• Characterisation of the expression of tumour antigens and biomarkers in myeloid leukaemia and ovarian cancer

      Khan, Ghazala (University of BedfordshireUniversity of Bedfordshire, 2016-12)
      Acute myeloid leukaemia (AML) and ovarian cancer (OVC) are two difficult to treat cancers. AML is often treatable however minimal residual disease (MRD) endures such that many patients who achieve remission eventually relapse and succumb to the disease. OVC affects approximately 7000 women in the U.K. every year. It can occur at any age but is most common after menopause. Diagnosis at an early stage of disease greatly improves the chances of survival however, patients tend to be diagnosed in the later stages of disease when treatment is often less effective. Immunotherapy has the potential to reduce MRD and delay or prevent relapse. In order for immunotherapy to work, tumour antigens need to be identified and characterised so they can be effectively targeted. Personalised treatments require the identification of biomarkers, for disease detection and confirmation, as well as to provide an indication of best treatment and the prediction of survival. PASD1 has been found to be frequently expressed in haematological malignancies and I wanted to determine if there was a correlation between the presence of antigen-specific T cells in the periphery of patients with AML and PASD1 protein expression in the leukaemic cells. The expression of other leukaemia antigens were concurrently examined as comparators. I performed RT-PCR on nine antigens and immunocytochemistry on PASD1 in 18 samples from AML patients. I found a correlation between PASD1 expression in AML samples and the presence of PASD1-specific T cells as detected on the pMHC array. OVC lacks suitable targets for immunotherapy with few CTAs having been identified. I examined the expression of SSX2IP and the CTAs PASD1 and SSX2 in OVC. I compared the protein expression of these known tumour antigens to the “gold standard” biomarker for the diagnosis of OVC, CA125 and two other proteins known to be promising in the diagnosis of OVC, HE4 and WT1. I analysed commercially available paraffin-embedded OVC multiple tissue arrays (MTAs) containing 191 samples, predominantly stage I (n= 166), II (n= 15) and III (n= 6) OVC as well as healthy donor (n= 8) and normal adjacent tissues (n= 8). Scoring was performed in a single blinded fashion. I found SSX2A to be expressed at a score level of 3 with a frequency (37/191) that exceeded that of CA125 (14/191), HE4 (14/191), WT1 (1//191) or PASD1 (0/191). To confirm this expression I used two additional commercially-available antibodies that recognise the region common to SSX2A and B, and an antibody specific for SSX2A. Using SSX2 peptides, I blocked the immunolabelling of SSX2 in SSX2-positive cell lines showing that the immunolabelling of SSX2 and SSX2A was specific. I demonstrated that the expression of SSX2 and specifically SSX2A was reproducible and restricted to ovarian cancer with little or no expression in endometrial tissues, or diseased or inflamed endometrial tissue. In summary, these studies demonstrated that PASD1 expression in leukaemia cells correlated with the presence of PASD1-specific T cells in the periphery of presentation AML patients. I have shown that PASD1 specific-T cells are present in AML patients at diagnosis and that immunotherapy targeting PASD1 could be used to break tolerance and clear residual leukaemia cells during first remission. Analysis of the expression of three antigens in OVC, identified the specific expression of SSX2, in particular SSX2A in OVC but not healthy or diseased endometrial tissues. The expression of SSX2A was more frequent and more specific to OVC, than HE4 and WT1, and more frequent at higher intensity, especially in early stage OVC, than CA125. SSX2 and explicitly SSX2A requires further investigation to determine whether the high level of background at score 2 can be reduced with better blocking of non-specific sites. This may require the use of different SSX2 antibodies or an improved staining protocol.
    • Development and charaterisation of 3 dimensional culture models for zebrafish (Danio rerio) skeletal muscle cells

      Vishnolia, Krishan Kumar (University of BedfordshireUniversity of Bedfordshire, 2013-09)
      Zebrafish (Danio rerio) have been extensively used over the past two decades to study muscle development, human myopathies and dystrophies, due to its higher degree of homology with human disease causing genes and genome. Despite its unique qualities, zebrafish have only been used as an in-vivo model for muscle development research, due to the limitations surrounding lack of a consistent isolation and culture protocol for zebrafish muscle progenitor cells in-vitro. Using different mammalian myoblast isolation protocols, a novel and robust protocol has been developed to successfully isolate and culture zebrafish skeletal muscle cells repeatedly and obtain differentiated long multi nucleated zebrafish myotubes. Commitment to myogenic lineage was confirmed by immuno-staining against muscle specific protein desmin, and expression pattern of different genetic markers regulating myogenesis. In order to recapitulate the in-vivo bio-physiological environment for zebrafish skeletal muscle cells in-vitro, these cells were successfully cultured in tissue engineered three dimensional (3D) constructs based on fibrin and collagen models. Maturation of tissue engineered collagen and fibrin based constructs was confirmed using the basic parameters described in the literature i.e. collagen three times greater contraction from the original width (Mudera, Smith et al. 2010) and fibrin constructs tightly coiled up to 4mm of diameter (Khodabukus, Paxton et al. 2007). In-vitro characterisation of zebrafish skeletal muscle cells showed hypertrophic growth of muscle mass compared to hyperplasic growth in-vivo as suggested for fish species in literature (Johnston 2006), which is different from human and other mammals. Comparative analysis of zebrafish muscle cells cultured in monolayer against cultured in 3D tissue engineered constructs showed significant increase in fusion index, nuclei per myotube (two-fold) and myotubes per microscopic frame (two-fold). Cells cultured in tissue engineered construct closely resembled in-vivo muscle in terms of their unidirectional orientation of myotubes. These tissue engineered 3D zebrafish skeletal muscle models could be used for various purposes such as drug screening, effect of different temperature extremes, studying underlined pathways involved in human diseases; and with further refinements it would potentially replace the need for studies on live fish in these areas.
    • Development of sensor systems for application in cryopreservation

      Jahangir, Jahanbeen (University of BedfordshireUniversity of Bedfordshire, 2014-12)
      This work describes the development, validation and application of sensor systems to monitor phase transition events of cryoprotectant mixtures in samples and cryopreservation profiles and post-thaw recovery of Lactobacillus delbrueckii subsp. bulgaricus CFL1. Ice nucleation and glass transition (Tg) temperatures influence cell viability during cryopreservation. Knowledge of these phase changes for cryoprotectant mixtures is an essential step in optimising cryopreservation protocols for cell survival. Differential scanning calorimetry (DSC) is used to determine Tg, but the expensive nature of such instrumentation limits its widespread use. Cost-effective sensor systems have been designed to monitor ice-initiation and Tg events in small volume samples of cryoprotectants solutions. Tg values were measured for glycerol, sucrose and Me2SO (with and without NaCl supplement and ice-nucleators) in cryotubes and cryostraws, using temperature and screen-printed impedance sensors. The effect of changes to ice-initiation temperature on Tg was also investigated at different cooling and warming rates by using a Grant Asymptote (EF600) controlled rate freezer. The resulting Tg values obtained by single-channel transition monitoring system (TMS 1) were not significantly different from the values obtained by DSC reported in the literature. However multiple channelled transition monitoring system (TMS 2) requires further circuit modification and multiple screen-printed temperature probes to study the phase-change temperatures and to determine transition events in more than one sample at a time. The lactic acid bacterium (LAB) Lactobacillus delbrueckii was investigated as a model system to monitor the effect of different cryopreservation protocols on post-thaw cell metabolic activity. An important parameter for monitoring the post-thaw quality of LAB for starter culture preparation is the change in pH of the culture medium during incubation at 40 oC. Glass pH combination electrodes are the most common and widely used sensors. However, they are fragile, must be conditioned before use and are not disposable. An alternative to conventional glass electrodes are screen-printed carbon-metal electrodes. Different percentage mixtures of ruthenium and antimony pastes were tested and 54.5% carbon-antimony electrodes gave the best sensitivity and consistency in potentials at fixed pH with a screen-printed salt-bridged Ag/AgCl reference. LAB cultures were cryo-preserved at very rapid, moderate and very slow cooling rates and their post-thaw metabolic activity after overnight incubation in MRS broth was determined using screen-printed pH electrodes. Back to back testing with conventional glass pH sensors was performed to compare responses. Results indicated that early ice-initiation (by means of nucleators) prevents the cells from extensive dehydration (during cooling) and enables maximum post-thaw recovery after incubation (due to equilibrium ice formation and ice melting). In future, screen-printed pH sensors require development with integrated salt-bridged Ag/AgCl reference to make it robust in signalling response. The availability of low cost, disposable, non-fragile sensors and sensor systems to monitor transition events allows the determination of Tg of cryopreservation media during both cooling and warming cycles. A combined screen-printed (impedance + temperature) sensor is proposed for this purpose. A combined screen-printed (pH + reference) sensor would allow the monitoring of metabolic activity in post-thaw and fresh starter cultures of LAB. At present the salt-bridged pH reference is manually attached to the screen-printed pH working electrode but it requires further modifications to the method of attachment. The two sensor systems would enable optimisation of cryopreservation protocols for LAB and could enable such measurements to become routine at commercial scale.