Typhoid vaccines based on protein-conjugated capsular Vi polysaccharide (TCVs) prevent typhoid in infants and young children. Analysis of the serum anti-Vi IgG response following immunisation against typhoid confirms the immunogenicity of TCVs and forms an important part of the pathway to licensing. Comparative studies could expedite the licencing process, and the availability of a standardised ELISA method alongside the 1st International Standard (IS) 16/138 for anti-typhoid capsular Vi polysaccharide IgG (human) will facilitate this process. To this end, a non-commercial ELISA based on a coat of Vi and poly-l-lysine (Vi-PLL ELISA) was evaluated by 10 laboratories. Eight serum samples, including IS 16/138, were tested in the standardised Vi-PLL ELISA (n = 10), a commercial Vi ELISA (n = 3) and a biotinylated Vi ELISA (n = 1). Valid estimates of potencies relative to IS 16/138 were obtained for all samples in the Vi-PLL ELISA and the commercial ELISA, with good repeatability and reproducibility evident from the study results and concordant estimates obtained by the two ELISA methods. The study demonstrates that the Vi-PLL ELISA can be used in clinical trial studies to determine the immunogenicity of TCVs.
Intestinal dysbiosis involves a shift in microbial composition and abundance within the gut and compelling evidence has highlighted the pivotal role dysbiosis plays in the onset and pathogenesis of numerous diseases, including inflammatory bowel disease (IBD), allergies and even mental health disorders. The intestinal microbiota is largely defined by host diet; a recent mouse model of total parenteral nutrition (TPN) showed that a dysbiotic shift in microbial dominance occurs following enteral nutrient depravation. Furthermore, metabolomics studies have identified that IBD patients can be discriminated from healthy based on their urinary metabolite profiles, but whether such profiles are accountable to intestinal dysbiosis remains uncertain. The research presented herein employed two human models; a novel TPN model in loop ileostomy patients, and patients with IBD, to assess microbial shifts and associated physiological consequences as well as determine whether urinary metabolite profiles are reflective of the intestinal microbiota. Using 16S rDNA-qPCR and -DGGE methods we revealed extensive variations in the microbiota of functional and defunctioned ileum following enteral nutrient deprivation, with a significant relative decrease in the Firmicutes phylum and concomitant increases in γ-Proteobacteria. Immunohistochemical techniques exposed a distinct physiological environment associated with a dysbiotic microbiota. Such environment was defined by reduced epithelial cell proliferation and mucosal atrophy that is likely due to altered host-microbiota interactions at the epithelial surface. We also observed post-operative complications that were potentially dysbiosis-mediated in almost 50% of the study cohort. Urinary NMR and Illumina 16S next-generation sequencing multi-omics statistical analyses identified correlations between dietary-associated urinary metabolites, particularly epicatechin, and distinct enterotype-like microbiota profiles. Application of this principle to prediction of IBD, as an example of a dysbiosis-associated disease, proved to be difficult due to limited sample numbers confounding interpatient variability. This research furthers the utilisation of intestinal microbiota as a therapeutic target, possibly via novel prebiotic administration to the defunctioned ileum with the potential to restore microbiota function prior to reanastomosis and reduce post-operative complications. Furthermore, it also provides promise for inexpensive, non-invasive detection of dysbiosis as a risk-factor of associated diseases. Further research, employing greater numbers of participants, is required to fully evaluate the potential predictive value of dysbiosis.
Abstract The human gut microbiome has been shown to be associated with a variety of human diseases, including cancer, metabolic conditions and inflammatory bowel disease. Current approaches for detecting microbiome associations are limited by relying on specific measures of ecological distance, or only allowing for the detection of associations with individual bacterial species, rather than the whole microbiome. In this work, we develop a novel hierarchical Bayesian model for detecting global microbiome associations. Our method is not dependent on a choice of distance measure, and is able to incorporate phylogenetic information about microbial species. We perform extensive simulation studies and show that our method allows for consistent estimation of global microbiome effects. Additionally, we investigate the performance of the model on two real-world microbiome studies: a study of microbiome-metabolome associations in inflammatory bowel disease, and a study of associations between diet and the gut microbiome in mice. We show that we can use the method to reliably detect associations in real-world datasets with varying numbers of samples and covariates.
Patients who develop colorectal cancer or chronic colitis often require surgical gut resection. To facilitate healing a temporary redirection of luminal contents, via stoma formation upstream of the surgical site, is often used. Reanastamosis of the stoma at a later date reinstates luminal flow through the entire intestine. Stoma reversal is associated with post-operative complications and therefore a proportion of patients (~20%) experience a reduced quality of life and ~5% of cases prove to be irreversible. Our research aims to investigate the pathological consequences of stoma formation. Functional and defunctioned limbs were compared using histological, flow cytometric, 16s rDNA PCR-DGGE and qPCR techniques, to assess morphology, immune and microbial populations respectively. Histological examination revealed villous atrophy in the defunctioned limb but no significant difference in overall inflammation score, indicating that severe immunopathology was not contributing to intestinal changes. However, a shift the in the distribution of inflammatory macrophages in the mucosa of defunctioned vs. functional bowel suggests immunological dysregulation. DGGE and qPCR analyses of major bacterial Phyla revealed variations in microbial profiles between the functional and defunctioned intestinal limbs, suggesting that stoma-associated nutrient deprivation results in intestinal dysbiosis. This data may provide a rationale for the use of prebiotic preparations to sustain intestinal microbes, to maintain mucosal homeostasis and improve the success of stoma reversal surgery. Bowel Cancer Research
This study aimed to identify the challenges facing teachers in their professional development across the primary, post-primary and tertiary educational sectors. Through the literature six main areas of investigation were identified: motivation, 21st century competencies, peer coaching, knowledge-sharing, professional learning communities and measurement/ assessment.
The data was collected through 12 semi-structured interviews with teachers across the three sectors. A thematic analysis of the data was conducted. A number of themes were extracted, for example: environment, systems, culture and time. The findings of the study are discussed through interpretation of teacher’s experiences.
The results identified challenges around individualistic work cultures, lack of purposeful systems of measurement, trust and fear issues, all of which were underpinned by time constraints. It was observed that there were more organisational structures at tertiary level. There was more commonality between primary and post-primary sectors. Despite the differences all three sectors have common challenges, such as effective knowledge-sharing and timely measurement to inform effective 21st century professional development.
In this work, we explore the effects of O-acetylation on the physical and immunological characteristics of the WHO International Standards of Vi polysaccharide (Vi) from both Citrobacter freundii and Salmonella enterica serovar Typhi. We find that, although structurally identical according to NMR, the two Vi standards have differences with respect to susceptibility to de-O-acetylation and viscosity in water. Vi standards from both species have equivalent mass and O-acetylation-dependent binding to a mouse monoclonal antibody and to anti-Vi polyclonal antisera, including the WHO International Standard for human anti-typhoid capsular Vi PS IgG. This study also confirms that human anti-Vi sera binds to completely de-O-acetylated Vi. Molecular dynamics simulations provide conformational rationales for the known effect of de-O-acetylation both on the viscosity and antigenicity of the Vi, demonstrating that de-O-acetylation has a very marked effect on the conformation and dynamic behavior of the Vi, changing the capsular polysaccharide from a rigid helix into a more flexible coil, as well as enhancing the strong interaction of the polysaccharide with sodium ions. Partial de-O-acetylation of Vi revealed hidden epitopes that were recognized by human and sheep anti-Vi PS immune sera. These findings have significance for the manufacture and evaluation of Vi vaccines.
Abstract Dupuytren’s disease is a common fibroproliferative disease of the palmar fascia of the hand with advanced cases treated surgically. Anti-tumour necrosis factor (TNF) injection has undergone phase 2 trials and may be effective in slowing early-stage disease progression. Here we sought to determine how new synthesis of type I collagen in Dupuytren’s differs from normal palmar fascia samples and to analyse the role of TNF in aberrant collagen synthesis. Model non-fibrotic, but fibrous connective tissues, were used to analyse active type I collagen protein synthesis in development, ageing and degenerative disease, where it was restricted to early development and ruptured tissue. Dupuytren’s tissue was shown to actively synthesise type I collagen, including abnormal type I collagen homotrimer. TNF-α reduced COL1A2 gene expression only in the presence of serum in 2D cell culture and had opposing effects on collagen protein production in the presence or absence of serum. TNF-α had only limited effects in 3D tendon-like constructs. Anti-TNF did not reduce type I collagen synthesis in 3D tendon-like constructs or prevent type I collagen homotrimer synthesis in Dupuytren’s tissue. Hence, modulation of the TNF-α pathway in Dupuytren’s disease is unlikely to prevent the pathological collagen accumulation that is characteristic of fibrosis.
Loop ileostomy is a common surgical procedure to allow downstream tissue healing, with the aim of re-joining the bowel approximately 12 months later. The reversal procedure is associated with a substantial morbidity up to 40%. Our previous research demonstrated that defunctioned ileum becomes atrophied, with extensive microbial dysbiosis. This study sought to investigate the potential influence of delaying ileostomy reversal surgery upon both clinical and pathological outcomes. Post-operative clinical data was recorded, including routine blood test results, duration of hospital stay, length of time with stoma and incidence of post-operative complications. We measured ileal fibrosis and atrophy and assessed whether these, or dysbiosis, were impacted by the length of time a stoma was in place, or were linked to clinical outcomes. Associations between clinical data were investigated using scatterplot matrix analysis and t-tests. We found no differences in time between ileostomy formation and reversal in patients experiencing complications vs. individuals with no complications. Furthermore, there were no correlations between days with stoma and pathological measures, such as atrophy or fibrosis, and no ongoing increases in collagen production at the time of reversal surgery. This data suggests that the length of time a stoma is in place does not impact on the likelihood of complications. The incidence of complications is associated with increased loss of microbiota in the defunctioned ileum, but importantly, the decrease in bacteria is not linked to time with stoma. Microbiota diversity in the functional and defunctioned limb correlated within an individual, and was not significantly different between those who experienced complications following surgery vs. those that didn't. Microbiota diversity was also not significantly impacted through delay (>365 days) in stoma reversal. We propose that methods to restore intestinal microbiota numbers, and not necessarily their composition, prior to reversal should be explored to improve the clinical outcomes of ileostomy reversal surgery.
Abstract Motivation The human gut microbiome has been shown to be associated with a variety of human diseases, including cancer, metabolic conditions and inflammatory bowel disease. Current statistical techniques for microbiome association studies are limited by relying on measures of ecological distance, or only allowing for the detection of associations with individual bacterial species, rather than the whole microbiome. Results In this work, we develop a novel Bayesian multi-task approach for detecting global microbiome associations. Our method is not dependent on a choice of distance measure, and is able to incorporate phylogenetic information about microbial species. We apply our method to simulated data and show that it allows for consistent estimation of global microbiome effects. Additionally, we investigate the performance of the model on two real-world microbiome studies: a study of microbiome-metabolome associations in inflammatory bowel disease (Beamish, 2017), and a study of associations between diet and the gut microbiome in mice (Turnbaugh et al ., 2009). We show that we can use the method to reliably detect associations in real-world datasets with varying numbers of samples and covariates. Availability Our method is implemented using the R interface to the Stan Hamiltonian Monte Carlo sampler. Software for running our methods is available at https://github.com/FrankD/MicrobiomeGlobalAssociations . Contact f.dondelinger@lancaster.ac.uk
Loop ileostomy is an effective procedure to protect downstream intestinal anastomoses. Ileostomy reversal surgery is often performed within 12 months of formation but is associated with substantial morbidity due to severe post-surgical complications. Distal ileum is deprived of enteral nutrition and rendered inactive, often becoming atrophied and fibrotic. This study aimed to investigate the microbial and morphological changes that occur in the defunctioned ileum following loop ileostomy-mediated fecal stream diversion. Functional and defunctioned ileal resection tissue was obtained at the time of loop-ileostomy closure. Intrapatient comparisons, including histological assessment of morphology and epithelial cell proliferation, were performed on paired samples using the functional limb as control. Mucosal-associated microflora was quantified via determination of 16S rRNA gene copy number using qPCR analysis. DGGE with Sanger sequencing and qPCR methods profiled microflora to genus and phylum level, respectively. Reduced villous height and proliferation confirmed atrophy of the defunctioned ileum. DGGE analysis revealed that the microflora within defunctioned ileum is less diverse and convergence between defunctioned microbiota profiles was observed. Candidate Genera, notably Clostridia and Streptococcus, reduced in relative terms in defunctioned ileum. We conclude that Ileostomy-associated nutrient deprivation results in dysbiosis and impaired intestinal renewal in the defunctioned ileum. Altered host-microbial interactions at the mucosal surface likely contribute to the deterioration in homeostasis and thus may underpin numerous postoperative complications. Strategies to sustain the microflora before reanastomosis should be investigated.