layer. Upon emergence of inflammation, dysbiosis was evident in stool and at this time point, the spatial segregation between microbiota and host tissue was also disrupted, correlated with worsened inflammation. Our results reveal that microbial dysbiosis is detectable prior to changes in the stools. Importantly, dysbiosis in the mucus layer preceded development of colitis. Conclusions: Our data reveal the importance of mucus sampling for understanding the underlying etiology of IBD and fundamental process underlying disease progression.

ObjectivesSeasonal allergies, hay fever and asthma affect approximately one in four people and the incidence is increasing. Whilst the causes are unknown, hypotheses propose associations with environmental changes, exposure to pollutants and decreased exposure to childhood infections. High resolution datasets on pollen count and pollution are available; however there is no equivalent for incidence of seasonal allergy symptoms. We planned a national citizen science project using smartphones to gather data from the general population on seasonal allergy symptoms, and where and when they occur. The resulting dataset will be linked with other publicly available data, enabling better understanding of allergy triggers. This is a joint project between the Royal Society of Biology, the British Society for Immunology, and The University of Manchester. ApproachIn spring 2015, two codesign workshops were held for members of the public with seasonal allergies and/or asthma (n=33). Guided by researchers, attendees used paper prototyping techniques to illustrate the functionalities of a mobile application. They also prioritised functions for inclusion within the app, discussed data sharing options and suggested material for the accompanying project website (www.britainbreathing.org). Following codesign workshops, designs and requirements were collated, refined and used to build the first version of the application in Android. ResultsWorkshop feedback indicated that potential users prioritised simple, personal tracking. They also valued the ability to access information about symptom frequencies among other users locally. Support for academic research was high, although most wanted some control over data sharing. People were comfortable with GPS data being collected, provided it did not impinge on privacy. We agreed to make data openly available via an interactive widget on the project website. The resulting first version of the application enables personal symptom tracking and will be released in March 2016 via the Google Play store (free of charge). A national media campaign will drive recruitment, alongside inclusion in the European City of Science 2016 programme in Manchester. Emerging data on the incidence of allergy symptoms by location will be presented. ConclusionCitizen science can be more than simply crowdsourcing data. We demonstrated that paper prototyping was a feasible and useful technique for codesigning an application with members of the public. Furthermore, workshop feedback indicated a high level of support for citizen science, provided users gained simple, personalised feedback. Further research is required to determine how codesign processes influence subsequent participant recruitment and engagement in citizen science projects.

Motivation: As a natural consequence of being a computer-based discipline, bioinformatics has a strong focus on database and software development, but the volume and variety of resources are growing at unprecedented rates. An audit of database and software usage patterns could help provide an overview of developments in bioinformatics and community common practice, and comparing the links between resources through time could demonstrate both the persistence of existing software and the emergence of new tools. Results: We study the connections between bioinformatics resources and construct networks of database and software usage patterns, based on resource co-occurrence, that correspond to snapshots of common practice in the bioinformatics community. We apply our approach to pairings of phylogenetics software reported in the literature and argue that these could provide a stepping stone into the identification of scientific best practice. Availability and implementation: The extracted resource data, the scripts used for network generation and the resulting networks are available at http://bionerds.sourceforge.net/networks/ Contact: robert.stevens@manchester.ac.uk

Mice represent the most commonly used species for preclinical in vivo research. While incisional and excisional acute murine wound models are both frequently employed, there is little agreement on which model is optimum. Moreover, current lack of standardization of wounding procedure, analysis time point(s), method of assessment, and the use of individual wounds vs. individual animals as replicates makes it difficult to compare across studies. Here we have profiled secondary intention healing of incisional and excisional wounds within the same animal, assessing multiple parameters to determine the optimal methodology for future studies. We report that histology provides the least variable assessment of healing. Furthermore, histology alone (not planimetry) is able to detect accelerated healing in a castrated mouse model. Perhaps most importantly, we find virtually no correlation between wounds within the same animal, suggesting that use of wound (not animal) biological replicates is perfectly acceptable. Overall, these findings should guide and refine future studies, increasing the likelihood of detecting novel phenotypes while reducing the numbers of animals required for experimentation.

Part 2: Regular Papers; International audience; Information sharing is a vital element of a successful business. However, technological, organisational and human challenges obstruct the effective movement of data. In this paper, we analyse a collection of case studies from healthcare describing failing information systems developments. A set of 32 failing factors were extracted showing that data movement, either between systems, people or organisations, is a key indicator of IT failure. From this examination, we derived anti-patterns for data movement in which some key differences between the source and target location of the movement caused high costs to the developments. Finally, we propose data journey modelling as a lightweight technique that captures the movement of data through complex networks of people and systems, with the aim of improve go/no-go decision making for new IT developments, based on the anti-patterns we have identified.

Transcriptome analyses were performed in the anterior raphe area of mutant mice deficient in the serotonin transporter (5-HTT KO) or overexpressing this protein (5-HTT TG), which exhibit opposite changes in anxiety-related behavior. Among genes with altered expression, the gene encoding the neuropeptide urocortin 1 was down-regulated in 5-HTT KO and up-regulated in 5-HTT TG mice. Expression of the gene encoding cocaine-and-amphetamine-related-peptide, which colocalizes with urocortin 1, was also increased in 5-HTT TG mutants. Real-time RT-PCR confirmed these data and immunoautoradiographic labeling showed that parallel changes in neuropeptide levels were confined to the non-preganglionic Edinger-Westphal nucleus. Thus, 5-HTT expression correlates with that of urocortin 1, suggesting that this peptide can be involved in the behavioral changes observed in 5-HTT mutant mice.