Bacterial inhabitants of tumours: methods for exploration and exploitation

The presence of bacteria in patient tumours of various types has been reported by numerous groups since 2014, but the findings of these and many similar studies remain contentious. Tumour samples provide many obstacles to carrying out robust and reliable microbial surveys, primarily the anticipated low biomass of these samples, which leaves them vulnerable to environmental contamination. While the debate over the presence or absence of bacterial communities in these tumours continues, it impedes any research into how such bacteria might be utilised in medicine. Larger sample numbers are required, from diverse tumour tissues within the human body, and these must be analysed in a reproducible and accurate manner to allow for the drawing of definitive conclusions in this debate. To accommodate this requirement, the primary methodological aspects of this thesis were: i) The assembly and validation of a contamination control pipeline using recent advances is bioinformatic contamination control detection. ii) The development and validation of a bacterial DNA extraction protocol for formalin fixed, paraffin embedded (FFPE) samples, with accompanying FFPE biological standards for use as controls. A key aim of this thesis was to increase the accuracy and reproducibility of research into clinical tissue biopsies by eliminating the role of contamination, and to expand the applicability of FFPE tissues which represent an invaluable resource of samples for analysis. In this thesis, ecological surveys of a variety of related environments were conducted with the common goal of characterising a detectable bacterial community and identify potential bacterial biomarkers unique to these host environments. Regardless of whether or not a consistently present and detectable tumour microbiome exists, tumours possess several phenotypes making them hospitable environments for bacteria to colonise. Where the unique physiology of tumours is seen as an obstacle for traditional cancer treatments, they represent an opportunity for bacterial-mediated solutions. Therefore, findings from sequencing-based research of host environments have potential to be translated into the use of administered bacteria as delivery vehicles to locally produce biomolecules. There are two considerations in this context, requiring two very different applications of bioinformatics. i) The first is to identify which bacteria colonize the desired niche in body; this can be a ‘foreign’ body such as a tumour (Chapter 3 and 4), or parasite (Chapter 5), or a distal niche such as the gut. ii) The second, often under-considered parameter, relates to what these bacteria produce. Synthetic biology presents enormous scope for sophisticated medical therapy mediated by novel synthetic proteins. However, the task of getting a bacterial cell to successfully express and secrete a stable protein that it does not produce naturally is far from trivial, and is becoming a key aspect of the synthetic biology field. To facilitate this synthetic protein aspect, a novel strategy for the performance prediction of designed protein constructs was developed. This tool was able to predict the overall performance of a protein construct in vitro using only in silico derived data. This thesis aimed to develop novel strategies for the analysis of bacterial communities within tumours by i) increasing the sample sizes available to future projects by enabling the use of FFPE samples and ii) improving the accuracy of analysis by designing bioinformatics analysis pipelines appropriate for these samples. This enabled further research concerned with finding differentially present taxa between the tumour and surrounding environment, which have the potential for use as therapeutic vectors. As the key aim is to establish the presence of potential bacterial therapeutic vectors rather than to establish the role these bacteria play in tumorigenesis, this approach is easily translatable to other foreign bodies, and could therefore be validated in a parasitic nematode model

The journal 'chiropractic & osteopathy' changes its title to 'chiropractic & manual therapies'. a new name, a new era

Chiropractic & Osteopathy changes its title to Chiropractic & Manual Therapies in January 2011. This change reflects the expanding base of submissions from clinical scientists interested in the discipline of manual therapy. It is also in accord with the findings of a review of the journal content and a joint venture between the original parent organisation the Chiropractic and Osteopathic College of Australasia and a new partner the European Academy of Chiropractic, which is a subsidiary body of the European Chiropractors' Union. The title change should encourage submissions from all professionals interested in manual therapy including chiropractors, osteopaths, physiotherapists, medical doctors and scientists interested in this field

Microbiome analysis as a platform R&D tool for parasitic nematode disease management

The relationship between bacterial communities and their host is being extensively investigated for the potential to improve the host’s health. Little is known about the interplay between the microbiota of parasites and the health of the infected host. Using nematode co-infection of lambs as a proof-of-concept model, the aim of this study was to characterise the microbiomes of nematodes and that of their host, enabling identification of candidate nematode-specific microbiota member(s) that could be exploited as drug development tools or for targeted therapy. Deep sequencing techniques were used to elucidate the microbiomes of different life stages of two parasitic nematodes of ruminants, Haemonchus contortus and Teladorsagia circumcincta, as well as that of the co-infected ovine hosts, pre- and post infection. Bioinformatic analyses demonstrated significant differences between the composition of the nematode and ovine microbiomes. The two nematode species also differed significantly. The data indicated a shift in the constitution of the larval nematode microbiome after exposure to the ovine microbiome, and in the ovine intestinal microbial community over time as a result of helminth co-infection. Several bacterial species were identified in nematodes that were absent from their surrounding abomasal environment, the most significant of which included Escherichia coli/Shigella. The ability to purposefully infect nematode species with engineered E. coli was demonstrated in vitro, validating the concept of using this bacterium as a nematode-specific drug development tool and/or drug delivery vehicle. To our knowledge, this is the first description of the concept of exploiting a parasite’s microbiome for drug development and treatment purposes

Association of HLA-DRB1-restricted CD4+ T cell responses with HIV immune control

The contribution of HLA class II-restricted CD4+ T cell responses to HIV immune control is poorly defined. Here, we delineated novel peptide-DRB1 restrictions in functional assays and analyzed the host genetic effects of HLA-DRB1 alleles on HIV viremia in a large cohort of HIV controllers and progressors (n=1085). We found distinct stratifications in the effect of HLA-DRB1 alleles on HIV viremia, with DRB1*15:02 significantly associated with low viremia (P=0.003, q=0.04) and DRB1*03:01 significantly associated with high viremia (P=0.004, q=0.04). Interestingly, a sub-group of HLA-DRB1 alleles linked with low viremia showed the ability to promiscuously present a larger breadth of peptides with lower functional avidity when compared to HLA-DRB1 alleles linked with high viremia (p=0.018). Our data provide systematic evidence that HLA-DRB1 allele expression significantly impacts the durable control of HIV replication, an effect that appears to be mediated primarily by the protein-specificity of HIV-specific CD4+ T cell responses to Gag and Nef

A Multivalent and Cross-Protective Vaccine Strategy against Arenaviruses Associated with Human Disease

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8+ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8+ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8+ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies

Outcomes for the First Year of Ontario's Birth Center Demonstration Project

Introduction In 2014, Ontario opened 2 stand‐alone midwifery‐led birth centers. Using mixed methods, we evaluated the first year of operations for quality and safety, client experience, and integration into the maternity care community. This article reports on our study of safety and quality of care. Methods This descriptive evaluation focused on women admitted to a birth center at the beginning of labor. For context, we matched this cohort (on a 1:4 basis) with similar low‐risk midwifery clients giving birth in a hospital. Data sources included Ontario's Better Outcomes Registry and Network (BORN) Information System, the Canadian Institute for Health Information, Ontario census data, and birth center records. Results Of 495 women admitted to a birth center, 87.9% experienced a spontaneous vaginal birth, regardless of the eventual location of birth, and 7.7% had a cesarean birth. The transport rate to a hospital was 26.3%. When compared with midwifery clients with a planned hospital birth, rates of intervention (epidural analgesia, labor augmentation, assisted vaginal birth, and cesarean birth) were significantly lower in the planned birth center group, even when controlled for previous cesarean birth and body mass index. Markers of potential morbidity were identified in about 10% of birth center births; however, there were no short‐term health impacts up to discharge from midwifery care at 6 weeks postpartum. Care was low in intervention and safe (minimal negative outcomes and transport rates comparable to the literature). Discussion In the first year of operation, care was consistent with national guidelines, and morbidity and mortality rates and intervention rates were low for women with low‐risk pregnancies seeking a low‐intervention approach for labor and birth. Further evaluation to confirm these findings is required as the number of births grows.Ontario Ministry of Health and Long‐Term Car

Heterozygote advantage at HLA class I and II loci and reduced risk of colorectal cancer

Objective: Reduced diversity at Human Leukocyte Antigen (HLA) loci may adversely affect the host's ability to recognize tumor neoantigens and subsequently increase disease burden. We hypothesized that increased heterozygosity at HLA loci is associated with a reduced risk of developing colorectal cancer (CRC). Methods: We imputed HLA class I and II four-digit alleles using genotype data from a population-based study of 5,406 cases and 4,635 controls from the Molecular Epidemiology of Colorectal Cancer Study (MECC). Heterozygosity at each HLA locus and the number of heterozygous genotypes at HLA class -I (A, B, and C) and HLA class -II loci (DQB1, DRB1, and DPB1) were quantified. Logistic regression analysis was used to estimate the risk of CRC associated with HLA heterozygosity. Individuals with homozygous genotypes for all loci served as the reference category, and the analyses were adjusted for sex, age, genotyping platform, and ancestry. Further, we investigated associations between HLA diversity and tumor-associated T cell repertoire features, as measured by tumor infiltrating lymphocytes (TILs; N=2,839) and immunosequencing (N=2,357). Results: Individuals with all heterozygous genotypes at all three class I genes had a reduced odds of CRC (OR: 0.74; 95% CI: 0.56-0.97, p= 0.031). A similar association was observed for class II loci, with an OR of 0.75 (95% CI: 0.60-0.95, p= 0.016). For class-I and class-II combined, individuals with all heterozygous genotypes had significantly lower odds of developing CRC (OR: 0.66, 95% CI: 0.49-0.87, p= 0.004) than those with 0 or one heterozygous genotype. HLA class I and/or II diversity was associated with higher T cell receptor (TCR) abundance and lower TCR clonality, but results were not statistically significant. Conclusion: Our findings support a heterozygote advantage for the HLA class-I and -II loci, indicating an important role for HLA genetic variability in the etiology of CRC

Effects of Impact and Target Parameters on the Results of a Kinetic Impactor: Predictions for the Double Asteroid Redirection Test (DART) Mission

The Double Asteroid Redirection Test (DART) spacecraft will impact into the asteroid Dimorphos on 2022 September 26 as a test of the kinetic impactor technique for planetary defense. The efficiency of the deflection following a kinetic impactor can be represented using the momentum enhancement factor, β, which is dependent on factors such as impact geometry and the specific target material properties. Currently, very little is known about Dimorphos and its material properties, which introduces uncertainty in the results of the deflection efficiency observables, including crater formation, ejecta distribution, and β. The DART Impact Modeling Working Group (IWG) is responsible for using impact simulations to better understand the results of the DART impact. Pre-impact simulation studies also provide considerable insight into how different properties and impact scenarios affect momentum enhancement following a kinetic impact. This insight provides a basis for predicting the effects of the DART impact and the first understanding of how to interpret results following the encounter. Following the DART impact, the knowledge gained from these studies will inform the initial simulations that will recreate the impact conditions, including providing estimates for potential material properties of Dimorphos and β resulting from DART’s impact. This paper summarizes, at a high level, what has been learned from the IWG simulations and experiments in preparation for the DART impact. While unknown, estimates for reasonable potential material properties of Dimorphos provide predictions for β of 1–5, depending on end-member cases in the strength regime