Despite high remission rate in acute myeloid leukaemia (AML) after chemotherapy, relapse of the underlying disease remains a major challenge and one of the most frequent causes of treatment failure. In this study, the presence of leukaemiaassociated phenotypes (LAPs) was first studied retrospectively using our standard diagnostic protocol with 3-colour flow cytometry. LAPs were present in 54 (64%) of 84 AML patients analysed between 2002 to 2004. The presence of LAPs was correlated with failure to respond to induction chemotherapy (p <0.05) in univariate analysis. Presence of LAPs was shown to be an independent predictor for failure to respond to induction chemotherapy with a relative risk ratio of 1.6 (p < 0.05, 95% CI, 1.0-2.6) in multivariate analysis. Subsequently, in a prospective study, we used 5-colour multiparametric flow cytometry (MFC) for detection of LAPs to determine if LAPs could be detected in a greater proportion of leukaemic patients and minimal residual disease (MRD) detection could therefore be applied in more patients. In 54 consecutive, newly diagnosed AML patients from 2005 to 2007, LAPs were identified in 51 (94%). Thus, MRD studies were potentially applicable to virtually all patients. The sensitivity and specificity of MFC technique was improved by analysing 10 normal and 5 regenerating bone marrows (BM) for the presence of these LAPs and by determining maximum log difference (LD). CD7, CD19, CD2, CD11b and CD56 were the most sensitive and reliable markers for MRD studies. LAPs were rarely detected in either normal or regenerating BMs. Through dilutional experiments from 50% LAPs to 0.001%, it was determined that 1 leukaemic in 104 and 105 normal cells could be detected using the improved techniques. Of the 54 patients, 31 received chemotherapy, with 27 achieving complete remission (CR). Two were LAP negative and thus 25 were evaluable for MRD post induction and 22-post consolidation chemotherapy. Detection of MRD >0.15% was able to distinguish between two groups of patients according to relapse status. Although, the number of patients was small, detection of MRD post induction > 0.15% was shown to be an independent predictor of adverse prognosis for both relapse free survival (RFS) and overall survival (OS) in a multivariate analysis [p = 0.037 and 0.026, 95% CI (1.1-20.5 and 1.2-22.2), hazard ratio 4.7 and 5.2 respectively]. Post consolidation, there was a trend for patients with higher MRD values to show shorter RFS (p = 0.06). MFC using 5-colour allows us to detect LAPs in virtually all AML patients and our preliminary results suggest the technique is a suitable approach for MRD analysis. However, 5-colour MFC is technically challenging, resource intensive, and may not be feasible in a routine diagnostic laboratory. This led us to assess whether we could identify other potential markers for LAPs. Interleukin-3 alpha receptor- chain IL-3_ (CD123) has been suggested to be a marker of leukaemic stem cells (LSC). These cells are thought to be responsible for initiating and maintaining leukaemic cell growth post chemotherapy and hence to give rise to relapse of the disease. Therefore, we analysed 34 AML patients for expression of CD123 in the blast population and defined a population containing leukaemic stem cells using the immunophenotypic markers CD123+/CD34+/CD38-. Thirty-two (94%) of AML patients expressed CD123. We then used a molecular marker to determine whether CD123 expression was confined to the LSC. Thirtynine patients were screened for the presence of FMS-like tyrosine kinase 3 - internal tandem duplication (FLT3/ITD) as the most common molecular abnormality in AML patients. Of those, 12 (31%) were FLT3/ITD positive. In seven of them, CD34+/CD38-/CD123+ and CD34+/CD38-/CD123- populations were sorted to homogeneity by Fluorescence Activated Cell Sorting (BD FACSAriaTM Cell Sorter) and tested for FLT3/ITD. In six of seven patients with FLT3/ITD positive AML, we could not detect the mutation in the CD34+/CD38-/CD123- fraction, but the mutation was detected in the CD34+/CD38-/CD123+ fraction in all seven patients. This novel finding demonstrates that, the oncogenic event occurs in CD123 positive cells, thus supporting the concept that CD123 is a marker of the LSC in CD123 positive AML. This observation suggests novel treatment approaches employing surface marker CD123-targeting antibodies may be of use in the treatment of AML. In conclusion, we demonstrate that using five-colour MFC improves LAP detection in AML and enables MRD studies using immunophenotyping to be applied to virtually all AML patients. Additionally, it increases the sensitivity of the technique for detecting LAP populations. Moreover, evaluation of MRD post induction chemotherapy is the most sensitive time point for detection of MRD, with MRD levels >0.15% predicting relapse and worse prognosis. As an alternative to using individualised LAPs specific to each patient, CD34+/CD38-/CD123+ cells may in the future serve as a better marker for MRD studies. This marker identifies the putative LSC, which is responsible for regrowth of leukaemia and relapse of the disease. Thus, instead of looking at whole “blast” population which results in huge data analysis and interpretation for the different LAPs which may have different underlying biology, it may be more informative to look at the frequency of LSC after achieving CR using CD34+/CD38-/CD123+ as the single LAP for MRD studies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317088 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Karlstad : Karlstads universitet, 2008. / Härtill 4 uppsatser.

Myeloid precursor cell reprogramming into a myeloid-derived suppressor cell (MDSC) contributes to high mortality rates in mouse and human sepsis. S100A9 mRNA and intracellular protein levels increase during early sepsis and remain elevated in Gr1+CD11b+ MDSCs after pro-inflammatory sepsis transitions to the later chronic anti-inflammatory and immunosuppressive phenotype. The purpose of this study was to determine whether intracellular S100A9 protein might sustain Gr1+CD11b+ MDSC repressor cell reprogramming during sepsis. We used a chronic model of sepsis in mice to show that S100A9 release from MDSCs and circulating phagocytes decreases after early sepsis and that targeting the S100a9 gene improves survival. Surprisingly, we find that intracellular S100A9 protein translocates from the cytosol to nucleus in Gr1+CD11b+ MDSCs during late sepsis and promotes expression of miR-21 and miR-181b immune repressor mediators. We further provide support of this immunosuppression pathway in human sepsis. This study may inform a new therapeutic target for improving sepsis outcome.

Indiana University-Purdue University Indianapolis (IUPUI) / Acute Myeloid Leukemia (AML) is the most common hematologic malignancy. Malglycemia is a disorder of glucose metabolism and includes hyperglycemia, hypoglycemia and the combination of hyperglycemia and hypoglycemia. Malglycemia has been shown to occur frequently during hospitalization among critical care patients and has been associated with increased risk of sepsis and mortality. Little is known, however, about the prevalence and role of malglycemia on the health outcomes of AML patients hospitalized for initial induction therapy. Malglycemia may be of particular importance to the patient with AML because, researchers have found that malglycemia may promote cellular changes which facilitate the progression of cancer, alter treatment response, and attenuate immune response. The purpose of this study was to determine the prevalence of malglycemia (hyperglycemia, hypoglycemia or the combination) and to examine its role on a comprehensive set of health outcomes (neutropenic days, infection, and septicemia, and sepsis, induction hospital length of stay, complete remission and mortality) in AML patients hospitalized for initial induction therapy. A retrospective cohort study design was used. Records of 103 AML patients, hospitalized for initial induction chemotherapy were reviewed. Results of the study showed that 98% of the AML patients had at least one episode of hyperglycemia, with a prevalence rate of 33% over the entire induction inpatient hospitalization for this population. All patients noted with hyperglycemia also had hypoglycemia and thus, the prevalence rate of hypoglycemia alone could not be determined. Prevalence of the combination of hyperglycemia and hypoglycemia was 1.4 %. Although not statistically significant, a trend was noted for AML patients with hyperglycemia to experience more days with neutropenia, greater numbers of infection, sepsis, septicemia and death (mortality) than patients without hyperglycemia during induction treatment. Patients with the combination of hyperglycemia and hypoglycemia also experienced an increased risk of developing septicemia (p = .025) and sepsis (p =.057). Future studies with larger sample sizes are needed to confirm these findings.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24522号 / 医博第4964号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 萩原 正敏, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Tumour initiation and progression occur due to oncogenic mutations that also contribute to therapeutic resistance in many human tumours. Mutations activating the "PI3K/AKT" signalling pathway and inactivating the "TP53" tumour suppressor gene are common mechanisms that cancer cells require to proliferate and escape pre-programmed cell death. p53 mutant (p53mut) tumours not only fail to respond to DNA damaging therapy, but are also described to promote therapeutic resistance by dominant negative suppression of p53 dependent promoter activity. Our work identifies the crucial interaction between the PI3K/AKT pathway and p53 mutations that regulate treatment sensitivity in tumours. Using a combination of in vitro and in vivo techniques we demonstrate that AKT inhibition promotes reduced cellular levels of p53mut via a novel Nucleophosmin 1 (NPM) mediated regulation of the tumour suppressor p14ARF and promotes re-engagement of cell cycle arrest, senescence and increased sensitivity to ionising radiation in both in vivo and in vitro systems. We show that the PI3K/AKT pathway plays an important role in the regulation of p53mut and inhibitors of this pathway can re-sensitise treatment resistant tumours. This has helped us to simultaneously highlight the cohort of patients where the greatest efficacy may be achieved in clinical practise. We further show that the AKT mediated regulation of NPM that we describe in solid tumours is relevant in Acute Myeloid Leukaemia (AML) with mutated NPM, albeit showing physiologically different effects. This further highlights the necessity for rational treatment planning with the newer targeted agents that inhibit specific signalling pathways in AML patients.

The absence of invariant Natural Killer T cells (iNKT cells) in mice infected with Influenza A virus (flu) has previously been shown to augment the expansion of Myeloid-derived suppressor cells (MDSCs), a bone marrow derived population that powerfully suppress the development of viral and tumor immune responses. Moreover, iNKT cell adoptive transfer into flu-infected mice has been shown to abolish the expansion and flu-induced suppressive activity of the MDSCs in a CD1d- and CD40-dependent manner. However, the mechanisms by which this relatively small subset of T cells influence myelopoiesis and MDSC differentiation remain largely unknown. In this manuscript we firstly better define the MDSCs found in flu-infection as IL-10-secreting neutrophils that can suppress T cell proliferation. We then go further to show that the flu-induced ability to suppress T cells is acquired as early as the level of the Granulocyte-Macrophage Progenitors (GMPs) in the bone marrow and that iNKT cells can not only abrogate the suppressive activity of the IL-10-secreting neutrophils in the periphery but also that of the GMPs by a direct CD1d-dependent GCSF-mediated crosstalk. MDSC expansion has previously been shown to be associated with the expression of the myeloid-related protein S100A9, and the mechanism of action of granulocytic-MDSCs shown to be ARG1-dependent. We built upon both these findings to show that iNKT cells influence the expansion and function of the MDSCs in part by regulating S100A9 and ARG1 expression. Following this we then showed for the first time that the acute phase protein Serum Amyloid A (SAA), shown to increase during flu-infection, has a dual reciprocal role: having the ability to up-regulate S100A9 and ARG1 in myeloid cells and differentiate IL-10-secreting suppressive neutrophils, while simultaneously facilitating the ability of the MDSCs to crosstalk with iNKT cells in a CD1d-dependent GCSF-mediated manner to abrogate the SAA-induced suppressive activity. All together the data highlights the complexity of the immune response and the role iNKT cells play in influencing the differentiation of MDSCs during demand-driven myelopoiesis. More importantly however, it further affirms that research into harnessing the immunomodulatory capacity of iNKT cells remains an exciting prospect in bolstering future vaccination strategies and should continue to be pursued.

Localized Aggressive Periodontitis (LAgP) is an aggressive, early onset form of periodontitis characterized by a unique myeloid cell phenotype. In addition to its bacterial origin, the unique phenotype of the myeloid cell contributes to disease pathogenesis and progression through mechanisms mediating host inflammatory and immune responses. LAgP monocytes synthesize increased levels of the potent proinflammatory lipid mediator, Prostaglandin E2 (PGE2), preferentially differentiate into dendritic cells, and lead to increased IgG2 production. In addition, levels of Platelet Activating Factor (PAF) have shown to be elevated in the gingival tissue and gingival crevicular fluid of subjects with periodontitis. The aim of this study was to further characterize the unique phenotype of the myeloid cell by investigating its role in the increased levels of PAF in periodontitis subjects, examining differences in gene expression of the immune response gene, STATl which is involved in IFN-γ signaling, and by examining the differential expression and function of the scavenger receptor CD36. LAgP monocytes have exhibited decreased activity of the PAF-acetylhydrolase (PAFAH), the catalytic enzyme that breaks down PAF. Since PAF levels are regulated by synthesis and degradation, we hypothesized that synthesis by myeloid cells, monocytes or PMN, also contribute to the increased PAF levels in LAgP. We also hypothesized, based on initial microarray data that myeloid cells have decreased gene expression of STATl and downstream IFNy related genes in LAgP. In addition, based on the initial microarray results, we hypothesized that LAgP monocytes have increased CD36 expression with increased capacity for the binding and uptake of chemically modified versions of LDL. Monocytes were isolated from the peripheral blood of LAgP and NP control subjects over a Ficoll gradient. A radiolabeled PAF assay was used to quantify total PAF synthesis in both resting monocytes and PMN, and in monocytes and PMN stimulated with calcium ionophore A23 187. Quantitative RT-PCR was used to quantify STATl and CD36 gene expression from RNA isolated from adherent monocytes, and CD36 expression and AcLDL (acetylated LDL) uptake was quantified using flow cytometry. Our results indicate that PAF synthesis is increased in LAgP PMN but not in monocytes. LAgP monocytes synthesize less PAF compared to NP control, and their response to calcium ionophore A23 187 (IoA), expressed as fold increase, was blunted. LAgP and NP monocytes did not differ in STATl gene expression as determined by quantitative RT-PCR, and CD36 experiments suggest the possibility that dendritic cells express increased scavenger receptor CD36 than macrophage cells. In conclusion, LAgP myeloid cells are unique in their response to A23 187, and LAgP PMN contribute to increased PAF primarily through synthesis, whereas the LAgP monocytes contributes to elevated PAF through decreased catabolism. STAT1 gene expression did not differ between LAgP and NP monocytes, however this does not rule out the possibility of differential STATl signaling in LAgP monocytes though inhibitory proteins or differential phosphorylation of STATl. Finally, CD36 expression appears from preliminary data to be increased in dendritic cells. These findings add to the current understanding of the unique phenotype of the LAgP monoctye and further experiments will continue to expand our understanding of how unique biology of myeloid cells and their ability to facilitate crosstalk between the innate and adaptive immune system, and the host inflammatory system.

Influenza A virus (IAV) infection is a highly contagious respiratory disease, which can cause substantial morbidity and occasionally death. Invariant natural killer T (iNKT) cells, a subset of CD1d-restricted T lymphocytes, have been identified as important modulators of immunity, mediating both pro- and anti-inflammatory responses. We show that iNKTs play an important role for the generation of protective innate and adaptive immune responses to IAV, and enhance heterotypic immunity to influenza virus following vaccination with a novel pseudotyped virus, S-FLU, which lacks HA expression. iNKT-deficient mice (J&alpha;18^-/-) showed increased susceptibility and lung pathology following IAV infection, which correlated with an exaggerated accumulation of inflammatory monocytes and neutrophils in the lung. Consistent with these findings, we demonstrated in IAV-infected CD1d^-/-:CD1d^{&plus;/&plus;} mixed bone marrow chimeric mice, that the lack of CD1d expression on myeloid cells purified from the lungs of IAV-infected mice significantly increased expression of genes linked to cell activation, survival and polarisation between pro- and antiinflammatory responses. We extended these results by examining the role of chemokine signalling during IAV infection, and identified a novel role for fractalkine (CX3CL1) and its receptor (CX3CR1) in innate immune cell recruitment. The use of CX3CR1-iNKT cell double knockout mice revealed that, although upregulated in J&alpha;18^-/- mice, CX3CR1-CX3CL1 signalling is not required for cell migration during exacerbated IAV-responses. Finally, we showed that iNKT-deficient mice displayed reduced longevity of peripheral IAVspecific CD8^&plus; T cells following S-FLU vaccination, compared with wild-type mice. S-FLU vaccination protected mice following 5 day heterotypic challenge, however vaccinated mice exhibited reduced viral clearance, and importantly a significant reduction in IAV specific memory T cell response, suggesting a possible role of iNKT cells during T cell priming in modulating the lifespan of antigen-specific T cell responses. Although additional experiments are warranted, these results suggest that harnessing iNKT cells should be considered to modulate the innate and adaptive immune response to optimise heterotypic vaccine design and for therapeutic intervention against acute influenza infection.