As 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.

N-WASP activates the Arp2/3 complex to stimulate actin polymerisation during a number of cellular processes. These include, phagocytosis, endocytosis, and invadopodia and podosome formation. In addition, it is also frequently recruited by pathogens to assist in their actin-dependent spread. During its egress from the cell, vaccinia virus fuses with the plasma membrane and promotes the activation of Src and Abl family kinases. This event leads to phosphorylation of tyrosine 112 and tyrosine 132 of the viral transmembrane protein, A36, triggering a signalling cascade that stimulates actin polymerisation. The phospho-tyrosine residues provide docking sites for the cellular adaptors Nck and Grb2, with Nck recruiting WIP-N-WASP and in turn the Arp2/3 complex. I found that clathrin is transiently recruited to vaccinia in the moments before actin polymerisation. Clathrin performs an organisational role, clustering A36 beneath the virus to enhance robust actin tail formation. Loss of clathrin recruitment resulted in a more disperse localisation of A36 and N-WASP, which led to an altered actin tail morphology, indicating that the spatial organisation of N-WASP is a parameter affecting actin polymerisation. During actin tail formation, I additionally found that Cdc42 could regulate N-WASP activity. Cdc42 supported the main signalling nexus of Nck-WIP-N-WASP, with its major role attributed to stabilising N-WASP beneath the viral particle. I further found that Cdc42 acts in a feed-forward loop with N-WASP and the RhoGEF intersectin-1. This pathway is conserved in FcγR-mediated phagocytosis, validating the use of pathogens to understand the molecular detail of Arp2/3-dependent actin-signalling pathways.

Persistent high-risk human papillomavirus infection (HPVI) of the uterine cervix is associated with squamous intraepithelial lesions (SIL) and cervical cancer. Low-grade SIL (LSIL) is common but factors determining the outcome are incompletely understood and the management is controversial. The infecting HPV type and the host response to HPVI are thought to be important. Genetic polymorphisms in the regulatory sequences of genes have been identified that are associated with different levels of cytokine expression. These include interieukin 1 alpha (IL-1alpha, 46 base pair VNTR in intron 6), interieukin 1 receptor antagonist (IL-1Ralpha, 86 bp VNTR in intron 2) and tumour necrosis factor alpha (TNFalpha, single nucleotide polymorphisms at -308 and -238). Cytokine gene polymorphisms and HPV types were investigated in 209 women presenting to colposcopy clinic with smears suggesting LSIL and compared to controls with normal cervix and those with high-grade SIL (HSIL). Twelve month follow up showed 9% progression to HSIL, 15% persistence of LSIL, 56% regression and 21% loss to follow up. High risk HPVI was common (62%) in women with smears suggesting LSIL but more so in those with co-existing HSIL (85%). HPV 16 was the most common infecting type but 14 infecting high-risk HPV types were seen. The risks of progressing or regressing were not influenced by the presence of HR HPV in this study. Screening for HPV types 16 and 18 alone would miss 36% of disease progression. The low secretor phenotype of TNFalpha -308 was associated with all grades of SIL but most strongly with LSIL (p=0.004). The low secretor phenotype of IL-1alpha was associated with LSIL (p=0.031) but not with HSIL. No associations were demonstrated between IL-1Ralpha polymorphism and cervical disease. No associations were shown between cytokine polymorphisms and HPVI or disease outcome.

Adoptive (Natural killer) NK cell therapy relies on the use of a large amount of NK cells that are cytotoxic, and yet not exhausted. For this purpose, NK cells can be isolated from cord blood, peripheral blood or generated in vitro from haematopoietic stem cells (HSC). In vitro generation of high numbers of activated NK cells using HSC would facilitate multiple infusions and treatment of patients with large tumour burden, allowing to by-pass the limitations of NK cell numbers and activation state of blood-derived NK cells. However, comprehensive studies about the use of fresh or cryopreserved HSC and of different HSC sources for protocols of NK cell production in vitro have yet to be performed. The aim of this thesis was to investigate these variables and establish an optimal protocol for the generation of NK cells in vitro. This work investigated the use of a published protocol and a modified version using only IL-15 for the last weeks of culture; moreover, the comparison of NK cells derived from fresh cord blood stem cells (CBSC) and frozen CBSC and a different HSC source, mobilised peripheral blood stem cells (PBSC), was performed. Using this protocol, we showed that frozen CBSC generated higher NK cell numbers expressing activating receptors, lacking killer-cell immunoglobulin-like receptor expression but with better immunoregulatory and cytotoxic properties compared to NK cells from fresh CBSC and PBSC cultures. More than half of the NK cells generated in vitro from all HSC types expressed the myeloid-marker CD33; blocking of this marker did not impact on NK cell functions. Finally, CBSC and PBSC showed a different threshold for NK cell activation with interleukin 12. In conclusion, our study provides evidence that frozen CBSC are a suitable source of HSC for NK cell generation in vitro compared to fresh CBSC and frozen PBSC.

Objectives: To investigate the prevalence of haemostatic techniques at myomectomy. To investigate the effectiveness of modified triple tourniquets at open myomectomy and for the first time to pilot their use at laparoscopic myomectomy. Design: A questionnaire based postal survey. A randomised controlled trial (RCT) and a feasibility study Population All UK Consultant Obstetricians Gynaecologists. 38 patients with symptomatic fibroids undergoing open or laparoscopic myomectomy. Methods: A standard questionnaire postal survey. At open myomectomy a number 1 polyglactin suture was tied around the cervix to occlude the uterine arteries, and polythene (polyglactin at laparoscopic myomectomy) tourniquets were tied around the ovarian vessels. At the end of the procedure the ovarian ties were released but the uterine artery suture remained in situ. Outcome measures Use of haemostatic techniques at open, laparoscopic and hysteroscopic myomectomy. Intra-operative blood loss, post-operative blood loss, blood transfusion rates, operative morbidity, uterine blood flow, ovarian function, quality of life and effect on menstruation. Results: 59% responded to the survey. At open myomectomy 90% regularly used a haemostatic technique, with 85% prescribing pre-operative gonadotrophin releasing hormone agonists. Open myomectomy RCT there was less blood lost in the tourniquet group than in the control (p 0.0001). The volume in the pelvic drain postoperatively failed to reach statistical significance between the two groups. There were no differences in uterine artery Doppler resistance indices and ovarian function was unaffected by the tourniquets. Open myomectomy significantly improved menorrhagia and quality of life. In the laparoscopic study triple tourniquets were applied successfully and appeared effective. Conclusions: Modified triple tourniquets are effective in reducing bleeding at open myomectomy and appear safe with no obvious effect on uterine perfusion or ovarian function. Our feasibility study would suggest they can also be successfully used at laparoscopic myomectomy.

No description available.

No description available.

No description available.

Standard therapy for patients with chronic lymphocytic leukaemia involves the use of DNA damaging drugs. If the cellular DNA damage response has been compromised then these standard therapies will be ineffective at inducing apoptosis. Central to this response is the protein, p53. Loss of the chromosome containing the gene TP53, which encodes the protein p53 and/or mutation of the gene, occurs in approximately 5-10% of newly diagnosed CLL patients. This rises to over 30% in patients who relapse after therapy. In addition another important protein within the DNA damage response, the protein-kinase ATM, may be deleted and/or mutated in a further 20-30% of CLL cases. Loss of the chromosomal loci containing either TP53 or ATM has been shown to predict a poor response to treatment and shorter overall survival. It is now known that detecting loss alone is insufficient as miss-sense or non-sense mutation of these genes alone can lead to an equally poor outcome. Screening these genes for mutations is not carried out routinely and can be technically challenging. In addition to this it is not fully understood to what degree other genes may be involved in dysfunction of the DNA damage response. Functional assays provide an alternative method for testing the integrity of the p53-dependant DNA damage response without needing to focus upon specific genomic abnormalities. A flow-cytometry based assay, utilising etoposide/nutlin3a and developed in our laboratory, has already been shown to detect and discriminate between p53 and ATM abnormalities in a small series of patients. This project aims to assess the clinical value of the etoposide/nutlin3a functional assay for detecting abnormalities of the p53- dependant DNA damage response which would contra-indicate treatment with DNA damaging drugs in samples referred from around the UK . A total of 472 samples from participating centres around the UK have been tested using the functional assay and also assessed for deletion of ATM and p53. The assay is largely unaffected by transport of whole blood samples across the UK if transit time is no greater than 48 hours and the sample is not stored above room temperature. Dysfunctional assay results have been shown to have a highly significant association with deletion of the genes for p53 and ATM. The use of nutlin3a has been shown to discriminate between dysfunctional cases with deletion of ATM or loss/mutation of p53. The assay has been shown to detect patients with mutation of the p53 gene who do not have concurrent deletion of the second allele. A novel dysfunctional response pattern involving p21 expression has been shown to associate with abnormalities of p53 rather than single nucleotide polymorphisms. The results also show that deletion of p53 and particularly ATM does not per se lead to a dysfunctional response as currently detected by the assay. This suggests that other mechanisms or genes are involved in resistance to DNA damage induced apoptosis.

Patients with advanced gastric cancer have limited therapeutic options to which response rates are low. Very few targeted therapies are available. There is an urgent need for novel targeted inhibitors for patients who are either ineligible for the existing targeted therapies or have developed resistance. Several studies have supported the importance of the IGF signal transduction pathway in cancer cell survival and proliferation and inhibitors of the type I IGF and insulin receptors have reached clinical trials in various cancer types. To date, the role of the IGF signal transduction pathway in gastric cancer has not been investigated thoroughly. The aim of the present study is to investigate the importance of the IGF signal transduction pathway in gastric cancer cells and the effect of inhibition of IGF signal transduction in cell survival and proliferation. IGF-1 was protective against gastric cancer cell death induced by protein kinase inhibition and disruption of cell attachment to the extracellular matrix. The survival effect was mediated by the PI3K/Akt pathway. The survival effect of IGF-1 was even more prominent in ex vivo cultures of gastric cancer cells established from patient ascites. IGF-1 stimulated proliferation of gastric cancer cells, which was mediated by activation of the Ras/Raf/ERK pathway. IGF-2 and insulin induced also gastric cancer cell survival and proliferation. Inhibition of the type I IGF receptor by siRNA knockdown reduced proliferation of gastric cancer cells and ex vivo cultures by inhibition of cell mitosis and DNA synthesis. Inhibition of the insulin receptor by siRNA knockdown resulted in the induction of apoptosis. Combined inhibition of the two receptors with a small molecule tyrosine kinase inhibitor reduced effectively cell growth and induced apoptosis. Our results suggest that inhibition of the type I IGF and insulin receptors could be a valid therapeutic strategy for advanced gastric cancer patients who are not eligible for the currently available targeted treatments.