Na ortopedia veterinária, o enxerto ósseo é comumente utilizado em uma variedade de procedimentos ou afecções ortopédicas como artrodeses, fusões espinhais, reparo de fraturas, tratamentos de união atrasada e não-união de fraturas, preenchimento de falhas ósseas, osteotomias corretivas e reconstruções após a ressecção de neoplasia óssea. O uso de enxerto ósseo alógeno, comparado com o enxerto autógeno, tem como vantagens menor morbidez e dor pós-cirúrgica, diminuição do tempo cirúrgico e, principalmente, fornecimento de maior volume ósseo para a reconstrução adequada de uma grande falha óssea. As desvantagens do seu uso são os riscos de reação imunológica e transmissão de doenças, além da necessidade da formação de um banco de ossos, o que aumenta os custos e necessita de espaço apropriado. O objetivo do presente trabalho foi avaliar a taxa e a forma de incorporação do aloenxerto ósseo cortical submetido ao congelamento em nitrogênio líquido e inserido em tíbias de ovelhas, visando à preservação do membro locomotor. Seis ovelhas clinicamente sadias, da raça Corriedale, com idade estimada entre dois e três anos, foram submetidas à avaliação radiográfica pré-operatória das tíbias para que fosse descartada qualquer alteração óssea. Simultaneamente, realizou-se a ostectomia da diáfise tibial de duas ovelhas para a retirada de um segmento de 7 cm que, após a remoção do periósteo e da medula óssea, foi imerso em nitrogênio líquido, tendo como finalidade a desvitalização óssea, e implantado imediatamente no outro paciente. A estabilização entre os fragmentos e o implante ósseo foi realizada com placa cirúrgica de compressão dinâmica (PCD). Após os procedimentos cirúrgicos, foram realizadas avaliações clínicas e radiográficas a cada 30 dias até o 180º dia de pós-operatório. Aos 60 dias de pós-operatório já se observava o completo uso funcional do membro operado. A união radiográfica das interfaces proximal e distal e a consolidação óssea ocorreram em tempo médio de 95 dias em todos os animais. Conclui-se que o aloenxerto ósseo cortical submetido ao congelamento em nitrogênio líquido é um método apropriado para a preservação do membro locomotor, tendo em vista que a incorporação do mesmo ocorreu gradualmente e a taxa foi de 100%. / Bone grafting is employed in veterinary orthopedics with a variety of purposes as for arthrodesis, spinal fusions, fracture repairs, treatment of delayed or non-unions, filling bone defects, supporting corrective osteotomy, and as replacement in limb sparing techniques for tumor resection. The allograft bone, in contrast to the autologous bone graft, is associated with less site-donor morbidity and pain, reduced surgical time, and adequate bone supply to fill greater defects. Still, the allogeneic bone, besides requiring the establishment of a bone bank, can be a source of diseases and may induce immunogenic response. This study evaluated the allogeneic cortical bone graft incorporation after submission of the harvested fragment to a bout freezing in liquid nitrogen. Six adult clinically healthy sheep were submitted to a 7cm ostectomy of the tibial diaphysis. After removal of periosteum and bone marrow, the fragment was submersed in liquid nitrogen and implanted in another sheep missing a same-sized segment at the corresponding bone. Stabilization of the allograft in the host bone was accomplished by a dynamic compressive plate (DCP). Clinical and radiographic evaluations were performed in the immediate post-operatory period and in every 30 days for six months after surgery. The proximal and distal host-graft interfaces showed radiographic union at a mean postoperative time of 95 days in all the animals. The cortical bone allograft submitted to liquid nitrogen freezing provided adequate bone healing in the sheep model, representing a feasible alternative for limb preservation techniques.

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No. of bitstreams: 2 Dissertação - Brunno Câmara Lopes Costa - 2018.pdf: 3761382 bytes, checksum: 9df697870cea1a53a75ea6dad69c9e91 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-08-17 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Human Bocavirus (HBoVs) are classified in the Parvoviridae family and are associated with respiratory and gastrointestinal symptoms. Viral infections are an important cause of morbimortality in immunocompromised patients such as allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. The aim of the present study was to evaluate the positivity rate and loads of HBoVs in clinical samples (feces and sera) of patients who were subjected to allo-HSCT at a reference center for bone marrow transplantation in Goiânia, Goiás. A total of 105 fecal samples and 145 sera samples were collected from 21 consecutive patients, during October 2012 to October 2014. Samples were screened by qPCR TaqMan assay, with specific probe and primers targeting all HBoVs genotypes (HBoV-1 to -4), and viral loads were determined using serial dilutions of a recombinant plasmid, targeting the NP1 gene. The results showed that 53.4% (11/21) of the patients were male, aged between four and 61 years-old (mean 35 years). The most observed hematologic malignancy was myeloid leukemia (acute or chronic), accounting for 57.1% (12/21) of the cases. The HBoVs were detected in 42.9% (9/21) of the patients and 77.7% (7/9) were positive in both fecal and serum samples. The viral load in fecal samples were higher than in the sera samples and a prolonged fecal shedding were observed, with two patterns: one intermittent and another continuous. Of all HBoV positive patients, six (66.6%) had the first positive sample before the transplantation, and a rise of the viral loads after the allo-HSCT occurred when comparing to the loads before the allo-HSCT. Furthermore, on most cases the highest viral loads were detected during the first 100 days after the allo-HSCT. Considering the symptoms presented by the patients, 66.6% (6/9) had diarrhea at the same period of the viral genome detection in feces, but no statistical significance was observed. Three fecal samples were characterized as being HBoV-1, with more than 99% of nucleotide identity among them. The present data shows a high occurrence and loads of HBoVs in allo-HSCT recipients, with first positivity in fecal samples and later viral detection in sera. These results suggest that fecal samples could be the sample of choice in HBoV monitoring of these patients both before and after the transplant. / Os Bocavírus humanos (HBoVs) pertencem à família Parvoviridae e têm sido associados a sintomas respiratórios e gastroentéricos. As infecções virais são uma importante causa de morbimortalidade em pacientes imunocomprometidos, como os pacientes submetidos a transplante alogênico de células progenitoras hematopoiéticas (TACPH). Dados sobre a ocorrência de HBoV nesses pacientes ainda são escassos. Os objetivos deste estudo foram avaliar a frequência e a carga de HBoVs em amostras clínicas (fezes e soro) de pacientes submetidos a TACPH e avaliar as características gerais e sintomas apresentados, e caracterizar as amostras positivas. Foram incluídos no estudo 21 pacientes consecutivos, dos quais foram coletadas 105 amostras fecais e 145 amostras de soro, em um centro de transplante de medula óssea em Goiânia, Goiás, durante o período de outubro de 2012 a outubro de 2014. As amostras foram testadas por qPCR TaqMan®, com sonda e iniciadores específicos para todos os genótipos de HBoVs (HBoV-1 a -4), e a carga viral nas amostras foi determinada pela construção de uma curva padrão de diluições seriadas de um plasmídeo recombinante, contendo como inserto a região NP1. Os resultados mostraram que 53,4% (11/21) dos pacientes eram do sexo masculino, com idade entre quatro e 61 anos de idade (mediana 35 anos). A neoplasia hematológica mais observada foi a leucemia mieloide (aguda e crônica), totalizando 57,1% (12/21) dos casos. Os HBoVs foram detectados em 42,9% (9/21) dos pacientes, sendo que em 77,7% (7/9) desses houve positividade em ambas amostras de soro e fezes. A carga de HBoV nas amostras fecais foi significativamente maior do que nas amostras séricas, sendo observado dois padrões principais de excreção nas fezes, um intermitente e outro contínuo. Dos nove pacientes, seis (66,6%) tiveram a primeira amostra positiva antes de serem submetidos ao transplante, sendo observado aumento nas cargas de HBoV pós-TACPH em comparação às cargas pré-TACPH e, na maioria dos casos, os picos da carga viral foram detectados durante os 100 primeiros dias após o TACPH. Considerando os sintomas apresentados pelos pacientes, 66,6% (6/9) desses apresentavam diarreia no mesmo período da detecção de HBoV nas amostras fecais, porém não houve significância estatística entre a positividade e os sintomas gastroentéricos. Três amostras fecais foram caracterizadas como sendo o genótipo HBoV-1, com mais de 99% de identidade nucleotídica entre elas. Os dados apresentados mostram uma alta ocorrência e elevada carga de HBoVs nos pacientes submetidos ao TACPH, além de detecção inicial nas fezes com posterior positividade no soro. Esses resultados sugerem que as fezes poderiam ser a amostra clínica de escolha para o monitoramento desses pacientes, tanto antes quanto após o transplante.

Na ortopedia veterinária, o enxerto ósseo é comumente utilizado em uma variedade de procedimentos ou afecções ortopédicas como artrodeses, fusões espinhais, reparo de fraturas, tratamentos de união atrasada e não-união de fraturas, preenchimento de falhas ósseas, osteotomias corretivas e reconstruções após a ressecção de neoplasia óssea. O uso de enxerto ósseo alógeno, comparado com o enxerto autógeno, tem como vantagens menor morbidez e dor pós-cirúrgica, diminuição do tempo cirúrgico e, principalmente, fornecimento de maior volume ósseo para a reconstrução adequada de uma grande falha óssea. As desvantagens do seu uso são os riscos de reação imunológica e transmissão de doenças, além da necessidade da formação de um banco de ossos, o que aumenta os custos e necessita de espaço apropriado. O objetivo do presente trabalho foi avaliar a taxa e a forma de incorporação do aloenxerto ósseo cortical submetido ao congelamento em nitrogênio líquido e inserido em tíbias de ovelhas, visando à preservação do membro locomotor. Seis ovelhas clinicamente sadias, da raça Corriedale, com idade estimada entre dois e três anos, foram submetidas à avaliação radiográfica pré-operatória das tíbias para que fosse descartada qualquer alteração óssea. Simultaneamente, realizou-se a ostectomia da diáfise tibial de duas ovelhas para a retirada de um segmento de 7 cm que, após a remoção do periósteo e da medula óssea, foi imerso em nitrogênio líquido, tendo como finalidade a desvitalização óssea, e implantado imediatamente no outro paciente. A estabilização entre os fragmentos e o implante ósseo foi realizada com placa cirúrgica de compressão dinâmica (PCD). Após os procedimentos cirúrgicos, foram realizadas avaliações clínicas e radiográficas a cada 30 dias até o 180º dia de pós-operatório. Aos 60 dias de pós-operatório já se observava o completo uso funcional do membro operado. A união radiográfica das interfaces proximal e distal e a consolidação óssea ocorreram em tempo médio de 95 dias em todos os animais. Conclui-se que o aloenxerto ósseo cortical submetido ao congelamento em nitrogênio líquido é um método apropriado para a preservação do membro locomotor, tendo em vista que a incorporação do mesmo ocorreu gradualmente e a taxa foi de 100%. / Bone grafting is employed in veterinary orthopedics with a variety of purposes as for arthrodesis, spinal fusions, fracture repairs, treatment of delayed or non-unions, filling bone defects, supporting corrective osteotomy, and as replacement in limb sparing techniques for tumor resection. The allograft bone, in contrast to the autologous bone graft, is associated with less site-donor morbidity and pain, reduced surgical time, and adequate bone supply to fill greater defects. Still, the allogeneic bone, besides requiring the establishment of a bone bank, can be a source of diseases and may induce immunogenic response. This study evaluated the allogeneic cortical bone graft incorporation after submission of the harvested fragment to a bout freezing in liquid nitrogen. Six adult clinically healthy sheep were submitted to a 7cm ostectomy of the tibial diaphysis. After removal of periosteum and bone marrow, the fragment was submersed in liquid nitrogen and implanted in another sheep missing a same-sized segment at the corresponding bone. Stabilization of the allograft in the host bone was accomplished by a dynamic compressive plate (DCP). Clinical and radiographic evaluations were performed in the immediate post-operatory period and in every 30 days for six months after surgery. The proximal and distal host-graft interfaces showed radiographic union at a mean postoperative time of 95 days in all the animals. The cortical bone allograft submitted to liquid nitrogen freezing provided adequate bone healing in the sheep model, representing a feasible alternative for limb preservation techniques.

Introduction: Acute myeloid leukemia (AML) is a highly heterogeneous disease which renders risk stratification at diagnosis of high importance to personalize therapy. Allogeneic hematopoietic stem cell transplantation (HSCT) offers the highest chance for sustained remission in most AML patients, but usually comes at the risk of a significant treatment-related mortality. The red cell distribution width (RDW) is an universally accessible parameter that identifies individuals with a higher mortality in many diseases, including some hematological entities. However, the impact of diagnostic RDW levels in AML – especially in the context of a HSCT consolidation - has not been evaluated so far. Purpose: To evaluate the prognostic impact of RDW levels at AML diagnosis. Methods: A total of 294 newly diagnosed AML patients (median age 60.6, range 14.3-76.5 years), with available diagnostic RDW levels were retrospectively included in this analysis. All patients received a consolidation therapy with an allogeneic HSCT in curative intention between August 2007 and December 2020 at the University Medical Center Leipzig. The RDW was measured in all patients at AML diagnosis before the start of cytoreductive therapies. Results: RDW levels at diagnosis were highly variable (median 16.6%, range 12%-30.6%) and above the upper level of normal (>15%) in 73% of the analyzed AML patients. Patients with RDW levels above 15% did not have worse outcomes compared to patients with low diagnostic RDW levels. However, when the cohort was dichotomized according to a receiver operating characteristic (ROC)-based optimal cut-point (20.7%), patients with high RDW levels had a significantly higher non-relapse mortality (NRM), shorter overall survival and a trend for shorter event-free survival, while the risk of relapse or disease progression was similar in both groups. In multivariate analyses, the RDW remained an independent prognostic factor for higher NRM after adjustment for the body mass index at diagnosis. Patients with a higher RDW were more likely to harbor a secondary AML, as well as to harbor secondary AML-associated gene mutations (i.e. JAK2, ASXL1, or spliceosome mutations, especially SRSF2). Conclusion: High RDW levels at diagnosis represent an independent risk marker for a higher mortality following allogeneic HSCT. When confirmed in prospective clinical trials, the RDW might help to personalize AML consolidation therapy including conditioning regimens before allogeneic HSCT.:1. Bibliographische Beschreibung 2. Abkürzungsverzeichnis 3. Einführung / Introduction 3.1. Acute Myeloid Leukemia 3.1.1. Definition 3.1.2. Epidemiology and etiology 3.1.3. Clinical presentation 3.1.4. Diagnosis of AML 3.1.4.1. Morphology 3.1.4.2. Immunophenotyping 3.1.4.3. Cytogenetic and molecular analyses 3.1.5. AML classification according to WHO classification 3.1.6. Prognostic factors in AML 3.1.6.1. Patient-related risk factors 3.1.6.2. Genetic risk factors 3.1.6.3. Measurable residual disease 3.1.7. Treatment of AML 3.1.7.1. Induction therapy in curative intention 3.1.7.2. Consolidation therapies 3.1.7.3. Palliative treatment approaches 3.1.7.4. New substances 3.2. Allogeneic HSCT 3.2.1. Principles of allogeneic HSCT 3.2.2. Conditioning regimens 3.3. Red cell distribution width 4. Aufgabenstellung / Objectives 5. Materialien und Methoden / Materials and Methods 5.1. Patients and treatments 5.1.1. Treatment protocols 5.1.2. Allogeneic HSCT and immunosuppression 5.1.3. Assessment of GvHD 5.2. Disease characterization 5.2.1. Evaluation at AML diagnosis 5.2.1.1. Morphology 5.2.1.2. Flow cytometry 5.2.1.3. Genetic analyses 5.2.1.4. Evaluation of RDW levels 5.2.2. Evaluation at HSCT 5.2.2.1. Definition of remission status at HSCT 5.2.2.2. Evaluation of measurable residual disease at HSCT 5.3. Statistical Analyses 5.3.1. Associations 5.3.2. Clinical endpoints 5.3.3. Definition of an optimal cut-point for RDW levels 5.3.4. Multivariate analyses 6. Ergebnisse / Results 6.1. Overall outcomes of the patient cohort 6.2. RDW levels at AML diagnosis regarded as continous parameter 6.3. The role of RDW levels at diagnosis as a predictor for outcomes after allogeneic HSCT 6.4. Associations of RDW levels at diagnosis 7. Diskussion / Discussion 8. Zusammenfassung / Summary 9. Literaturverzeichnis / References 10. Erklärung über die eigenständige Abfassung der Arbeit 11. Curriculum Vitae 12. Komplette Publikationsliste (Peer-reviewed) 13. Danksagung

Introduction: In acute myeloid leukemia (AML), leukemia initiating cells exist within the CD34+/CD38- cell compartment. They are assumed to be more resistant to chemotherapy, enriched in minimal residual disease cell populations, and responsible for relapse. Purpose: We evaluated clinical and biological associations and the prognostic impact of a high diagnostic CD34+/CD38- cell burden in AML patients receiving an allogeneic stem cell transplantation (HSCT) in complete remission. Here, the therapeutic approach is mainly based on immunological graft-versus-leukemia effects. Methods: Percentage of bone marrow CD34+/CD38- cell burden in 169 AML patients at diagnosis was measured using flow cytometry. The optimal cutoff of 6% was applied and used to evaluate the impact of a high CD34+/CD38- cell burden on outcome. Results: The CD34+/CD38- cell burden and was highly variable (median 0.5%, range 0-89% of all mononuclear cells). A high CD34+/CD38- cell burden at diagnosis associated with worse genetic risk and secondary AML. Patients with a high CD34+/CD38- cell burden had shorter relapse-free and overall survival, which may be mediated by residual leukemia initiating cells in the CD34+/CD38- cell population, escaping the graft-versus-leukemia effect after allogeneic HSCT. Conclusion: Evaluating the CD34+/CD38- cell burden at diagnosis may help to identify patients at high risk of relapse after allogeneic HSCT. Further studies to understand leukemia initiating cell biology and develop targeting therapies to improve outcomes of AML patients are needed.:Bibliographische Beschreibung / Bibliographic description 1 Einleitung / Introduction 2 Epidemiology and AML diagnosis 2 Therapeutic options in AML 3 Genetic risk classification for therapeutic decisions in AML 6 Immunophenotyping in AML 10 Leukemia Initiating Cells 11 Objectives of the here presented study 13 Publikation / Publication 14 Anlage / Supplemental Material 23 Zusammenfassung / Summary 48 Weiterführende Arbeiten / Future developments GPR56 as new LIC marker 52 Referenzen / References 55 Referenz der Publikation / Reference of the publication 60 Erklärung über die eigenständige Abfassung der Arbeit 61 Curriculum Vitae 62 Komplette Publikationsliste 65 Danksagung 74

CD133, a pentaspan glycoprotein, has long been known to represent aggressive, stem-like populations across various human malignancies. While its expression correlates with numerous clinical outcomes including disease progression, metastasis, recurrence, and poor overall survival in numerous cancers, little is currently known about its function. In the brain cancer glioblastoma (GBM), CD133-expressing cells have previously been shown to initiate tumours, evade therapy and interestingly, self-renew, a key property of cancer stem cells. With an implied signalling role in driving self-renewal, we aim to elucidate the role of CD133 in glioblastoma. To understand the role of CD133, we aim to study its protein-protein interactions using the proximity-dependent labelling technique known as miniTurboID. By tagging proteins of interest with a promiscuous biotin ligase at both protein termini, potential interactors can be biotinylated and identified by subsequent mass spectrometry. While miniTurboID has traditionally been performed by synthetic transgenes expressing the tagged proteins of interest in commercial cell lines, overexpression may not recapitulate its native function. Thus, using CRISPR technology, we aim to insert the miniTurboID ligase at both the N- and C-terminus of CD133 in patient-derived human GBM lines. Although little is currently known about CD133 function, development of targeted therapies has presented a promising strategy in pre-clinical studies. In the Singh Lab, we previously developed a chimeric antigen receptor T-cell, or CAR-T, comprised of a T-cell expressing a synthetic receptor capable of recognizing a tumor-associated antigen and activating cytolytic-killing directed towards the target cell. Currently, CAR-T therapies are autologous, or patient-derived, in nature which may host a myriad of concerns including patient-specific qualitative and quantitative T-cell dysfunction, inconsistent generation of CAR products, and availability to rapidly progressing patients. To circumvent this concern, “off-the-shelf”, donor-derived or allogeneic CAR-T products may be generated for use in GBM patients. However, in addition to CAR integration, allogeneic products must be additionally modified to eradicate expression of the endogenous TCR, as this would induce a phenomenon known as graft versus host disease, in which healthy tissues are targeted. Thus, in this thesis, we show gene editing potential in human GBMs to perform an endogenous genomic knock-in of miniTurboID. With the identification of interacting proteins, defining the subsequent functionality of CD133 may elucidate oncogenic cellular programs, and highlight common nodes of interaction within divergent cell signaling pathways. To develop an allogeneic CAR-T product, we designed a two-step approach in which the CAR sequence was integrated into the TCR gene for simultaneous knock-out. We later show early pre-clinical efficacy in comparison to traditional autologous CAR-T in our patient-derived models of human GBM. Thus, by using CD133 as a centralizing concept in this thesis, we ultimately hope to develop our biological understanding of CD133, while testing the therapeutic development of a donor-derived CAR-T therapy. / Thesis / Master of Science (MSc) / Glioblastoma (GBM) is one of the most common malignant brain tumors in adults. Despite an aggressive therapy regimen, almost all patients relapse 7-9 months post-diagnosis. Therapy failure and poor patient outcome may be attributed to a small population of cells known as glioblastoma stem cells, or GSCs, that are able to escape therapy and seed disease recurrence. GSCs are most notably identified by the cell surface protein CD133, which has previously been shown to associate with pro-tumor properties including treatment resistance, tumor growth, maintenance, progression and metastasis. While expression of CD133 in cancer has been heavily characterized, little is currently known about its function. One such avenue to understand its mechanism of action in cancer, and more particularly GBM, is to define its interactions with other proteins. Protein-protein interactions play a pivotal part as the backbone of signalling pathways that drive tumor development and growth. Therefore, defining and mapping the CD133 interaction network may help us understand how this protein governs regulation of GSCs, and ultimately, GBM progression. While the biology of CD133 has yet to be elucidated, targeting CD133 on GSCs has presented a promising therapeutic strategy for patients with GBM. Previously in the Singh Lab, we developed an engineered T-cell therapy, known as a CAR-T, that can recognize CD133 to induce tumor cell death. While this showed success in our animal models of human GBM, other considerations must be addressed on its path to clinical development. As of current, CAR-T therapies are generated from T-cells taken from cancer patients. This hosts a myriad of concerns including the quality of patient T-cells, the time and cost to manufacture, and its availability for patients with rapidly progressing disease. To circumvent this issue, donor-derived CAR-T cells can be genetically engineered for safe usage in GBM patients as a readily available, “off-the-shelf” therapy. To define the function of CD133, we have attempted to use a technique known as BioID, which tags the protein of interest with a smaller biotin ligase. This biotin ligase can subsequently tag proteins that come within the vicinity of CD133, that may later be identified by sequencing as potential interactors. As current use of BioID may not reliably mimic the interaction of CD133, we sought to genetically engineer human GBM lines with the BioID protein to more closely resemble tumor-relevant behaviours of CD133. To develop a donor-derived CAR-T therapy, we similarly used genetic engineering of T-cells to ensure specific targeting of tumor cells with CD133, while sparing healthy tissues. By using CD133 as a centralizing concept in this thesis, we ultimately hope to develop our biological understanding of CD133, while testing the therapeutic development of a donor-derived CAR-T therapy.

Allogeneic hematopoietic stem cell transplantation (alloHCT) represents the only potentially curative treatment in high-risk AML patients, but up to 40% of patients suffer from relapse after alloHCT. Treatment of overt relapse poses a major therapeutic challenge and long-term disease control is achieved only in a minority of patients. In order to avoid post-allograft relapse, maintenance as well as pre-emptive therapy strategies based on MRD-detection have been used. A prerequisite for the implementation of pre-emptive therapy is the accurate identification of patients at risk for imminent relapse. Detection of measurable residual disease (MRD) represents an effective tool for early relapse prediction in the post-transplant setting. However, using established MRD methods such as multicolor flow cytometry or quantitative PCR, sensitive MRD monitoring is only applicable in about half of the patients with AML and advanced MDS undergoing alloHCT. Donor chimerism analysis, in particular when performed on enriched leukemic stem and progenitor cells, e.g. CD34+ cells, is a sensitive method and has emerged as an alternative option in the post alloHCT setting. In this review, we will focus on the current strategies for lineage specific chimerism analysis, results of pre-emptive treatment using this technology as well as future developments in this field.

Allogeneic Hematopoietic stem cell transplants (HSCT) are used for the treatment of bone marrow aplasias. Allogeneic HSCT is performed by treating the patient with chemotherapy drugs and irradiation and then transplanting hematopoietic stem cells from a healthy donor to restore the immune system and hematopoietic cells. Allogeneic HSCTs has the added benefit of the graft vs leukemia effect (GvL), whereby donor allogeneic T cells are able to mount immune responses against any residual cancer cells. However, alloreactivity towards the mismatched minor and major histocompatibility antigens the patient's healthy tissues leads to graft vs host disease (GvHD). This process is also mediated by Macrophages, Dendritic cells, B cells. Furthermore, a decrease in the number of NK, B, and T regulatory cells exacerbates GvHD. This leads to a state of systemic inflammation, tissue damage and multiorgan fibrosis. Current therapies designed to suppress the immune system have been shown to be efficacious in preventing GvHD but patients become susceptible to infection or experience cancer relapse through the elimination of the GvL response as well. In this thesis, we explore two strategies for targeting T cell activation in two mouse models of GvHD. In the first model, we examined the contribution of donor-derived complement C5 on the induction GvHD. We observed that recipient mice were only protected from GvHD when donor cells were deficient for complement protein C5. Our second strategy involves selective targeting of alloreactive T cells using peptide immunotherapy. For this approach, we first developed a humanized mouse model of GvHD whereby cells from donor mice expressing human class II HLA were reconstituted into recipient mice expressing human class I HLA. We then tested peptide immunotherapy using peptides derived from the human class I HLA. Our initial results were inconclusive and require further optimization. / Thesis / Doctor of Philosophy (PhD) / Graft vs Host Disease is an unwanted side effect of mismatched bone marrow transplant. Donor T cells recognize and attack mismatched tissues of the recipient and this leads to systemic inflammation and tissue scarring. Current treatments primarily target T-cell activation by suppressing the immune system, however, this leaves the patients susceptible to recurrent infections. In this thesis we describe the creation of two mouse models of Graft vs Host Disease and then examine two ways of specifically targeting donor T cell activation that is designed not to affect normal immune responses.

No description available.

Le rôle de l'interleukine 2 (IL-2) dans les cellules dendritiques (DC) humaines n’est pas bien défini. Dans ce travail, nous avons exploré les effets de l'IL-2 sur des DC dérivées de monocytes. Nous démontrons une expression constitutive des chaines des β et γ du récepteur de l’IL-2 sur les DC ainsi qu’une une expression inductible de la chaîne α en réponse à des agents ‘maturants’ tels le LPS et le TNFα. L’IL-2 induit la phosphorylation du facteur de transcription STAT5 provoquant l’augmentation de la synthèse d'IFN-γ par les DC sans modifier leur phénotype ou leur survie. En revanche, nous n'avons pas mis en évidence d’activation des autres voies de signalisation du récepteur de l’IL-2. Nous avons également démontré que l'IL-2 augmente les capacités des DC à activer les lymphocytes T CD4+ allogéniques et les lymphocytes T CD8+ indépendamment des lymphocytes T auxilliaires. Nous n’avons pas pu mettre en évidence la sécrétion endogène d'IL-2 par les DC cependant les anticorps anti-CD25 diminuent les capacités allostimulatrices de DC en absence d’IL-2 exogène. Ainsi nos travaux indiquent que l’expression de CD25 par les DC matures est un événement clé menant la DC à un nouvel état d’activation. / Human dendritic cells (DC) express interleukin 2 (IL-2) receptor α-chain (CD25), but the role of IL-2 in DC is poorly understood. In this work, we explored the effects of IL-2 on monocyte-derived DC. First, we demonstrated the constitutional expression of β and γ chain of IL-2R on DC, while the α-chain was inducible by LPS and TNFα. Then we found that IL-2 does not affect DC phenotype and apoptosis but increases IFN-γ synthesis in DC through activation of transcription factor STAT5. Moreover we reported that IL-2 increases the ability of DC to activate allogeneic CD4+ T cells and helpless CD8+ T lymphocytes, most likely because of IL-2–triggered IFN-γ synthesis. We have not been able to demonstrate the endogenous secretion of IL-2 by DC, however anti-CD25 decreased allostimulatory capacity of DC in the absence of exogenous IL-2.In summary, we disclose that IL-2 induces DC functional maturation and activation. Interestingly, our study suggests a direct effect of anti-CD25 monoclonal antibodies on DC and that CD25 expression regulation on human DC could be used to control immune response in vivo.