The failure of the immune system to provide efficient protection against tumour cells has been considered as a major issue in immunology. It is now well established that inadequate function of the host immune system is one of the main mechanisms by which tumours escape from immune control contributing to the limited success of cancer immunotherapy. Several cell populations have been described which display immunosuppressive properties and may impede tumor-specific immunity. Among them, GR1+CD11b+ immature myeloid suppressor cells and CD4+CD25+ regulatory T cells seem to play an important role. These cells accumulate in the spleens of tumour bearing mice and patients with cancer and contribute to immunosuppression by inhibiting the function of CD8+ T cells and/or by promoting tumour angiogenesis. The aim of our work was to define the mechanisms by which a single dose of cyclophosphamide (CTX), a chemical agent commonly used in chemotherapy treatment, induces the rejection of established P815 mastocytoma. Our data show that CTX treatment leads to the selective loss of GR1medCD11b+ splenic myeloid cell producing TGF-â, a cytokine which is known to suppress antitumoral response. Furthermore, injection of CTX causes a decrease in the number of naturally occurring regulatory T cells (CD4+CD25+Foxp3+) in the spleen and the tumor. Finally, CTX treatment induces the differentiation of GR1highCD11b+ splenic myeloid cells into mature GR1highCD11b+CD11c+ (possibly dendritic cells?) which express high levels of CD11c, MHC class II and CD86 molecules. Of note, these cells are mainly detected in tumour necrosis areas. Collectively, these results suggest that CTX prevents suppressive mechanisms and induces a population of CD11c+ myeloid cells which may present tumor antigens and activate T lymphocytes, an hypothesis in line with the requirement for CD4+ cells in CTX-induced long term resistance.

I have studied the calpain system in acute myeloid leukemia using the 32D and 32Dkit cell lines. Specifically, I characterized the calpain system in the cell lines, and performed calpastatin overexpression and knockdown studies. I found that calpain activity is elevated in the 32D and 32Dkit cells, and calpain inhibition causes apoptosis. Both μ- and m-calpain contribute to the calpain activity in these cell lines. The 32Dkit cells have higher calpain activity than the 32D cells, which I have shown is partially attributed to basal ckit activation. Calpastatin was present in both cell lines, but exists mainly in a degraded form. Calpastatin overexpression lowered calpain activity and provided a growth disadvantage to the 32Dkit cells, but had no effect on 32D cells. Calpastatin knockdown caused a significant increase in calpain activity in the 32D cells, which changed the cell cycle distribution but had no other major effects.

published_or_final_version / Medicine / Master / Master of Philosophy

I have studied the calpain system in acute myeloid leukemia using the 32D and 32Dkit cell lines. Specifically, I characterized the calpain system in the cell lines, and performed calpastatin overexpression and knockdown studies. I found that calpain activity is elevated in the 32D and 32Dkit cells, and calpain inhibition causes apoptosis. Both μ- and m-calpain contribute to the calpain activity in these cell lines. The 32Dkit cells have higher calpain activity than the 32D cells, which I have shown is partially attributed to basal ckit activation. Calpastatin was present in both cell lines, but exists mainly in a degraded form. Calpastatin overexpression lowered calpain activity and provided a growth disadvantage to the 32Dkit cells, but had no effect on 32D cells. Calpastatin knockdown caused a significant increase in calpain activity in the 32D cells, which changed the cell cycle distribution but had no other major effects.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2009. / Includes bibliographical references (p. 140-165).

Epigenetic regulators are commonly mutated in cancer. Activating mutations and overexpression of EZH2 occur in lymphoma and other malignancies, while loss-of-function mutations are found in myeloid malignancies. This thesis is a study of this apparent contradiction, examining the importance of cellular context for Ezh2 loss during the evolution of a single malignancy, Acute Myeloid Leukaemia (AML). This work demonstrates diametrically opposite functions for Ezh2 at early and late stages during the evolution of leukemias generated by disparate AML-associated fusion-oncogenes. Ezh2 functions as an oncogene that may be therapeutically targeted during disease maintenance. In contrast, Ezh2 behaves as a tumour suppressor gene during AML induction. Integrated genomic analysis demonstrates that largely different expression programmes are de-repressed during disease induction and maintenance following Ezh2 loss. Studying disease induction, Ezh2 represses a subset of bivalent promoters that resolve towards gene activation upon Ezh2 loss, inducing a feto-oncogene programme including genes like Plag1, whose overexpression phenocopies Ezh2 loss to accelerate AML induction in mouse models. This data highlights the importance of cellular context and phase of disease evolution for Ezh2 function. Moreover, the work herein identifies EZH2 as a potential target in AML, whilst providing reassurance of the safety of this therapeutic strategy.

The classical myeloproliferative disorders (MPD), comprising essential thrombocythaemia (ET), polycythaemia vera (PV) and idiopathic myelofibrosis (IMF), are clonal premalignant haematopoietic neoplasms associated with activating mutations in signalling pathway molecules and a variable tendency to develop acute myeloid leukaemia (AML). This thesis examined genotype-phenotype associations of JAK2 and MPL mutations, the presence of clonal diversity in the MPD and the genetic events associated with progressive disease. Mutations in MPL were identified in 4% of ET and 7% of IMF but not in PV. Three different acquired MPL mutations were identified, one of which had been reported as an inherited allele. Although MPL mutations did not delineate a distinct clinical or histopathological subtype of ET, molecular testing provides an important new tool in the diagnostic armamentarium. Clones homozygous for the JAK2 V617F mutation were identified in female but not male patients with ET, suggesting that gender differences may be important in the determination of disease phenotype. In patients with two acquired genetic alterations, a signalling pathway mutation and a cytogenetic abnormality were usually present within the same clone. By contrast, coexistence of two signalling pathway mutations indicated the presence of biclonal disease that in two patients had arisen independently and not from a shared founder clone. RAS mutations were identified as potential cooperating events in patients with JAK2 or MPL mutant IMF. In patients developing AML following a JAK2 V617F-positive MPD, those with V617F-positive leukaemia had progressed via an accelerated phase of disease and harboured acquired alterations of RUNX1 or EVI1. V617F-negative leukaemias tended to follow directly from ET or PV, and loss of the JAK2 mutation by reversion to wild-type due to mitotic recombination, gene deletion or gene conversion was excluded. The thesis concludes with a discussion of how clonal heterogeneity can be integrated into current models of MPD disease pathogenesis.

A leukemia Differentiation Factor (DF), that induced differentiation of rat leukemia MIA C51 cells, was isolated from endotoxin-stimulated rat lung conditioned media.

Background: HIV/AIDS remains a global concern that, although manageable using anti-retroviral therapy (ART), is still eluded by a cure with paucity of knowledge regarding its acquisition and spread especially through the male genital tract (MGT)1–4. Several authors have shown the human foreskin to be an effective mucosal effector site with heterogenous populations of innate and adaptive immune cells, that are permissive to HIV infection5–8. In support of this, medical male circumcision (MMC), has been reported to confer up to 60 % risk reduction in HIV acquisition9–17. Most studies have focused on investigating blood lymphoid immune cells and their interaction with HIV-1, this study sought to elucidate the myeloid cell composition of the inner and outer foreskin, and to investigate the susceptibility of these cells to ex vivo HIV infection by (i) Isolating migratory and non-migratory Langerhans cells (LCs) and “macrophage-like” cells from the foreskin epidermis (ii) Immunophenotyping and characterising foreskin LCs and “macrophage-like” cells using CD4+CCR5+ as proxy for HIV susceptibility, HLA-DR+CD80/86+ for maturation, and the mannose receptor, DC-SIGN and Siglec-1 as HIV attachment factors and (iii) Investigating the HIV susceptibility of foreskin epidermal cells using an optimised ex vivo pluricellular foreskin infection model of suspension cells. Methodology: Foreskin specimen were obtained from 60 seronegative adult South African men (aged 18-35 years) undergoing voluntary medical male circumcision (vMMC). Migratory and non-migratory foreskin cells were isolated from the inner and outer foreskin using spontaneous migration and enzymatic digestion of remnant epidermal tissue respectively, and subsequently immunophenotyped using multiparameter flow cytometry (n=31). The optimal HIV infection model was determined through assessment of different infection models inclusive of i) epidermal sheets, ii) foreskin explants and iii) pluricellular suspension cells (n=5). Using the ex vivo pluricellular foreskin infection model of suspension cells (n=17), Subtype C transmitted founder (T/F) and chronic infection derived (CC) infectious molecular clones (IMCs) were used alongside Subtype B NL4-3 IMCs with CCR5, CXCR4 and BaL envelopes. The extent of HIV infection was quantified by measurement of p24 in different immune cell subsets over a time-course. The different HIV infected cell subsets were characterized using CD45, CD207, CD1a, CD11c, CD14, CD3, HLA-DR, CD80/86, CD209, CD206, CD169, CD4 and CCR5. Results: Foreskin myeloid cells contained a rare population of LCs (1.11 % ± 1.02 %;) that was predominantly migratory (p = 0.0084) and “macrophage-like” cells (9.87 % ± 9.64 %) that, in addition to being 8-fold more abundant (p

No description available.