Lam Audrey Carmen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 166-195). / Abstracts in English and Chinese. / Acknowledgements --- p.vi / Publications --- p.vii / Abbreviations --- p.x / Abstract --- p.xiii / Chapter Chapter One - --- Introduction --- p.1 / Chapter Section 1.1 --- Hematopoietic Stem Cells --- p.1 / Chapter 1.1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.2 --- Hematopoietic Stem and Progenitor Cells --- p.1 / Chapter Section 1.2 --- Stem Cell Transplantation --- p.4 / Chapter 1.2.1 --- Stem Cell Transplantation --- p.4 / Chapter 1.2.2 --- Sources of Hematopoietic Stem Cells for Transplantation --- p.4 / Chapter 1.2.3 --- Cord Blood as a Source of Hematopoietic Stem Cells --- p.6 / Chapter 1.2.3.1 --- Advantages of Cord Blood Transplant --- p.6 / Chapter 1.2.3.2 --- Disadvantages of Cord Blood Transplant --- p.7 / Chapter Section 1.3 --- Ex Vivo Expansion --- p.8 / Chapter 1.3.1 --- Optimization of Expansion Conditions --- p.10 / Chapter 1.3.1.1 --- CD34+ Cell Selection --- p.10 / Chapter 1.3.1.2 --- Cytokines --- p.11 / Chapter 1.3.1.2.1 --- Thrombopoietin --- p.12 / Chapter 1.3.1.2.2 --- Stem Cell Factor --- p.14 / Chapter 1.3.1.2.3 --- Flt-3 Ligand --- p.15 / Chapter 1.3.1.2.4 --- Granulocyte-Colony Stimulating Factor --- p.16 / Chapter 1.3.1.2.5 --- Interleukin-3 --- p.17 / Chapter 1.3.1.2.6 --- Interleukin-6 --- p.18 / Chapter 1.3.1.2.7 --- Comparison of Flt-3 Ligand and Stem Cell Factor --- p.20 / Chapter 1.3.1.3 --- Culture Medium --- p.20 / Chapter 1.3.2 --- Mannose-Binding Lectin --- p.22 / Chapter 1.3.3 --- Ex Vivo Expansion for Clinical Transplantation --- p.23 / Chapter Section 1.4 --- Non-Obese Diabetic/Severe Combined Immunodeficient Mouse Transplantation Model --- p.29 / Chapter Chapter Two - --- Objectives --- p.32 / Chapter Chapter Three - --- Materials and Methodology --- p.34 / Chapter Section 3.1 --- Collection of Cord Blood Samples / Chapter Section 3.2 --- Cryopreservation and Thawing of Cord Blood --- p.34 / Chapter Section 3.3 --- Enrichment of CD34+ Cells --- p.35 / Chapter Section 3.4 --- Ex Vivo Expansion --- p.38 / Chapter 3.4.1 --- Effects of Flt-3 Ligand and stem Cell Factor on the Expansion of Megakaryocytic Progenitor Cells --- p.39 / Chapter 3.4.1.1 --- Ex Vivo Expansion of Cord Blood CD34+ Cells with Flt-3 Ligand or Stem Cell Factor --- p.39 / Chapter 3.4.1.2 --- Flt-3 Receptor Assay --- p.40 / Chapter 3.4.2 --- Effects of Mannose-Binding Lectin on the Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells --- p.41 / Chapter 3.4.2.1 --- Ex Vivo Expansion of Cord Blood CD34+ Cells with Mannose-Binding Lectin --- p.41 / Chapter 3.4.2.2 --- Effects of Mannose-Binding Lectin on the Preservation of Early Stem and Progenitor Cells --- p.41 / Chapter 3.4.2.3 --- Transplantation of Expanded Cells into NOD/SCID Mice --- p.42 / Chapter 3.4.3 --- "Optimization of Culture Duration, Culture Media, Autologous Plasma and Cytokine Combinations for the Preclinical Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells" --- p.42 / Chapter 3.4.3.1 --- "Comparison of Culture Duration, Culture Media and Cytokine Combinations" --- p.42 / Chapter 3.4.3.2 --- Effects of Autologous Cord Blood Plasma --- p.43 / Chapter 3.4.3.3 --- Effects of Flt-3 Ligand and Dosage of Thrombopoietin and Stem Cell Factor --- p.43 / Chapter 3.4.3.4 --- Transplantation of Expanded Cells into NOD/SCID Mice --- p.44 / Chapter Section 3.5 --- Progenitor Colony-Forming Assays --- p.44 / Chapter 3.5.1 --- Colony-Forming Unit Assay --- p.44 / Chapter 3.5.2 --- Colony Forming Unit Megakaryocyte --- p.46 / Chapter 3.5.3 --- Calculations of CFU --- p.46 / Chapter Section 3.6 --- Flow Cytometry Analysis --- p.47 / Chapter Section 3.7 --- Transplantation of Non-Obese Diabetic/Severe Combined Immunodeficient Mice --- p.48 / Chapter Section 3.8 --- Assessment of Human Cell Engraftment in Transplanted NOD/SCID Mice --- p.49 / Chapter 3.8.1 --- Flow Cytometry Analysis --- p.49 / Chapter 3.8.2 --- PCR Analysis --- p.50 / Chapter Section 3.9 --- Statistical Analysis --- p.52 / Chapter Chapter Four - --- Effects of Flt-3 Ligand and Stem Cell Factor on the Expansion of Megakaryocytic Progenitor Cells --- p.53 / Chapter Section 4.1 --- Results --- p.53 / Chapter 4.1.1 --- Ex Vivo Expansion of CD34+ Cells --- p.53 / Chapter 4.1.2 --- Identification of Flt-3 Receptors --- p.55 / Chapter Section 4.2 --- Discussion --- p.55 / Chapter Chapter Five- --- Effects of Mannose-Binding Lectin on the Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells --- p.68 / Chapter Section 5.1 --- Results --- p.68 / Chapter 5.1.1 --- Ex Vivo Expansion of CD34+ Cells with Mannose-Binding Lectin --- p.68 / Chapter 5.1.2 --- Effects of Mannose-Binding Lectin on the Preservation of Early Stem and Progenitor Cells --- p.72 / Chapter 5.1.3 --- Transplantation of Expanded Cells into NOD/SCID Mice --- p.75 / Chapter Section 5.2 --- Discussion --- p.76 / Chapter Chapter Six - --- "Optimization of Culture Duration, Culture Media, Autologous Plasma and Cytokine Combinations for the Preclinical Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells" --- p.111 / Chapter Section 6.1 --- Results --- p.111 / Chapter 6.1.1 --- Kinetics of Expansion --- p.111 / Chapter 6.1.2 --- Assessment of Culture Media --- p.113 / Chapter 6.1.3 --- Effects of Autologous Cord Blood Plasma --- p.115 / Chapter 6.1.4 --- Effects of Granulocyte-Colony Stimulating Factor --- p.117 / Chapter 6.1.5 --- Effects of Interleukin-6 --- p.118 / Chapter 6.1.6 --- Effects of Increased Dosage of Thrombopoietin and Stem Cell Factor --- p.119 / Chapter 6.1.7 --- Effects of Flt-3 Ligand --- p.120 / Chapter 6.1.8 --- Transplantation of Expanded Cells into NOD/SCID Mice --- p.121 / Chapter Section 6.2 --- Discussion --- p.123 / Chapter Chapter Seven- --- General Discussion and Conclusion --- p.163 / Bibliography --- p.166

In dieser Arbeit wurden die Daten von 90 konsekutiven Patienten mit Multiplem Myelom, welche im Zeitraum vom 11.04.2005 bis zum 08.11.2010 mit einer Hochdosischemotherapie und autologer Stammzelltransplantation in den Kliniken Essen Süd behandelt wurden, retrospektiv untersucht und statistisch ausgewertet. Es konnte gezeigt werden, dass ein gutes Ansprechen nach den Induktionstherapien mit einem guten Ansprechen sechzig Tage nach der Transplantation korreliert. Einen Zusammenhang zwischen dem initialen Ausmaß der Endorganschäden und dem Verlauf oder dem Ansprechen der Hochdosischemotherapie mit autologer Stammzelltransplantation konnte nicht gefunden werden. Das kalendarische Alter spielt im Gegensatz zum Allgemeinzustand und den Vorerkrankungen bei der Einschätzung der zu erwartenden Toxizität eine untergeordnete Rolle. Die beiden Hauptfaktoren, die den Verlauf einer Hochdosischemotherapie mit anschließender peripherer Stammzelltransplantation beeinflussten, waren die Dauer der G-CSF Therapie und die Anzahl der übertragenen Stammzellen. Während die unterschiedlich lange G-CSF Gabe (ab Tag „+3“ vs. Tag „+7“) nur zu einer schnelleren Regeneration der Leukozyten führt und keinen relevanten Effekt auf die untersuchten klinischen Parameter Fieber, Dauer der intravenösen Antibiotikatage, Ausmaß der Mukositis und die Aufenthaltsdauer der Patienten hatte, führte die Steigerung der Anzahl der übertragenen Stammzellen zu einer signifikant schnelleren Regeneration von Thrombozyten und Leukozyten, einem Rückgang der Transfusionshäufigkeit an Erythrozyten und einem geringeren Verbrauch an intravenösen Antibiotika. Zusammenfassend ist die G-CSF Gabe ab Tag „+7“ nach Hochdosistherapie ausreichend, eine längere Gabe erbringt keinen relevanten klinischen Vorteil. Zudem sollte auf eine ausreichende Menge an übertragenen Stammzellen geachtet werden. Zur Beurteilung der zu erwartenden Toxizität ist die Anwendung des HCT-CI-Score einfach und praktikabel. / In this study the data of 90 consecutive patients with multiple myeloma were analyzed retrospectively. There is a correlation between a good response after the induction therapy and sixty days after high-dose chemotherapy with autologous stem cell transplantation. There is no correlation between organ damages (CRAB) and the progression or the response of the high-dose chemotherapy with autologous stem cell transplantation. The chronological age is not as important as the general condition and medical history of the patient to expect the toxicity of the high-dose chemotherapy. The two main factors that influenced the process of high dose chemotherapy followed by peripheral stem cell transplantation were the duration of G-CSF therapy and the number of transplanted stem cells. The different duration of G-CSF leads only to a faster recovery of leukocytes with no relevant effect on the investigated clinical parameters temperature, duration of intravenous antibiotic days and the extent of mucositis. The increase of transplanted stem cells results in a significant faster recovery of platelets and leukocytes, a decrease in frequency of transfusion of red blood cells and a reduced use of intravenous antibiotics. In summary, the G-CSF administration from the 7th day after transplantation is sufficient for high-dose therapy, a longer administration provides no relevant clinical advantage. One should take care for having got sufficient quantity of transplanted stem cells.

Medications that treat diseases such as Parkinson???s disease work by regulating dopamine transmission at synapses. Surprisingly, little is known about the mechanisms regulating dopamine release at synapses. In this thesis, we study mechanisms that regulate vesicle recycling in axons and dendrites of dopamine neurons. Key questions we addressed were: (1) Are vesicles in axons and dendrites associated with the same regulatory proteins, and thus by implication the same regulatory mechanisms, as in excitatory neurons; (2) Do vesicles undergo recycling, and (3) if so, are they characterised by a distinct pool size and rate of recycling. To study this, we cultured dopamine neurons and used immunocytochemistry to detect vesicular monoamine transporter 2 (VMAT2) and identify axons, dendrites and synaptic proteins, combined with labelling of recycling vesicles using FM 1-43. Vesicles in axons, but not in dendrites, were associated with presynaptic proteins such as Synaptophysin and Bassoon. We identified two kinds of presynaptic sites in axons: ???synaptic??? (located close to soma and dendrites??? and ???orphan???. The recycling vesicle pool size was smaller at orphan sites than at synaptic sites, and the initial rate of vesicle pool release was also lower at orphan sites. Both synaptic and orphan sites exhibited lower rates of vesicle pool release compared to hippocampal synapses, suggesting functional differences in presynaptic physiology between dopamine neurons and hippocampal neurons. In somatodendritic regions, VMAT2 was localised to the endoplasmic reticulum, Golgi, endosome, and large dense-core vesicles, suggesting that these vesicles might function as a part of the regulated secretory pathway in mediating dopamine release. None of the synaptic vesicle proteins we studied were detected in these regions, although some preliminary evidence of vesicle turnover was detected using FM 1-43 labelling. This thesis provides a detailed analysis of neurotransmitter release mechanisms in dopamine neurons. Our data suggests that presynaptic release of dopamine is mediated by mechanisms similar to those observed in excitatory neurons. In somatodendritic regions, our data suggests that VMAT2 is localised to organelles in secretory pathways, and that distinct mechanisms of release might be present at somatodendritic sites to those present in presynaptic sites. This thesis provides novel methods for analysing vesicle recycling in dopamine neurons, which provides the basis for further studies examining presynaptic function of dopamine neurons in normal brain function, disease, and therapeutic approaches.

Modulation of corneal transplant rejection using gene therapy shows promise in experimental models but the most appropriate vector for gene transfer is yet to be determined. The overarching aim of the thesis was to evaluate the potential of a lentiviral vector for use in human corneal transplantation. Specific aims were: (i) to assess the ability of an HIV-1-based lentiviral vector to mediate expression of the enhanced yellow fluorescent protein (eYFP), and a model secreted protein interleukin-10 (IL10), in ovine and human corneal endothelium; and (ii) to examine the influence of lentivirus-mediated IL10 expression on the survival of ovine corneal allografts. Four lentiviral vectors expressing eYFP under the control of different promoters, were tested: the simian virus type-40 (SV40) early promoter, the phosphoglycerate kinase (PGK) promoter, the elongation factor-1alpha (EF) promoter, and the cytomegalovirus (CMV) promoter. Two lentiviral vectors expressing IL10 were tested: one containing the SV40 promoter and another containing a steroid-inducible promoter (GRE5). Lentivirus-mediated expression in transduced ovine and human corneal endothelium was assessed by fluorescence microscopy, real-time quantitative RT-PCR and ELISA, following alterations of transduction period duration (2–24 hr) and vector dose, as well as in the presence or absence of polybrene or dexamethasone (GRE5 vector). It was also compared to expression mediated by adenoviral vectors. Orthotopic transplantation of ex vivo transduced donor corneas was performed in outbred sheep. Allografts were reviewed daily for vascularisation and signs of immunological rejection. Lentivirus-mediated eYFP expression was delayed in ovine corneal endothelium compared to human. However, in both species the final transduction rate was greater than 80% and expression was stable for at least 14 d in vitro. Lentivirus-mediated expression in ovine and human corneal endothelium was higher with the viral promoters in comparison to the mammalian promoters. A 24 h transduction of ovine corneal endothelium with the lentiviral vector encoding IL10 resulted in expression levels which were increasing after 15 d of organ culture but logarithmically lower than those achieved by adenovirus. Shortening the lentiviral transduction period to 2 h led to a reduction in expression, but the addition of polybrene (40 micrograms / ml) to the transduction mixture restored expression to levels comparable to those attained after a 24 h transduction period. Lentivirus-mediated IL10 expression was higher and more rapid in human corneal endothelium compared to ovine corneas. Dexamethasone-responsive transgene expression was observed in both ovine and human corneal endothelium using the lentiviral vector containing the GRE5 promoter. Lentivirus-mediated expression in ovine corneal endothelium was stable for 28 d in vivo. A modest prolongation of ovine corneal allograft survival (median of 7 d) was achieved by transduction of donor corneas for 2–3 h with the lentivirus expressing IL10. Attempts to increase the expression of IL10 by the addition of polybrene (40 micrograms / ml) to the transduction mixture, resulted in a toxic effect on corneal allografts which abrogated the beneficial effect of IL10. The lentiviral vector shows potential for the stable expression of therapeutic transgenes in human corneal transplantation. However, the mechanisms underlying the species-specific differences in HIV-1-mediated transgene expression will need to be elucidated and overcome if the ovine preclinical model is to provide justification for a clinical trial.

"December 2003" / Includes bibliographical references (leaves 183-224) / xvii, 236 leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 2004

Primary graft non-function is one of the major limitations of organ transplantation increasing the risk of rejection and early graft failure. A major cause of primary non-function is ischemia reperfusion injury (IRI), an obligatory insult in transplantation. During procurement, the donor is subjected to a period of ischemia inducing the release of tissue-damaging factors such as nitric oxide and reactive oxygen species. Upon engraftment and reperfusion with the recipient blood, these ischemia-induced factors cause rapid cell death and amplification of the inflammatory response leading to further tissue damage. / CD39 is an integral vascular and immune ectonucleotidase. CD39 hydrolyses extracellular nucleotides ATP and ADP into AMP, which is then hydrolysed into adenosine by CD73. Extracellular adenosine produced by the concerted action of CD39 and CD73 has potent anti-inflammatory and anti-coagulation effects acting principally via the purinergic adenosine receptor A2a. / NKT cells have only recently been recognised and constitute an important subset of T lymphocytes that display both effector and suppressive functions. NKT cells are found in high proportion in the liver of mice and are implicated by depletion studies in protection against hepatic IRI. / We have generated mice transgenic for human CD39 (hCD39) and have shown they have an anti-coagulant phenotype. As CD39 is also critical to immune regulation we hypothesised that transgenic expression of hCD39 would modify lymphocyte development and/or function and consequently impact on ischemia reperfusion injury. / Flow cytometric analysis was used to assess the number and phenotype of lymphocytes within the thymus and in the periphery of hCD39 transgenic mice. In vitro and in vivo assays were used to test the function of CD4+ T cells and invariant NKT cells from hCD39 transgenic mice. Bone marrow adoptive transfers experiments defined the role of hCD39 expression on bone marrow progenitor cells in comparison to tissue expression. The importance of adenosine signalling through the A2a receptor was studied by crossing hCD39 transgenic mice with A2a receptor knock-out (KO) mice. The effect of hCD39 expression on ischemia reperfusion injury was evaluated in a model of murine liver transplantation / A high level of hCD39 expression in the transgenic thymus resulted in lymphocyte maturation blockade and peripheral lymphopenia of CD4+ T cells and invariant NKT cells. Both lymphocyte populations were functionally deficient. The observed phenotype resulted from the expression of hCD39 on bone marrow progenitor cells but was independent of A2a receptor signalling. Over-expression of hCD39 in transgenic livers was protective against ischemia reperfusion injury induced by cold storage and liver transplantation.

München, Techn. Universiẗat, Diss., 2007.

Human mesenchymal stem cells (MSCs) are being largely studied for their differentiation potential and immunological properties. In the present study, we evaluated the ability to reliably differentiate mesenchymal lineages into hepatocyte-like cells both in vitro and in vivo. For this purpose, we handled several tissue sources and compared typical MSCs from bone marrow (BM) or umbilical cord, to liver-derived mesenchymal-like cells and to fibroblasts. We observed that hepatocyte differentiation of BM-MSCs was incomplete and variable with elective expression of some specific markers. These mesenchymal-derived hepatocyte-like cells (MDHLCs) were also chimerical in their phenotype as they expressed mesenchymal markers while these were down-regulated. We therefore designed differentiation cocktails with an aim to improve MDHLC phenotype and some unexpected results were obtained with LIF cytokine whose action on stem cells for hepatocyte differentiation was not documented. Nevertheless, we observed a limitation in the acquisition yield of hepatic features. Furthermore, the hepatocytelike phenotype of MDHLCs completely disappeared when the cells were incubated into growth medium. However, we showed that hepatic functionality of these cells, as urea secretion and gluconeogenesis, could be increased under specific conditions, suggesting the potential to improve MDHLC phenotype. In vivo, MSCs were able to express hepatic markers into SCID-mice livers while their chimerical phenotype remained. In contrast, MDHLCs down-regulated their hybrid phenotype after transplantation suggesting a beneficial influence of in vitro differentiation step. MSCs were also able to engraft and even partially differentiate into wild-type mice which was a strong argument for their low immunogenicity. Surprisingly, fibroblasts showed highly similar potential than MSCs to differentiate into hepatocyte-like cells both in vitro an in vivo and these results underlined the difficulty to accurately distinguish between both cell types using current techniques. Umbilical cord-derived stem cells (UCMSCs) and adult-derived human liver stem cells (ADHLSCs) were different in nature and displayed a native hybrid phenotype while their differentiation allowed high levels of hepatocyte-like feature acquisition. Together all these data suggest the current possibility to engineer mesenchymal-derived hepatocyte-like cells owning specific features acquisition while remaining limited in their commitment. This highlights the need for further investigations to evidence the usefulness of these mesenchymal lineages for liver cell therapy.

Currently, in most situations, the autologous immune system is unable to eradicate the residual leukemic burden persisting after chemo-radiotherapy, but a balance can be established between leukemic and immune cells leading to a clinical remission for several months or years. If this balance is broken, a clinical relapse can occur. The high incidence of relapses in human cancers demonstrates the frequent inefficacy of the immune system to control these residual cells. In this context, allogeneic hematopoietic stem cell transplantation (HSCT) has been proven to be the most effective way to reinforce the immune reaction against leukemia, graft-versus-leukemia (GVL) effect and, so, achieve a definitive eradication of the residual disease in a significant proportion of patients. Indeed, the whole concept of HSCT evolved from an organ transplant concept (to replace a defective ill organ with a new healthy one) to the concept of creating an extraordinary immunotherapeutic platform in which the donor immune system contributes to the eradication of the residual leukemic cells. Thus, the past and present issues remain those of finding the best immunomodulatory modalities to achieve a full engraftment, a powerful GVL effect and no or moderate graft-versus-host disease (GVHD). Different ways to reach this goal, such as post transplant cytokine modulation, specific or global cellular depletion of the graft and post transplant global or specific donor immune cell add-backs, are still extensively studied. Nevertheless, the persistent high relapse rate (RR) observed in leukemia patients after HSCT remains the most important cause of death before transplant-related toxicities. Moreover, since only about 40 to 70% (depending on the ethnic context) of patients with high-risk hematological malignancies, eligible for allogeneic HSCT, have a fully HLA-matched sibling or matched unrelated donor (MUD), a great deal of effort has been invested to make the use of an alternative haploidentical sibling donor feasible. The advantage of this procedure is the immediate availability of a donor for almost all patients. The aim of the work described in this thesis has been to implement a strategy to transplant a patient using a HLA haploidentical donor. The strategy is to try to improve DFS that could be applied both in the autologous or allogeneic context: first, by using nonspecific immune manipulation post transplant and then, by developing specific strategies directed against leukemia antigens. Particularly in the allogeneic situation, the aim was to increase the GVL effect without inducing or aggravating the deleterious GVHD. The first part of this thesis described our own clinical results, consisting of three consecutive phase I/II studies, in which we tried to determine the feasibility of giving prophylactic donor lymphocyte infusions (DLI) post transplant and the effect of replacing granulocyte colony-stimulating factor (G-CSF), typically used to speed up neutrophil recovery, with granulocyte macrophage colony-stimulating factor (GM-CSF), which is known for its immunomodulatory properties. The slow immune reconstitution in haploidentical transplant is chiefly responsible for the high incidence of early lethal viral and fungal infections, and most probably for early relapses; therefore, we sought to accelerate and strengthen the post transplant immune reconstitution without increasing the GVHD rate. Thus, we have studied the impact of post transplant growth factor administration and of unselected DLI in haploidentical transplant. We have also implemented, in our center, anti-cytomegalovirus (CMV) specific T cell generation and infusion to improve anti-CMV immune reconstitution. Since then, our results have been pooled in a multi-center analysis performed by the European Bone Marrow Transplantation group (EBMT) allowing us to compare our results with those of the entire group. We have also participated in the design of an ongoing study aimed at selectively depleting the graft from alloreactive T cells, and improving post transplant T cell add-backs. In our attempts to generate and expand ex vivo lymphocytes (directed against pathogens (CMV) and leukemia-associated antigens, Wilms' tumor gene 1 (WT1) and to use them in vivo, we found inconsistent results (in the case of WT1) using classical clinical grade dendritic cells (DC) generated and matured in bags, as was the case for the majority of the teams worldwide. This led us to question the full functionality of these DC and we undertook a thorough comparative analysis of DC generated and differentiated in bags and in plates (typical for most pre-clinical studies). This analysis showed us that one cannot transpose pre-clinical studies (using culture plates) directly to clinical protocols (generally using clinical grade culture bags) and that DC generated in bags are functionally deficient. We learned that, if we want to use a DC vaccine to improve the GVL effect in haploidentical transplant, we will have to be careful about the technique by which they are generated. To improve immunotherapeutic approaches, the understanding of the mechanisms underlying tumor tolerance and how to manipulate them is critical in the development of new effective immunotherapeutic clinical trials. This is why we currently focus on how to obtain effective in vivo anti-leukemia immune reactions using an ex-vivo manipulated product to trigger the immunotherapeutic response. More specifically, we are analyzing the impact of regulatory T cell (Tregs) depletion and function for an adequate anti-leukemic immune response. This pre-clinical work aims at improving the outcome of leukemia patients who have relapsed and been put back into second remission and at decreasing the RR after HSCT, especially in the field of haploidentical transplantation. In conclusion, haploidentical transplantation has become a valuable tool. The results are at least similar to those obtained using MUD when performed in the same group of patients. Specific immunomodulation post transplant can affect events such as GVHD and GVL, but clinically we are still at the level of nonspecific manipulations. It is our hope that ongoing pre-clinical work will enable us to perform specific anti-pathogen and anti-leukemia immune manipulation that will favorably influence the patient outcome. / Dans la majorité des situations, le système immunitaire autologue est incapable d’éradiquer les cellules leucémiques résiduelles qui échappent à la radiothérapie et à la chimiothérapie, cependant un équilibre peut s’établir entre les cellules leucémiques et immunitaires aboutissant à une rémission pouvant durer plusieurs mois ou années. Si cet équilibre se rompt, une rechute clinique peut se déclarer. Dans ce contexte, il est prouvé que la greffe allogénique de cellules souches hématopoïétiques est le moyen le plus efficace de renforcer les réactions immunitaires contre la leucémie par la réaction du greffon contre la leucémie et ainsi d’obtenir une éradication définitive de la maladie résiduelle chez un nombre significatif de patients. En effet, le concept global de l’allogreffe de cellules souches hématopoïétiques a évolué du concept de transplantation d’organe (remplacement d’un organe malade par un nouvel organe sain) vers celui de créer une extraordinaire plateforme d’immunothérapie à travers laquelle le système immunitaire du donneur contribue à l’éradication des cellules leucémiques persistantes. Donc, la problématique reste celle de trouver les meilleures modalités d’immunomodulation pour achever une prise du greffon, un effet anti-leucémique puissant du greffon, et l’absence ou un minimum d’effet du greffon contre l’hôte. Différentes stratégies existent pour atteindre cet objectif, comme l’utilisation de cytokines pour moduler la reconstitution immunitaire, des déplétions cellulaires globales ou spécifiques du greffon et l’infusion de cellules immunes «globales» ou spécifiques du donneur après greffe. Ces stratégies sont encore largement à l’étude. Néanmoins, la persistance d’un taux de rechute élevé observé chez les patients leucémiques, après allogreffe reste la cause principale de décès, avant celle liée à la toxicité de la greffe. De plus, étant donné que seulement environ 40 à 70% (dépendant de l’origine ethnique) des patients avec une hémopathie à haut risque, éligibles pour une greffe allogénique, ont un donneur familial ou non familial complètement HLA compatible, des efforts importants ont été développés pour rendre faisable l’utilisation de donneurs familiaux alternatifs, haploidentiques. L’avantage de cette approche est l’accès immédiat à un donneur pour quasiment tous les patients. Le but du travail décrit dans cette thèse a été l’implémentation d’une stratégie d’allogreffe utilisant un donneur haploidentique. Le travail vise également à développer de façon plus large des stratégies qui peuvent améliorer le taux de survie sans rechute, non seulement dans le contexte des greffes haploidentiques, mais également dans le cadre des greffes allogéniques en général, ainsi que dans les situations autologues : premièrement, par la manipulation immunitaire non spécifique après greffe et ensuite par le développement de stratégies spécifiques dirigées contre des antigènes leucémiques. En particulier dans la situation allogénique, le but a été d’augmenter l’effet du greffon contre la leucémie sans induire ou aggraver l’effet délétère du greffon contre l’hôte. La première partie de la thèse décrit les résultats cliniques de notre propre protocole de greffe haploidentique, qui a consisté en trois études consécutives de phase I/II. Dans ces études, nous avons voulu déterminer la faisabilité de réaliser des infusions prophylactiques de lymphocytes du donneur après transplantation, et l’impact du remplacement du « granulocyte colony-stimulating factor » (G-CSF), largement utilisé pour permettre une récupération en polynucléaires neutrophiles plus rapide, par du « granulocyte-macrophage colony-stimulating factor » (GM-CSF), lequel est connu pour ses propriétés immunomodulatrices différentes. La reconstitution immunitaire très lente après greffe haploidentique est majoritairement responsable de l’incidence élevée de décès par infections virales et fungiques précoces, et très probablement des rechutes précoces. C’est pourquoi nous avons cherché à accélérer et à renforcer la reconstitution immunitaire post-greffe sans augmenter la fréquence de réaction du greffon contre l’hôte. Nous avons donc étudié l’impact de l’administration de facteurs de croissance et l’infusion de lymphocytes non sélectionnés du donneur en post greffe haploidentique. Nous avons également implémenté dans notre centre, la génération et l’infusion de lymphocytes T spécifiques anti-cytomégalovirus (CMV) afin d’améliorer la reconstitution immunitaire anti-CMV. D’autre part, nos résultats ont été regroupés dans une étude multicentrique menée par le groupe européen de transplantation de moelle osseuse (EBMT), ce qui nous a permis de comparer nos résultats avec ceux de l’entièreté du groupe. Nous avons parallèlement participé à la conception d’une étude actuellement en cours ayant pour but d’améliorer la reconstitution immunitaire après greffe par la déplétion sélective du greffon en lymphocytes T alloréactifs et par l’infusion après greffe de lymphocytes T du donneur également sélectivement déplétés en lymphocytes T alloréactifs. Afin d’optimaliser l’effet anti-leucémique du système immunitaire, nous avons débuté un protocole de vaccination par cellules dendritiques (DCs). Ces cellules dendritiques étaient chargées en lysat de blastes leucémiques dans le cas de patients présentant au diagnostic une leucémie aigue surexprimant l’oncogène 1 de la tumeur de Wilms (WT1). Néanmoins dans nos travaux de génération et d’expansion ex-vivo de lymphocytes T spécifiques de l’antigène WT1, utilisant les DCs de grade clinique, générées et maturées en poches, nous avons rencontré des résultats inconsistants, comme c’était le cas dans la majorité des protocoles cliniques internationaux de vaccination. Nous nous sommes alors posé la question de la fonctionnalité globale de ces cellules et nous avons entrepris une analyse comparative poussée des DCs générées et différenciées en poches ou en plaques. Les DCs générées en plaques sont celles utilisées dans la plupart des travaux précliniques. Cette analyse nous a montré que l’on ne pouvait pas directement transposer les résultats précliniques basés sur des DCs générées en plaques dans des protocoles cliniques basés sur des DCs générées en poches, car ces dernières présentent des déficits fonctionnels importants. Nous avons appris que si l’on voulait utiliser un vaccin à base de cellules dendritiques pour améliorer l’effet du greffon contre la leucémie dans les greffes allogéniques, nous devions être très attentifs quant au protocole utilisé pour la génération de ces vaccins cellulaires. Pour améliorer les approches immunothérapeutiques, la connaissance des mécanismes qui établissent la tolérance tumorale et des façons de manipuler ceux-ci, est critique dans le développement de nouveaux protocoles efficaces. C’est pourquoi nous nous concentrons actuellement sur les conditions nécessaires à l’obtention in vivo d’une réaction immune anti-leucémique efficace lors de l’utilisation d’un produit cellulaire manipulé ex vivo. Plus spécifiquement, nous analysons l’impact de la déplétion en lymphocytes T régulateurs (Tregs) sur la réponse anti-leucémique. Ce travail préclinique a pour but d’améliorer le devenir de patients leucémiques qui ont rechutés et ont été mis en seconde rémission, ainsi que de diminuer le taux de rechute après allogreffe, spécifiquement après greffe haploidentique. En conclusion, la transplantation haploidentique est actuellement un outil précieux pour de nombreux patients. Les résultats sont au minimum similaires à ceux qui sont obtenus par les greffes non-familiales HLA identiques lorsqu’elles sont pratiquées dans les mêmes groupes de patients. L’immunomodulation spécifique après greffe peut affecter des événements comme la réaction du greffon contre l’hôte et la réaction du greffon contre la leucémie, mais en pratique clinique nous en sommes encore au niveau de la manipulation aspécifique. Nous espérons que les travaux précliniques actuels vont nous permettre d’appliquer des stratégies spécifiques et d’obtenir une manipulation immune anti-leucémique qui aura une influence favorable significative sur le devenir des patients.

Background: The incidence of inadequate empiric antibiotic therapy (IET) and its clinical importance as a risk factor for hospital mortality in Canadian solid-organ transplant patients remains unknown. Methods: This retrospective cohort study evaluated all patients admitted to a transplant unit from May/2002-April/2004. Therapy was considered adequate when the organism cultured was found to be susceptible to an antibiotic administered within 24 hours of the index sample collection time. Univariate and multivariate regression analyses were conducted to determine associations between potential determinants, IET, and mortality. Results: IET was administered in 169/312 (54%) transplant patients. Regression analysis demonstrated that an increasing duration of IET (adjusted OR at 24h, 1.33; p < 0.001), ICU-associated infections (adjusted OR, 6.27; p < 0.001), prior antibiotic use (adjusted OR, 3.56; p = 0.004), and increasing APACHE-II scores (adjusted OR, 1.26; p < 0.001), were independent determinants of hospital mortality. Conclusions: IET is common and appears to be an important determinant of hospital mortality in the Canadian transplant population.