Clinical T Cell Receptor Repertoire Deep Sequencing and Analysis: An Application to Monitor Immune Reconstitution Following Cord Blood Transplantation

Spectratyping assays are well recognized as the clinical gold standard for assessing the T cell receptor (TCR) repertoire in haematopoietic stem cell transplant (HSCT) recipients. These assays use length distributions of the hyper variable complementarity-determining region 3 (CDR3) to characterize a patient's T cell immune reconstitution post-transplant. However, whilst useful, TCR spectratyping is notably limited by its resolution, with the technique unable to provide data on the individual clonotypes present in a sample. High-resolution clonotype data are necessary to provide quantitative clinical TCR assessments and to better understand clonotype dynamics during clinically relevant events such as viral infections or GvHD. In this study we developed and applied a CDR3 Next Generation Sequencing (NGS) methodology to assess the TCR repertoire in cord blood transplant (CBT) recipients. Using this, we obtained comprehensive TCR data from 16 CBT patients and 5 control cord samples at Great Ormond Street Hospital (GOSH). These were analyzed to provide a quantitative measurement of the TCR repertoire and its constituents in patients post-CBT. We were able to both recreate and quantify inferences typically drawn from spectratyping data. Additionally, we demonstrate that an NGS approach to TCR assessment can provide novel insights into the recovery of the immune system in these patients. We show that NGS can be used to accurately quantify TCR repertoire diversity and to provide valuable inference on clonotypes detected in a sample. We serially assessed the progress of T cell immune reconstitution demonstrating that there is dramatic variation in TCR diversity immediately following transplantation and that the dynamics of T cell immune reconstitution is perturbed by the presence of GvHD. These findings provide a proof of concept for the adoption of NGS TCR sequencing in clinical practice

Party finance reform as constitutional engineering? The effectiveness and unintended consequences of party finance reform in France and Britain

In both Britain and France, party funding was traditionally characterized by a laissez faire approach and a conspicuous lack of regulation. In France, this was tantamount to a 'legislative vacuum'. In the last two decades, however, both countries have sought to fundamentally reform their political finance regulation regimes. This prompted, in Britain, the Political Parties, Elections and Referendums Act 2000, and in France a bout of 'legislative incontinence' — profoundly transforming the political finance regime between 1988 and 1995. This article seeks to explore and compare the impacts of the reforms in each country in a bid to explain the unintended consequences of the alternative paths taken and the effectiveness of the new party finance regime in each country. It finds that constitutional engineering through party finance reform is a singularly inexact science, largely due to the imperfect nature of information, the limited predictability of cause and effect, and the constraining influence of non-party actors, such as the Constitutional Council in France, and the Electoral Commission in Britain

Detection of Low Frequency Multi-Drug Resistance and Novel Putative Maribavir Resistance in Immunocompromised Pediatric Patients with Cytomegalovirus.

Human cytomegalovirus (HCMV) is a significant pathogen in immunocompromised individuals, with the potential to cause fatal pneumonitis and colitis, as well as increasing the risk of organ rejection in transplant patients. With the advent of new anti-HCMV drugs there is therefore considerable interest in using virus sequence data to monitor emerging resistance to antiviral drugs in HCMV viraemia and disease, including the identification of putative new mutations. We used target-enrichment to deep sequence HCMV DNA from 11 immunosuppressed pediatric patients receiving single or combination anti-HCMV treatment, serially sampled over 1-27 weeks. Changes in consensus sequence and resistance mutations were analyzed for three ORFs targeted by anti-HCMV drugs and the frequencies of drug resistance mutations monitored. Targeted-enriched sequencing of clinical material detected mutations occurring at frequencies of 2%. Seven patients showed no evidence of drug resistance mutations. Four patients developed drug resistance mutations a mean of 16 weeks after starting treatment. In two patients, multiple resistance mutations accumulated at frequencies of 20% or less, including putative maribavir and ganciclovir resistance mutations P522Q (UL54) and C480F (UL97). In one patient, resistance was detected 14 days earlier than by PCR. Phylogenetic analysis suggested recombination or superinfection in one patient. Deep sequencing of HCMV enriched from clinical samples excluded resistance in 7 of 11 subjects and identified resistance mutations earlier than conventional PCR-based resistance testing in 2 patients. Detection of multiple low level resistance mutations was associated with poor outcome

Using Whole Genome Sequences to Investigate Adenovirus Outbreaks in a Hematopoietic Stem Cell Transplant Unit

A recent surge in human mastadenovirus (HAdV) cases, including five deaths, amongst a haematopoietic stem cell transplant population led us to use whole genome sequencing (WGS) to investigate. We compared sequences from 37 patients collected over a 20-month period with sequences from GenBank and our own database of HAdVs. Maximum likelihood trees and pairwise differences were used to evaluate genotypic relationships, paired with the epidemiological data from routine infection prevention and control (IPC) records and hospital activity data. During this time period, two formal outbreaks had been declared by IPC, while WGS detected nine monophyletic clusters, seven were corroborated by epidemiological evidence and by comparison of single-nucleotide polymorphisms. One of the formal outbreaks was confirmed, and the other was not. Of the five HAdV-associated deaths, three were unlinked and the remaining two considered the source of transmission. Mixed infection was frequent (10%), providing a sentinel source of recombination and superinfection. Immunosuppressed patients harboring a high rate of HAdV positivity require comprehensive surveillance. As a consequence of these findings, HAdV WGS is being incorporated routinely into clinical practice to influence IPC policy contemporaneously

Socio-Demographic Patterning of Physical Activity across Migrant Groups in India: Results from the Indian Migration Study

OBJECTIVE: To investigate the relationship between rural to urban migration and physical activity (PA) in India. METHODS: 6,447 (42% women) participants comprising 2077 rural, 2,094 migrants and 2,276 urban were recruited. Total activity (MET hr/day), activity intensity (min/day), PA Level (PAL) television viewing and sleeping (min/day) were estimated and associations with migrant status examined, adjusting for the sib-pair design, age, site, occupation, education, and socio-economic position (SEP). RESULTS: Total activity was highest in rural men whereas migrant and urban men had broadly similar activity levels (p<0.001). Women showed similar patterns, but slightly lower levels of total activity. Sedentary behaviour and television viewing were lower in rural residents and similar in migrant and urban groups. Sleep duration was highest in the rural group and lowest in urban non-migrants. Migrant men had considerably lower odds of being in the highest quartile of total activity than rural men, a finding that persisted after adjustment for age, SEP and education (OR 0.53, 95% CI 0.37, 0.74). For women, odds ratios attenuated and associations were removed after adjusting for age, SEP and education. CONCLUSION: Our findings suggest that migrants have already acquired PA levels that closely resemble long-term urban residents. Effective public health interventions to increase PA are needed

Monitoring Viral Infections and Immune Repertoires in Transplanted Children: A Statistical Approach

Transplantation in children is a relatively high-risk procedure, where pharmacological treatment and disease often render the recipient immunocompromised. This state allows typically non-serious infectious organisms such as cytomegalovirus (CMV) to grow uninhibited, leading to morbidity and mortality in the host. Underpinning the dynamics of these infections lies the immune response, driven in part by T cells and their innate ability to recognise pathogens via the T cell receptor (TCR). Applying deep sequencing techniques to the TCR repertoire allows researchers to explore this cellular response in unprecedented resolution. // This thesis explores the application of statistical modelling and data analysis techniques to two key areas of interest: the reconstituting immune response and the consequences of immunodeficiency within transplanted children. Specifically, the first part of this thesis focuses on the TCR repertoire, opening with an automated TCR next generation sequencing (NGS) data processing, subsampling, and analysis pipeline. This pipeline is then applied to NGS data from two distinct patient cohorts, facilitating a detailed statistical analysis on the clinical applicability of TCR deep sequencing, alongside an analysis of repertoire reconstitution. Lastly, open access complementarity determining region 3 (CDR3) epitope specificity data are integrated with the processed sequencing data to model antigen-specific CDR3 sequence profiles using profile hidden Markov models (PHMMs) and unsupervised CDR3 sequence clustering. This section of the thesis explores the utility of CDR3 sequencing for clinical TCR repertoire evaluation and demonstrates the wide-ranging potential applications of the sequencing data. // The second part of the thesis focuses on CMV infections, opening with a quantitative clinical audit on the treatment of CMV infections post-haematopoietic stem cell transplant (HSCT). The audit includes an analysis of the efficacy of anti-CMV drugs in children and a viral load-based time-to-event (survival) model. This work is then expanded with a Bayesian nonlinear mixed effects model for predicting CMV loads in infected transplant recipients, using a workflow that prioritises identifiability and biological plausibility of model parameters

Knowledge of gestational diabetes among a multi-ethnic cohort in Australia

Objective: to explore knowledge about gestational diabete(GDM)among a multi-ethnic sample of women who were receiving antenatal care in Melbourne,Australia

Using Whole Genome Sequences to Investigate Adenovirus Outbreaks, Including Five Deaths in a Haematopoietic Stem Cell Transplant Unit

Background Human mastadenoviruses (HAdV) are associated with significant morbidity and mortality amongst the immunocompromised population. A recent surge in HAdV cases, including five deaths, amongst a haematopoietic stem cell transplant population led us to use whole genome sequencing (WGS) to investigate. Methods To gain a complete transmission picture, we compared outbreak and non-outbreak sequences (54 sequences from 37 patients) with GenBank sequences and our own database of previously sequenced HAdVs (132 sequences from 37 patients). An improved bait set for WGS was used. Maximum likelihood trees and pairwise differences were used to evaluate genotypic relationships paired with epidemiological data from routine Infection, Prevention and Control (IPC) activity. Results Nine monophyletic clusters were identified, seven of which were corroborated by epidemiological evidence and by comparison of single nucleotide polymorphisms. Two incomplete patient clusters were identified by IPC over the same time period. Of the five patients who died, one had a mixed HAdV infection and two were the source of transmission events. Conclusions The clinical consequences of unmitigated HAdV transmission events are high. Focusing on two high risk wards using WGS we identified six transmission events, over prolonged periods, that would have gone unnoticed using traditional polymerase chain reaction and epidemiology. Mixed infection is frequent (10% of patients), providing a sentinel source of recombination and superinfection. Immunosuppressed patients harbouring a high rate of HAdV positivity require comprehensive surveillance. As a consequence of these findings, HAdV WGS is being incorporated routinely into a clinical algorithm to prevent transmission and influence IPC policy in real-time. Summary Whole genome sequencing of adenovirus, direct from clinical samples, can be used to identify cryptic health care associated transmission events, and to resolve transmission suspected by traditional epidemiology. It can also identify mixed genotype infections in immunocompromised patient populations