Deregulation of TGF-β Family Ligands in Cancer

The study of signalling dynamics is important for understanding how signalling pathways operate in health and disease. Previous work within the lab investigated how cells respond to TGF-β family ligands over time, in terms of pathway activity. Cells treated with TGF-β reach maximal signal induction at one hour which rapidly attenuates to a low-level steady state despite sustained ligand exposure. Additionally, cells become unresponsive to fresh ligand. This suggests that in diseases, including cancer, where there are sustained levels of pathway activity, another ligand may be responsible for signal propagation or there is re-wiring of mechanisms underlying TGF-β signalling dynamics. Further work in the lab demonstrated that reduced expression of components of the ESCRT machinery led to sustained pathway activity in cells exposed to TGF-β. Furthermore, it was shown that CAFs, a cell type in the tumour stroma, produce functional Activin ligand, which may also be responsible for sustained pathway activation. The work in this thesis aimed to investigate whether these two distinct possibilities could explain the sustained TGF-β family pathway activity observed in disease states by answering two questions: 1. Does compromised ESCRT signalling lead to enhanced TGF-β-mediated output? 2. Is CAF-sourced Activin responsible for pathway signal and output, previously ascribed to TGF-? I have demonstrated that compromised ESCRT function leads to an enhanced TGF-β-mediated epithelial-mesenchymal transition through elevated PSMAD signalling. I have also shown that CAFs from the MMTV-PyMT mouse model of breast cancer produce Activin, that is critical for their contractility whilst affecting their proteome and transcriptome. Furthermore, I have developed in vivo experiments to determine the role of CAF-sourced Activin during tumourigenesis

Impairments to Thermoregulation in the Elderly During Heat Exposure Events

Heat waves represent a public health risk to elderly people, and typically result in an increased rate of hospital admissions and deaths. Studies of thermoregulation in this cohort have generally focused on single elements such as sweating capacity. Sweating capacity and skin blood flow reduce with age, reducing ability to dissipate heat. Perception of effort during heat exposure is emerging as an area that needs further investigation as the elderly appear to lack the ability to adequately perceive increased physiological strain during heat exposure. The role of the gut and endotoxemia in heat stress has received attention in young adults, while the elderly population has been neglected. This shortcoming offers another potential avenue for identifying effective integrated health interventions to reduce heat illnesses. Increasing numbers of elderly individuals in populations worldwide are likely to increase the incidence of heat wave-induced deaths if adequate interventions are not developed, evaluated, and implemented. In this narrative-style review we identify and discuss health-related interventions for reducing the impact of heat illnesses in the elderly

Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender.

Cell therapies are becoming increasingly widely used, and their production and cryopreservation should take place under tightly controlled GMP conditions, with minimal batch-to-batch variation. One potential source of variation is in the thawing of cryopreserved samples, typically carried out in water baths. This study looks at an alternative, dry thawing, to minimise variability in the thawing of a cryopreserved cell therapy, and compares the cellular outcome on thaw. Factors such as storage time, patient age, and gender are considered in terms of cryopreservation and thawing outcomes. Cryopreserved leukapheresis samples from 41 donors, frozen by the same protocol and stored for up to 17 years, have been thawed using automated, water-free equipment and by conventional wet thawing using a water bath. Post-thaw viability, assessed by both trypan blue and flow cytometry, showed no significant differences between the techniques. Similarly, there was no negative effect of the duration of frozen storage, donor age at sample collection or donor gender on post-thaw viability using either thawing method. The implication of these results is that the cryopreservation protocol chosen initially remains robust and appropriate for use with a wide range of donors. The positive response of the samples to water-free thawing offers potential benefits for clinical situations by removing the subjective element inherent in water bath thawing and eliminating possible contamination issues

Using principles of authentic assessment to redesign written examinations and tests

Tests and examinations are widely used internationally. Despite their pervasiveness, they tend to measure lower order thinking skills in a decontextualized manner at a time when the literature frequently argues for the benefits of a richer, authentic approach to assessment. The focus of this paper is to improve authenticity in test assessment methods through promoting realism, cognitive challenge and evaluative judgement during the planning, administering and following up of assessment tasks. The article builds on a systematic literature review, in which the main principles of authentic assessment were outlined. In this paper, we posit how these principles can be implemented through the three chronological phases of the assessment process: before, during and after the act of assessment

Phosphatidylserine-dependent adhesion of T cells to endothelial cells

AbstractPhosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca2+, and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca2+-ATPase inhibitor 2,5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca2+ and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V–FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V–FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca2+ bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4+ and CD8+ antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed

Diagnosis of coeliac disease by flow cytometry of intraepithelial lymphocytes: a new 'gold' standard?

OBJECTIVE: The analysis of intraepithelial lymphocytes (IELs) by flow cytometry of duodenal biopsies-the 'IEL' lymphogram-has been proposed as a diagnostic test for coeliac disease. However, its clinical applicability has been limited due to variability in methods and definitions. This study set out to define useful parameters for the application of the IEL lymphogram to the diagnosis of coeliac disease. DESIGN: Flow cytometry was performed on 117 sets of duodenal biopsies in 107 adult patients with active coeliac disease, long-term coeliac disease on a gluten free diet and a control group. The initial 95 samples were used for hypothesis generation for the subsequent samples comprising 12 patients with coeliac disease and 10 controls. RESULTS: Rather than using single linear cut-offs for CD3 and T-cell receptor γδ (TCRγδ)+ve IELs, a discriminant function was identified as %CD3+ve IELs+2x(%TCRγδ+IELs)>100. This differentiated coeliac disease from control biopsies in the hypothesis generating group. These results were replicated in the validation group and found to be independent of histology in patients on long-term gluten free diet up to 12 years (combined sensitivity, 98.5%; specificity, 97.7%). CONCLUSIONS: Flow cytometric analysis of IELs is a highly sensitive and specific adjunct to serology and histological examination for the diagnosis of coeliac disease, even in individuals with coeliac disease following a gluten free diet who exhibit normal duodenal histology

Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies.

The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers