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Accurate quantification of rare genes from limited clinical samples is crucial for research purposes but is technically challenging, especially due to nucleic acid extraction. Using the commercially available genomic DNA (gDNA) extraction kits, which mostly include a DNA purification step through silica columns, magnetic beads or ethanol precipitation, are the preferred choice for many researchers. These kits, however, have a minimum cell number requirement for optimal DNA quality and yield. They are not ideal for use for clinical samples with limited cell numbers. Here, we report the development and validation of a novel crude lysate method for preparing DNA for the absolute quantification of rare genes, TRECs in our case, by droplet digital PCR (ddPCR), from infrequent cells, that removes the need for DNA extraction. Multiple optimization steps and analytical validation of this novel assay was performed on PBMCs extracted from the blood of healthy donors. The newly developed assay shows good agreement with standard ddPCR and has high accuracy, specificity, and reproducibility; additionally, it can also be applied to fixed and permeabilized cells. The assay has the potential to be used for quantification of other trace targets from limited cell samples.

In several murine models of autoimmune arthritis, Th17 cells are the dominant initiators of inflammation. In human arthritis the majority of IL-17–secreting cells within the joint express a cytokine phenotype intermediate between Th17 and Th1. Here we show that Th17/1 cells from the joints of children with inflammatory arthritis express high levels of both Th17 and Th1 lineage-specific transcription factors, RORC2 and T-bet. Modeling the generation of Th17/1 in vitro, we show that Th17 cells \"convert\" to Th17/1 under conditions that mimic the disease site, namely low TGFβ and high IL-12 levels, whereas Th1 cells cannot convert to Th17. Th17/1 cells from the inflamed joint share T-cell receptor (TCR) clonality with Th17 cells, suggesting a shared clonal origin between Th17 and Th17/1 cells in arthritis. Using CD161, a lectin-like receptor that is a marker of human Th17, we show synovial Th17 and Th17/1 cells, and unexpectedly, a large proportion of Th1 cells express CD161. We provide evidence to support a Th17 origin for Th1 cells expressing CD161. In vitro, Th17 cells that convert to a Th1 phenotype maintain CD161 expression. In the joint CD161+ Th1 cells share features with Th17 cells, with shared TCR clonality, expression of RORC2 and CCR6 and response to IL-23, although they are IL-17 negative. We propose that the Th17 phenotype may be unstable and that Th17 cells may convert to Th17/1 and Th1 cells in human arthritis. Therefore therapies targeting the induction of Th17 cells could also attenuate Th17/1 and Th1 effector populations within the inflamed joint.

A significant proportion of patients suffering from acute myeloid leukemia (AML) cannot be cured by conventional chemotherapy, relapsed disease being a common problem. Molecular targeting of essential oncogenic mediators is an attractive approach to improving outcomes for this disease. The hematopoietic transcription factor c-MYB has been revealed as a central component of complexes maintaining aberrant gene expression programs in AML. We have previously screened the Connectivity Map database to identify mebendazole as an anti-AML therapeutic targeting c-MYB. In the present study we demonstrate that another hit from this screen, the steroidal lactone withaferin A (WFA), induces rapid ablation of c-MYB protein and consequent inhibition of c-MYB target gene expression, loss of leukemia cell viability, reduced colony formation and impaired disease progression. Although WFA has been reported to have pleiotropic anti-cancer effects, we demonstrate that its anti-AML activity depends on c-MYB modulation and can be partially reversed by a stabilized c-MYB mutant. c-MYB ablation results from disrupted HSP/HSC70 chaperone protein homeostasis in leukemia cells following induction of proteotoxicity and the unfolded protein response by WFA. The widespread use of WFA in traditional medicines throughout the world indicates that it represents a promising candidate for repurposing into AML therapy.

This book provides the reader with an interactive experience of quantum optics, suitable for Final year undergraduate quantum optics students, and new graduate students in AMO physics.

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.

Purpose Severe Combined Immunodeficiency (SCID) is considered to be a paediatric emergency and unless identified promptly can be life-threatening. Frequently, infants are not diagnosed with SCID until they have become seriously ill with infection leading to treatment complications and a poorer prognosis. We aimed to test a newly available commercial duplex assay to measure T cell receptor excision circles (TRECs) to establish if this would be suitable for newborn screening for SCID in the UK. Methods Over 5000 anonymous retrospective dried blood spots (DBS) were used alongside 18 confirmed SCID positive DBS with a newly available duplex assay to measure TRECs levels and control gene levels. We also included testing of premature babies and babies from neonatal intensive care units (NICU) as these have been shown to have high false positive rates in other TREC screening assays. Results All 18 SCID DBS samples were successfully identified as SCID positives in the study. The number of presumptive positives detected was dependent on the TREC cut-off threshold settings. When analysed with five different TRECs cut-off values (20, 25, 30, 35 and 40 TREC copies/μl blood) the presumptive positive rate ranged from 0.04 to 1.00 % of samples tested. Premature infants and neonates from NICU did not show high presumed false positive rates in this assay. Conclusions The study demonstrated that this duplex assay kit will identify all newborns with SCID as presumptive positives. The data also shows that with suitable TREC cut-off settings the number of presumptive positives from non-SCID newborns will be manageable in the context of a national screening service.

This study aimed to assess the effectiveness of a systems-based secondary injury prevention intervention aimed at early detection and management of hamstring strain injury (HSI) and time-loss groin injury in an Australian male professional football club. Prospective cohort study. Australian male professional football club. Data were collected from male professional football players (n = 73) from a single football club. Unilateral knee flexion and side-lying hip adduction maximum voluntary isometric contractions were monitored routinely in-season, two days post-match (≥40 h [h]) during a three-season intervention period. Strength reductions greater than the tests’ minimal detectable change percentage prompted intervention. HSI and time-loss groin injury burdens were calculated per 1000 player hours and compared with those from an immediately preceding two-season control period, to assess the effectiveness of the intervention. Across the intervention period, there was a decrease in HSI (4.98 days absence/1000 h (19.8%) decrease) and time-loss groin injury burdens (0.57 days absence/1000 h (49.1%) decrease) when compared with the control period. A systems-based secondary injury prevention intervention shows preliminary positive findings in reducing HSI and time-loss groin injury burdens within a male professional football club, compared with usual care only. •Maximum voluntary isometric contractions may be safely monitored post-match play.•Indicated interventions may be individual- and position-specific.•Systems-based prevention may reduce hamstring and time-loss groin injury burden.

While the majority of children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) display mild or no symptoms, rare individuals develop severe disease presenting with multisystem inflammatory syndrome (MIS-C). The reason for variable clinical manifestations is not understood. Here, we carried out TCR sequencing and conducted comparative analyses of TCR repertoires between children with MIS-C (n = 12) and mild (n = 8) COVID-19. We compared these repertoires with unexposed individuals (samples collected pre-COVID-19 pandemic: n = 8) and with the Adaptive Biotechnologies MIRA dataset, which includes over 135,000 high-confidence SARS-CoV-2-specific TCRs. We show that the repertoires of children with MIS-C are characterised by the expansion of TRBV11-2 chains with high junctional and CDR3 diversity. Moreover, the CDR3 sequences of TRBV11-2 clones shift away from SARS-CoV-2 specific T cell clones, resulting in distorted TCR repertoires. In conclusion, our study reports that CDR3-independent expansion of TRBV11-2+ cells, lacking SARS-CoV-2 specificity, defines MIS-C in children.

Background and Objective Alemtuzumab (Campath ® ) is used to prevent graft-versus-host disease and graft failure following pediatric allogeneic hematopoietic cell transplantation. The main toxicity includes delayed immune reconstitution, subsequent viral reactivations, and leukemia relapse. Exposure to alemtuzumab is highly variable upon empirical milligram/kilogram dosing. Methods A population pharmacokinetic (PK) model for alemtuzumab was developed based on a total of 1146 concentration samples from 206 patients, aged 0.2–19 years, receiving a cumulative intravenous dose of 0.2–1.5 mg/kg, and treated between 2003 and 2015 in two centers. Results Alemtuzumab PK were best described using a two-compartment model with a parallel saturable and linear elimination pathway. The linear clearance pathway, central volume of distribution, and intercompartmental distribution increased with body weight. Blood lymphocyte counts, a potential substrate for alemtuzumab, did not impact clearance. Conclusion The current practice with uniform milligram/kilogram doses leads to highly variable exposures in children due to the non-linear relationship between body weight and alemtuzumab PK. This model may be used for individualized dosing of alemtuzumab.

Refractory cancers may arise either through the acquisition of resistance mechanisms or represent distinct disease states. The origin of childhood T-cell acute lymphoblastic leukaemia (T-ALL) that does not respond to initial treatment, i.e. refractory disease, is unknown. Refractory T-ALL carries a poor prognosis and cannot be predicted at diagnosis. Here, we perform single cell mRNA sequencing of T-ALL from 58 children (84 samples) who did, or did not respond to initial treatment. We identify a transcriptionally distinctive blast population, exhibiting features of innate-like lymphocytes, as the major source of refractory disease. Evidence of such blasts at diagnosis heralds refractory disease across independent datasets and is associated with survival in a large, contemporary trial cohort. Our findings portray refractory T-ALL as a distinct disease with the potential for immediate clinical utility. T-cell acute lymphoblastic leukemia is a highly aggressive disease with varying recurrence rates. Here, the authors build a single cell transcriptomic atlas of childhood T-cell acute lymphoblastic leukaemia (T-ALL). They identified a distinctive cancer cell state that correlates with high risk, treatment refractory T-ALL.