159 research outputs found
Non-MHC-restricted cytotoxic cells: Their roles in the control and treatment of leukaemias
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Autologous Cell Seeding in Tracheal Tissue Engineering
No full textPurpose of Review: There is no consensus on the best technology to be employed for tracheal replacement. One particularly promising approach is based upon tissue engineering and involves applying autologous cells to transplantable scaffolds. Here, we present the reported pre-clinical and clinical data exploring the various options for achieving such seeding. Recent Findings: Various cell combinations, delivery strategies, and outcome measures are described. Mesenchymal stem cells (MSCs) are the most widely employed cell type in tracheal bioengineering. Airway epithelial cell luminal seeding is also widely employed, alone or in combination with other cell types. Combinations have thus far shown the greatest promise. Chondrocytes may improve mechanical outcomes in pre-clinical models, but have not been clinically tested. Rapid or pre-vascularization of scaffolds is an important consideration. Overall, there are few published objective measures of post-seeding cell viability, survival, or overall efficacy. Summary: There is no clear consensus on the optimal cell-scaffold combination and mechanisms for seeding. Systematic in vivo work is required to assess differences between tracheal grafts seeded with combinations of clinically deliverable cell types using objective outcome measures, including those for functionality and host immune response
Translational process engineering for tissue engineered hollow organ advanced therapy investigational medicinal products
No full textTissue engineering has experienced increasing exposure and translational success in recent times, with tissue engineered products accounting for more than a quarter of approved advanced therapy medicinal products within Europe. Hollow organs represent a key target for developing novel therapies, typified by the recent success reported for tissue engineered hemilaryngeal replacements in a preclinical study. This thesis investigates the translational process engineering required to progress from a preclinical, good laboratory practice (GLP) process, to one that is compliant with good manufacturing practice (GMP) guidelines and suitable for clinical manufacture. A GLP decellularisation protocol was translated to conform to GMP-guidelines by modifying the existing standard operating procedure, and adapting an off-the-shelf bioreactor to form a closed-system for aseptic processing. The process was successfully validated for aseptic operation and decellularisation efficacy evaluated, relative to the preclinical process. The decellularised, human hemilarynx scaffolds produced were demonstrated to support bone marrow mesenchymal stromal cells (BM-MSCs) up to product release. A bespoke, modular bioreactor was designed and fabricated to enable manufacture of hemilarynges at scale. The bioreactor was successfully validated for aseptic use, whilst biocompatibility testing indicated no preclusion to use with BM-MSCs or epithelial cells. Proof-of-principle data supported the concept of epithelial sheet production inside the bioreactor, utilising a sheet-specific cassette. The bioreactor was retrospectively adapted to enable closed-system decellularisation processing of a third tissue-type, juvenile oesophagus. Acellular scaffold biomolecular composition and biomechanics were characterised, preceding implantation in a large-animal model. A second, bespoke bioreactor was designed, manufactured and employed to improve the manufacturing process. The combined human larynx data supported the award of a clinical trial authorisation, whilst the oesophageal work is now transitioning to a pivotal animal study. These findings support the application of bespoke bioreactor systems in process closure and translation towards robust, regulatory compliant, manufacturing processes for tissue engineered products
Human natural killer cell responses to tumour-priming
No full textAs one of the central components of host anti-tumour immunity, natural killer (NK) cells exert cellular cytotoxicity against tumour cells and secrete a milieu of immunoregulatory cytokines to inhibit tumour progression. NK cell-mediated cytotoxicity requires successful progression through discrete activation events that begin with NK cell adhesion to a target cell and culminate in the polarized release of cytotoxic granules into the immunological synapse. These activation events are tightly regulated by a complex array of signalling molecules, the engagement of which by ligands on target cells can determine susceptibility to NK cell-mediated killing. Resistance to NK cell cytotoxicity can be attributable to a deficiency in any of the signalling requirements for the events leading to granule exocytosis. Tumour resistance to NK cell lysis may be overcome by priming of NK cells with cytokines or by binding of NK cell activating receptors to ligands expressed on target cells. Here, the activation profiles of normal, freshly isolated human peripheral blood NK cells upon tumour-priming with the NK-resistant leukemic cell line, CTV-1 are defined, and candidate NK cell receptors involved in the delivery of the tumour-priming signal are identified. Results from this study demonstrate that NK cell responses to tumour-priming are distinct from those induced by the gold standard in immunotherapy, cytokine-priming. Tumour-priming of NK cells resulted in a significant downregulation of various activating NK cell receptors including CD16, NKp46, NK group 2, member D (NKG2D), and intracellular adhesion molecule (ICAM)-1. Tumour-primed NK cells (TpNKs) also exhibited a strong pro-inflammatory profile marked by ample secretion of macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation normal T cell expressed and secreted (RANTES), tumour necrosis factor (TNF)-α and interferon (IFN) -γ after short incubations with CTV-1. Their secretory profiles were distinct from the profiles of NK cells stimulated with exogenous cytokines or the NK-sensitive target cell line, K562. Collectively, data from this study demonstrates that NK cell responses can differ according to the type of stimulus, as well as the ligand combination presented by the target cell. The co-engagement of NK cell receptors LFA-1 and CD2 by their respective ligands on CTV-1 cells, ICAM-1 and CD15, seems to play an important role in the delivery of the tumour- priming signal
An investigation into the role of platelet- monocyte interaction and inflammation in coronary artery disease
No full textIntroduction: - Platelet monocyte complex (PMC) formation has been widely reported as a marker of platelet activation in vascular disease states and several studies have shown heightened systemic expression of PMC in stable and acute coronary disease. However, the relationship between intracoronary platelet and monocyte activation status and local intracoronary inflammation in acute coronary syndrome (ACS) remains unclear. Method:- Fifteen ST elevation myocardial infarction (STEMI), 8 non ST elevation myocardial infarction (NSTEMI) and 7 stable angina patients were recruited. PMC, P selectin positive PMC (activated platelet within the complex), tissue factor (TF) positive PMC (activated monocyte within the complex) were estimated with flow cytometry from blood samples aspirated from the coronary artery (distal to the lesion), aorta and right atrium . Plasma CRP, SAA, TNF –alpha and IL-6 were also measured. Results:- In ACS patients no significant transculprit lesion gradient of PMC expression was observed but significant gradients were found with P-selectin positive PMC (p= 0.01) and TF positive PMC expression (p=0.04). Overall median P- selectin positive PMC expression in ACS patients was significantly higher compared to stable angina (p= 0.006). Intracoronary P-selectin positive PMC was also found to be higher in the ACS group compared to stable group (p=0.003). Overall median CRP (p= 0.001), SAA (p=0.0007) and IL-6 (p= 0.03) levels were significantly higher in ACS. In STEMI intracoronary PMC correlated positively with intracoronary TNF-alpha (r= 0.68, p= 0.03). Positive correlation was also observed between intracoronary TF positive PMC (% monocyte) with TNF-alpha and IL-6 (r=0.66, p=0.05 & r=0.71, p= 0.05 respectively). Conclusion:- The work outlined in this thesis has demonstrated the importance of platelet and monocyte activation status of the PMC as a determinant of intracoronary inflammation. Beyond a local intracoronary role, PMC may contribute to systemic inflammation through P-selectin expression and local intracoronary inflammation through increased P-selectin and tissue factor expression
Immune reconstitution after T cell depleted autologous stem cell transplantation for the treatment of systemic sclerosis
No full textGenerating full-length Killer-cell Immunoglobulin-like Receptor (KIR) gene sequences using Third Generation long-amplicon sequencing to assess the impact of KIR polymorphism on Haematopoietic Cell Transplantation outcomes
No full textKiller-cell Immunoglobulin-like Receptor (KIR) polymorphism is extensive in both allelic and copy number variation. Although multiple assays have been designed to assess the latter form of polymorphism, KIR allelic diversity is less well understood owing to the homologous nature of different KIR genes and, until recently, limitations in sequencing technology. To better understand KIR allelic diversity in the UK, and its impact on haematopoietic cell transplant (HCT) outcomes, I have designed and validated a whole-gene, fully-phased allele sequencing strategy that encompasses third generation sequencing technology to deliver unambiguous genotypes for several different KIR genes. Subsequently, this strategy was applied to a novel, largely T cell deplete UK cohort of patients receiving HCT to treat acute myeloid leukaemia, and their respective donors. This assay utilises a semi-generic targeted polymerase chain-reaction amplification prior to multiplexed library preparation and sequencing, providing a relatively high-throughput methodology amenable to clinical laboratories. Initially, to assess the relevance of presence/absence KIR polymorphism on HCT outcomes, I utilised existing genotyping methods to establish baseline characteristics. In contrast to previous publications, relapse was largely unaffected by KIR polymorphism. However, striking differences related to preparative conditioning regimen were observed in KIR-mediated effects in other HCT outcomes. Presence of donor-encoded centromeric (Cen)-B motifs relayed increased risk of detrimental non-relapse mortality following myeloablative conditioning, whereas the opposite appeared to be true of reduced-intensity conditioning transplants. When the impact of allelic diversity at the KIR2DL1, KIR2DL2/3 and KIR3DL1/S1 loci on HCT outcomes was assessed in my cohort, allelic differences within the Cen-A, Cen-B and telomeric A haplotype motifs provided additional insight into the possible mechanisms of KIR-mediated influence on HCT outcomes. By estimating frequencies of different KIR alleles within a UK population, I have demonstrated that donor selection algorithms could feasibly incorporate allelic KIR genotypes that may improve quality of life after HCT
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