Search

Recent years have seen important advances in the area of T cell-based therapy for human malignancies. Epstein Barr virus-associated tumours like Hodgkin’s Lymphoma provide important models in this field. If a T cell-based therapy is to be effective however, T cells must be capable of trafficking to the tumour. To address this, the molecular mechanisms of T cell homing to Hodgkin’s Lymphoma were explored. Chemokine and adhesion receptors were examined on infiltrating T cells. CXCR3, CXCR4 and CCR7 were expressed on major T cell populations with CXCR5, CXCR6, CCR4 and CCR5 on minor populations. Tumour cells expressed CXCL10, CXCL12 and CCL21. Vessels expressed ICAM-1, CXCL12, CCL17 and CCL21. Tumour cell lines secreted factors that mediated chemotaxis of lymphoblasts in vitro and TIL demonstrated chemotaxis to CXCL12 but not CCL17. VAP-1 was expressed on vessels and a tissue-binding assay was evaluated to examine VAP-1 function. T cell clones generated as part of an existing clinical trial of adoptive T cell therapy were found to express a polarised Tc1 phenotype (CXCR3, CXCR6 and ccr5), which was typically independent of target antigen specificity, CD4/CD8 and donor status. However, lack of CCR7 expression and an inability to capture to VCAM-1 in a chemokine dependent manner suggested that clones expanded in vitro using existing protocols may be inefficient at trafficking to tumour tissue and thus may require modification of their homing phenotype.

This thesis explores research published between 2013-2017 and focuses on welfare issues in Italian pig production. This sector is characterized by animals (heavy pigs) slaughtered for dry-cured products at a minimum of nine months and 160 kg (compared to 110-115 kg of other EU systems). The first set of studies investigated the feasibility of avoiding tail docking under different husbandry conditions (Trial A and Trial B), besides the benefit of providing straw enrichment by racks (Trial A). Surgical tail docking within the first week of life is the routine solution in several EU countries to prevent tail biting, an abnormal behaviour causing stress, pain and tail injuries. In Trial A, tail presence did not significantly increase the risk of tail lesions, the level of acute phase proteins (i.e. blood markers of inflammation), or impair the health status (mortality, lung and gastric lesions) over the 30 weeks of fattening. Small amounts of straw (70 g/day per pig) increased the motivation for pigs to explore the environment, reduced serum haptoglobin (i.e. the inflammatory state) and reduced the risk of tail biting (at weeks 3, 9, 18) and ear biting (at weeks 3, 9). At slaughter, the straw group revealed a gastric ulcer risk ~ 70% lower than the one without straw (OR: 0.27). Males were at higher ulcer risk than females (OR: 1.52). In Trial B, tail presence had no effect on blood markers, conflicts, or ear and tail biting behaviours, both at the weaner and fattening phase. At fattening, however, undocked animals showed a higher prevalence of mild tail lesions (P<0.01) and a lower frequency of belly nosing behaviour (P<0.05). Blood samples taken from the animals in trial A and trial B were found to have a variable degree of spurious haemolysis. The release of free haemoglobin can bias the quantification of several analytes. Therefore, a further study evaluated the effect of physical haemolysis in 3 aliquots (on a scale of 1+ to 3+) of 30 non-haemolytic sera, in order to assess the threshold of acceptability for a panel of 27 blood analytes at increasing levels of haemolysis. A further issue explored was the assessment of heavy pig welfare at the time of slaughtering, in a European project which enabled the gathering of data with other Countries (Spain, Portugal, Finland, Brazil). Welfare Quality Protocol® was applied in nine Italian abattoirs, providing information for reference values of several animal-based indicators. Finally, the impact of sexual maturity in heavy female pigs was investigated, on the basis of previous studies on light pigs. They reported mounting and agonistic behaviour to affect animal welfare in both males and females, due to sexual activity. In the study, a reduction was found (P<0.05) in immunocastrated vs. entire females for aggressive interactions, haptoglobin levels, serum cortisol levels and back lesions at given timepoints throughout fattening. There was no effect on slaughter performances (back fat thickness and percentage of lean tissue). Taken together, the outcomes of all these studies highlighted the presence of specific welfare concerns in the Italian pig sector, due to the heavier final live weight (problematic during certain slaughtering phases) and the achievement of sexual maturity (leading to increased aggression of females). Similarly to other EU systems, tail biting was manageable with proper enrichment material; it occurred mostly at an early age and under poor health conditions. The studies have also brought practical implications in routine blood testing (by overcoming the unsuitability of blood measures due to haemolysis) and in investigating the role of gastric ulcers as an innovative stress marker.

The extensive genetic variation in the lipooligosaccharide (LOS) core biosynthesis gene cluster, a majority of which occurs in the LOS outer core biosynthesis gene content present between lgtF and waaV, have led to the development of a classification system; with 8 classes (I-VIII) for Campylobacter coli (C. coli) LOS region and four groups (1-4) with 23 classes (A-W) for Campylobacter jejuni (C. jejuni) LOS region. The aim of this work was to characterise the C. jejuni and C. coli LOS biosynthesis loci with special emphasis on their classes’ distribution and also to determine the role of LOS in mediating the host immune response. Analysis of the LOS locus gene content in 50 C. jejuni clinical isolates and 703 publicly available C. jejuni genome sequences revealed that the class B (Group 1) was the most abundant LOS locus class in C. jejuni. Two novel C. jejuni LOS types were identified from the GenBank database which may have arisen due to interspecies and intraspecies LOS gene recombination. In silico analysis of LOS locus gene content in 564 publicly available C. coli genome sequences identified previously unknown LOS inner core biosynthesis genes; all were located between waaF and gmhA and occurred in 5 C. coli LOS locus types (I, II, III, V, VIII). It was also determined that class III is the most abundant LOS locus type in C. coli and the environmental niches are the major reservoirs of C. coli. Moreover, this work highlighted that live and heat killed cells of both C. jejuni and C. coli, as well as, extracted LOS activate the NLRP3 [Nucleotide binding oligomerisation domain (NOD) like receptors with pyrin domain–containing 3] inflammasome dependent signalling in a human monocytic cell line, THP-1. However, C. jejuni 11168 LOS mutant live cells and its modified LOS with altered lipid A and lack of LOS core oligosaccharides both stimulated significantly reduced Caspase-1 and IL-1β compared to the wild-type 11168 live cells and LOS, which indicated that variation in LOS structure can alter NLRP3 inflammasome activation. This work extends the understanding of the Campylobacter LOS locus classification system and determines that LOS plays an important role in the development of host immune response during Campylobacter infection.

Epithelial ovarian cancer (EOC) represents the fifth most common cause of cancer mortality among women worldwide and accounts for the highest fatalities amongst gynaecological malignancies. The dysregulation of calcium-dependent peptidyl arginine deiminase 2 (PADI2) plays a key role in the tumorigenesis of several cancers; however, the role of PADI2 in the pathogenesis of EOC is yet to be investigated. Using RNA-seq and microarray data from primary serous ovarian cancers (The Cancer Genome Atlas data set) combined with survival data, the expression of PADI2 was assessed using each platform (n=262 and n=564). Kaplan-Meier analysis and Log-rank tests showed an association of PADI2 mRNA expression with overall survival using both platforms (RNA-seq cohort p=0.008 and microarray cohort p=0.0112). Low expression of PADI2 was associated with improved survival. Expression studies were used to examine the overexpression and knockout (CRISPR/Cas9 editing) of PADI2 expression, respectively, in the human-derived high-grade serous OVCAR-4 and mouse-derived EOC ID8-Luc2 cell lines. In OVCAR-4 cells, PADI2 overexpression reduced proliferation (22%) and cellular aggregation (94%), while increasing apoptosis (34%) and autophagy (38%), in a Ca2+- and citrullination-dependent manner, at 72 hours. PADI2 overexpression also induced cisplatin cytotoxicity by 10% at 72 hours. In ID8-Luc2 cells, PADI2 overexpression significantly decreased cisplatin cytotoxicity by 24%, independently of exogenous Ca2+ supplementation or induced citrullination, at 72 hours. In addition, qRT-PCR validation of TCGA gene co-expression data indicated that PADI2 overexpression correlated with expression of a number of genes. This was confirmed in functional studies where there was a 1.69-fold increase in ARHGEF10L and 0.75-fold decrease in FZD5 expression upon PADI2 overexpression. In this thesis, The Cancer Genome Atlas expression studies of PADI2 showed that higher PADI2 confers decreased survival of EOC patients. Conversely, PADI2 overexpression in vitro, induced apoptosis/autophagy and decreased proliferation/cellular aggregation possibly via deregulating FZD5 and ARHGEF10L. Collectively, this work suggests that PADI2 may serve as a potential therapeutic target in human EOC. Codon based selection analyses were used to test for evidence of positive selection in PADI2. The phylogenetic maximum likelihood analysis of PADI2 orthologues retrieved from 31 species indicated that there was no evidence of positive selection in PADI2 codons, but numerous residues were under evolutionary constraint.

Properdin, as the only positive regulator, amplifies complement activation and has been implicated in the tumour response in human lymphoma and carcinoma. This project investigated the role of properdin in a syngeneic orthotopic tumour model in mice engineered to be properdin deficient and their wildtype controls. The in vitro part of the project used macrophages differentiated from bone marrows of these mice and stimulated with conditioned medium of a syngeneic mouse melanoma cell line, B16F10. In comparison with macrophages from wildtype mice, macrophages from congenic properdin deficient mice showed skewing towards M2 profile, encompassing mRNA expression for genes involved in arginine metabolism, production of type 2 cytokines, and relatively lower surface expression of molecules needed for antigen presentation suggesting that properdin insufficiency promotes a tumour environment that helps the tumour evade the immune response. The in vivo part of this project established the immune profile of tumour bearing mice. MDSCs, C5a, CCL2, TGF-β and mRNA FOXP3 were significantly less abundant in tumours of properdin deficient compared to wildtype mice. Protein levels for CCL2, a chemokine associated with tumour progression, was higher in wildtype tumour bearing mice. In spleen, MDSCs, regulatory T cells, M2 macrophages (CD206+F4/80+) and TGF-β were decreased significantly in properdin deficient compared with wildtype mice (control) after subcutaneous injection with B16F10 cells, indicating that properdin may contribute to the accumulation of MDSCs in spleen, as well as to the migration of these cells into tumours. LDLR-/- mice group were analysed in parallel because of their inherently greater M2 skewing. An advanced bioimaging technique was applied to some of the experimental animals. Analysis of the level of serum properdin in response to treatment in a group of patients with pancreatic cancer showed high levels in this group. In conclusion, this project identified complement properdin as significant in the macrophage response to conditioned melanoma cell medium, in the composition of the tumour microenvironment and systemic response to tumour in vivo and as an acute responder to chemotherapy in patients with pancreatic cancer.