Dissertations / Theses on the topic 'Nonmyeloablative stem cell transplantation'

Hematopoietic stem cell transplantation (HSCT) is used to treat a wide range of hematologic and non-hematologic disorders. Recently, interest has grown in the potential of autologous HSCT coupled to gene therapy for the treatment of genetic blood disorders as a way of avoiding the severe immunologic reactions associated with allogeneic HSCT. However, the remaining risks in using myeloablative conditioning regimens to allow relatively small numbers of transplanted HSCs to be transplanted greatly limit the applicability of this approach. Nonmyeloablative regimens would be an appealing alternative but necessitate the generation of large numbers of genetically corrected HSCs to achieve therapeutic levels of chimerism. In this thesis I have explored the potential of forced overexpression of homeobox genes as a strategy to obtain the degree of HSC expansion required. In a first series of experiments, I found that HOXB4 and NUPHOX transduced and expanded HSCs maintain the ability of fresh HSCs to produce sustained, high level, polyclonal, lympho-myeloid chimerism when transplanted into mice given 2-2.5 Gy. I then tested the therapeutic efficacy of ex vivo expanded HSCs in nonmyeloablated mice with severe β-thalassemia caused by the homozygous deletion of the β-major globin gene (β-MDD). The results of these experiments showed that this approach could produce a dramatic improvement in the hematocrit, hemoglobin and RBC morphology and ultimately the cure of the thalassemic phenotype that was not achievable in recipients of equivalent numbers of unmanipulated BM cells or of cells transplanted immediately after transduction. Again, the cured mice displayed a sustained, high level of polyclonal chimerism. Together these data provide "proof of principle" of the curative potential of ex vivo expanded HSCs in a preclinical model of β-thalassemia treated with nonmyeloablative conditioning.
Medicine, Faculty of
Medical Genetics, Department of
Graduate

The Mucopolysaccharide (MPS) diseases are a group of lysosomal storage disorders, caused by a lack of the enzymes required for catabolism of glycosaminoglycans (GAGs), leading to severe neurological decline, skeletal deformities, organomegaly, cardiac and respiratory compromise, and premature death. The severe form of MPS I, Hurler syndrome, can be successfully treated using haematopoietic stem cell transplantation (HSCT), but the risks associated with myeloablation and immune suppression limit the broader application of HSCT to attenuated diseases. Successful engraftment in MPS I has been difficult to achieve, and requires fully myeloablative conditioning, whilst reduced intensity conditioning is a risk factor for graft rejection. Non-myeloablative conditioning generating reliable graft acceptance and high donor chimerism could increase safety and applicability of HSCT in genetic disease, therefore the aim of this research was to identify such a regimen in a clinically relevant mouse model of HSCT.Conditioning regimens developed in existing mouse models of HSCT have had limited clinical success, and often require clinically unachievable high cell doses or less stringent strain combinations to overcome allogeneic transplant rejection. To improve clinical relevance we used CBA donors and C57BL/6 recipients, which require full myeloablation with busulfan and immune suppression using non-depleting anti-CD4 and anti-CD8 monoclonal antibodies for engraftment of low cell doses across a major histocompatibility complex barrier. In syngeneic transplant donor chimerism was improved by generating a greater ratio of donor:recipient haematopoietic cells in the bone marrow initially, therefore we tested granulocyte colony stimulating factor (G-CSF), high cell dose and stem cell niche disruption and compared this to anti-CD40L costimulatory blockade in allogeneic transplant performed with a reduced dose of busulfan that was insufficient for graft acceptance. Despite improvements in initial engraftment with some of these treatments, only combined signal 1 and 2 T cell blockade were effective in reducing the dose of busulfan required for long-term graft acceptance. Early detection of MPS is important in treatment success; good disease biomarkers are vital, and biomarkers suitable for monitoring treatment outcome in MPS are lacking. We evaluated serum heparin cofactor II-thrombin (HCII-T) complex for MPS. We determined optimal sample collection and storage conditions, assay limitations and developed measurement in dried blood spots. Dermatan sulphate has a greater effect on in vivo HCII-T complex formation than heparan sulphate, thus in the MPS mouse models HCII-T is a reliable biomarker for MPS I, but not MPS IIIA or IIIB. HCII-T is greatly elevated in MPS I, II and VI patients, who all store dermatan sulphate, but it is also elevated by a small but significant amount in MPS III patients, who store heparan sulphate. HCII-T was also measured longitudinally in MPS I, II and VI patients, compared to an existing clinical biomarker, and validated against clinical outcomes to show that it is a good biomarker of short-term treatment outcomes and responds rapidly to perturbations in treatment. Finally, we determined whether an engraftment defect was observed in the MPS I mouse model, and show that this is present following both syngeneic and allogeneic HSCT. The effect of enzyme replacement therapy (ERT) and anti-inflammatory treatment prior to allogeneic HSCT was investigated, and initial results suggest that ERT, but not ibuprofen, may improve HSCT outcome. Overall, a clinically relevant mouse model of allogeneic HSCT has been developed and used to determine a non-myeloablative conditioning regimen that generates high levels of donor chimerism with a minimal dose of busulfan and blockade of both signal 1 and 2 of T cell activation. The conditions required to observe an engraftment defect in MPS I mice have also been defined, and preliminary studies have suggested that ERT, but not anti-inflammatory treatment, may overcome the engraftment defect in MPS I. Alongside this work, the HCII-T biomarker has been evaluated in MPS mouse models and patients, determining that it correlates well with short-term treatment outcomes. The techniques and models developed here will provide an excellent basis for further work in developing non-myeloablative conditioning for bone marrow transplant in MPS I.

Unsere Untersuchungen belegen, dass nichtmyeloablative Stammzelltransplantationen (NST) bei Patienten mit Hochrisiko-ALL oder -AML eine neue therapeutische Option darstellen. Die NST ermöglichte eine allogene Stammzelltransplantation bei Transplantationskandidaten mit Kontraindikationen gegen eine hochdosierte Strahlen- und Chemotherapie (Standardtransplantation). Nach NST kam es häufig zur Entwicklung von Infektionen und einer spät auftretenden akuten Transplantat-gegen-Wirt Reaktion (GvHD), diese waren aber mit einer geringeren Mortalität verbunden. Das erkrankungsfreie Überleben und das Gesamt-Überleben nach NST und nach Standardtransplantation waren fast gleich. Der höheren Rezidivrate nach NST stand eine höhere transplantationsassoziierte Mortalität nach Standardtransplantation gegenüber. Das Konditionierungsregime selber war ohne Einfluss auf das Überleben der Patienten. Sequenzielle Chimärismusuntersuchungen von Leukozytensubpopulationen erlaubten eine frühe Diagnose eines gemischten Chimärismus, der einen prädiktiven Wert für das Auftreten eines Rezidivs hatte. Ein stabiler gemischter Chimärismus wurde nicht beobachtet. Durch eine adoptive Immuntherapie mit Spenderlymphozyten (DLI) konnte bei der Mehrzahl der transplantierten Patienten mit gemischtem Chimärismus eine Chimärismuskonversion und eine lang anhaltende komplette Remission induziert werden, ein wichtiger Hinweis auf einen Transplantat-gegen-Leukämie Effekt (GvL) der Spenderzellen. Durch die NST rückt der immunologische Effekt der Transplantation gegenüber der Zytoreduktion bei der Standardtransplantation stärker in den Vordergrund. Unsere Ergebnisse sprechen für die Wirksamkeit eines GvL-Effektes bei akuten Leukämien auch nach NST. Die Erfahrungen mit der NST bei akuten Leukämien haben zu einer Anwendung bei refraktären Nierenzellkarzinomen geführt. Eine Tumorregression wurde erst nach Chimärismuskonversion und/oder Entwicklung einer GvHD beobachtet; diese Befunde sind vereinbar mit einem Transplantat-gegen-Tumor Effekt (GvT). Die transplantationsassoziierte Morbidität war allerdings bei diesen meist älteren Patienten erheblich. Die Therapie sollte ausschließlich im Rahmen klinischer Studien erfolgen, da es sich nach wie vor um ein experimentelles Therapieverfahren handelt. Die Analyse minimal residueller Erkrankung und der Einsatz hochauflösender Chimärismusuntersuchungen von Leukozytensubpopulationen sollte die Bedeutung des gemischten Chimärismus weiter klären und zu einem gezielteren Einsatz von DLI führen. Prospektive vergleichende Studien müssen in naher Zukunft den Stellenwert der NST untersuchen.
Our clinical investigations demonstrate, that nonmyeloablative stem cell transplantation (NST) is a novel therapeutic option in patients with high-risk ALL or AML. NST can be administered to patients eligible for allogeneic stem cell transplantation with contraindications against high-dose radio- and chemotherapy (standard SCT). After NST, infections and late onset acute graft-versus-host disease (GvHD) frequently occurred, but transplant-related mortality was low in contrast to standard SCT. Leukemia-free survival and overall survival were similar after NST and standard SCT. A higher relapse rate after NST was balanced by a higher transplant-related mortality after standard SCT. The conditioning regimen itself had no relevant impact on survival. Sequential chimerism analyses of leukocyte subpopulations resulted in early diagnosis of mixed chimerism, which proved to be predictive for later relapses. Stable mixed chimerism was not established in these patients. Adoptive immunotherapy in patients, who were in hematologic remission but had mixed chimerism after transplantation, induced conversion to complete donor chimerism and long-lasting complete remissions in the majority of patients, a strong hint to a graft-versus-leukemia-effect (GvL) of donor T-lymphocyte infusions. A change in the character of stem cell transplantation was achieved by NST with a shift from the predominantly cytoreductive effect of standard SCT towards an emphasis on the immunologic GvL-effect. Our results demonstrated the efficacy of a GvL-effect in acute leukemias after NST. The experiences with NST in acute leukemias have prompted studies in patients with refractory advanced renal cell cancer. Tumor regressions were observed only after chimerism conversion and / or development of GvHD, these results being compatible with a graft-versus-tumor effect (GvT). However, transplant-related morbidity was substantial in these mostly elderly patients. Therapy within clinical studies is mandatory, because allogeneic stem cell transplantation still has to be regarded an experimental procedure in these patients. The analysis of minimal residual disease and application of high-resolution chimerism analysis of leukocyte subpopulations by microarrays could lead to a more profound understanding of mixed chimerism and to a more rational use of DLI in the near future. Prospective randomized trials should be conducted to evaluate the role of NST.

Thesis (Ph. D.)--University of Sydney, 2009.
Title from title screen (viewed Apr. 24, 2008) Title from title screen (viewed Feb. 18, 2009) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Paediatrics and Child Health, Faculty of Medicine. Degree awarded 2009 ; thesis submitted 2008. Includes bibliographical references. Also available in print form.

Hematopoietic stem and progenitor cells (HSPCs) repopulate the blood system upon transplantation. A large-scale genetic approach to understand the factors that participate in successful engraftment has not been undertaken. In this thesis, I present the development of a novel live imaging-based competitive marrow repopulation assay in adult zebrafish, which allows fast and quantitative measurement of HSPC engraftment capability. Using this assay, a transplantation-based chemical screen was performed, which led to the discovery of 10 compounds that can enhance the marrow engraftment capability in zebrafish. Among them, the arachidonic acid-derived epoxyeicosatrienoic acids (EET), had conserved effects on both short- and long-term bone marrow engraftment in mice. Genetic analysis in zebrafish embryos demonstrated that EET acts through a \(G\alpha12/13\)-mediated receptor, which activates PI3K and induces transcription factors of the AP-1 family. This PI3K/AP-1 pathway directly induced the transcription of HSC marker, runx1, in embryos. The activation of PI3K by EET promoted HSPC migration and interactions with niche cells. Our studies define a role for EETs in the development of blood stem cells during embryogenesis, and in engraftment in adult vertebrates. The other compounds discovered in the screen implicate additional novel signaling pathways involved in the HSPC engraftment process, which require further investigation. In summary, this thesis elucidated an important role of bioactive lipids in regulating HSC engraftment in adults and during embryo development. Systematically mapping out the regulatory network will tremendously benefit both the basic understanding of stem cell biology and the clinical manipulation to generate better stem cells for transplantation.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for numerous haematological malignancies. Graft-versus-host disease (GVHD) is the major complication causing mortality and is classified into acute (aGVHD) and chronic. MicroRNAs play a significant role in inflammation and have reported potential as biomarkers of different diseases. This study has investigated the role of microRNAs in allo-HSCT outcomes and had two main aims; (1) identification of microRNAs specific to the aGVHD target organ, skin and (2) an investigation into the role of immune specific miRNAs (miR-146a and miR-155) in peripheral blood. Initially, pathway mining was performed on a list of 18 genes that were shown previously, to have deregulated expression levels with regards to GVHD. The pathway mining identified specific immunological pathways in relation to the genes and potential microRNA targets. Global microRNA profiling was performed on a discovery cohort that identified a signature microRNA list in skin biopsies obtained from patients at the time of cutaneous histopathological aGVHD onset (grades I-III) and healthy volunteers. Twelve microRNAs were selected for further validation and it was shown that miR-34a-5p, miR-34a-3p, miR-503-5p and let-7c-5p were elevated and significantly involved in allo-HSCT outcomes. There was an interaction between miR-34a-3p and miR-503-5p which was significantly diagnostic of aGVHD and let-7c-5p was significantly predictive of disease relapse. MiR-34a-5p protein targets; p53 and c-Myc were then evaluated in the same cohort. MiR-34a-5p expression levels and cells stained positively for p53 were significantly correlated in the epidermis. Preliminary optimization of miR-34a-5p knockdown study was successfully conducted which showed promising results in the reduction of T cell proliferation. The whole blood study showed that miR-146a-5p and its interaction with miR-155-5p was predictive of aGVHD incidence in pre-disease onset (Day+28) samples. Interestingly, the expression levels of miR-146a-5p and miR-155-5p negatively correlated with SPI1 (PU.1). In conclusion, these investigations showed that (1) the microRNAs studied in this investigation may regulate the expression levels of the selected 18 genes, (2) microRNA expression levels in clinical skin biopsies obtained at the time of aGVHD onset could potentially be used as diagnostic biomarkers for aGVHD and as predictive biomarkers for overall survival as well as relapse and (3) miR-146a-5p and miR-155-5p expression levels in whole blood could be used as predictive biomarkers for aGVHD incidence.

After traumatic injuries to the brachial plexus there is a risk that one or more of the spinal roots are torn from the spinal cord, known as avulsion injury. This often leads to paralysis and chronic pain, notoriously difficult to treat with current pharmacotherapy. Surgical treatment may improve motor function but sensory recovery is usually poor as sensory axons fail to establish functional connections inside the spinal cord. The aims of this thesis were to develop a model for dorsal root avulsion in rodents in order to investigate the potentials of stem cell therapy for enhancing sensory regeneration after spinal root avulsion. Two different types of stem cells, embryonic and neural crest stem cells, have been transplanted to the avulsion model and analysed using immunohistochemical methods. The results indicate that stem cells survive after transplantation to the avulsed dorsal root and associate with regenerating axons. Furthermore, the different stem cells display different phenotypes after transplantation where embryonic stem cells give rise to neurons located outside the spinal cord that could serve as projection neurons whereas the neural crest stem cells form elongated tubes outlining the avulsed dorsal root and are associated with regenerating neuronal fibers. We have also discovered that the neural crest stem cells migrate into the damaged spinal cord as single cells. The neural crest stem cells also display a diversity in generating both neuronal and glial cells that may have different beneficial effects in neural repair following dorsal root avulsion. To improve the survival of stem cell transplants, the potentials of co-transplanting embryonic stem cells together with nanoparticle delivered growth factor mimetics has been investigated. The results indicate that nanoparticle delivered growth factors improve both transplant survival and maturation in comparison to untreated controls and may be a promising strategy in stem cell transplantation.

Graft-versus-host disease (GvHD) remains the main complication associated with haematopoietic stem cell transplantation (HSCT). GvHD is caused by allo-reactive donor T cells mounting an attack against specific target tissues. CD4+CD25HiFoxp3+ regulatory T cells have been shown to modulate GvHD in vitro and also in vivo animal models. More recently early stage clinical trials have described the successful use of Treg to reduce the incidence of GvHD following HSCT. The aim of this study was to investigate further the suppressive mechanisms by which Treg are able to modulate GvHD and assess the influence of Treg on the beneficial graft-versus-leukaemia (GvL) effect therefore providing further insight into the use of Treg in the therapeutic management of GVHD. Data presented in this thesis demonstrates the successful isolation and expansion of a highly pure Treg population which maintained suppressive capacity throughout culture. We also confirmed that Treg retain suppressive capacity following cryopreservation resulting in reduced workload and increased consistency when used for in vitro functional studies. We also provide the first human in vitro evidence that Treg are able to prevent cutaneous GvH reaction by blocking the migration of effector T cells into the target tissues. The presence of Treg during allo-stimulation caused reduced effector cell activation, proliferation, IFNγ secretion and decreased skin homing receptor expression. Further investigation into the Treg modulation of dendritic cells demonstrated, for the first time in experimental in vitro human GvHD, that this was due to ineffective effector T cell priming in the presence of Treg caused by impairment of dendritic cell functions. Comprehensive phenotypic and functional analysis of Treg treated moDC showed their decreased antigen processing ability and allostimulatory capacity, resulting in a less severe GvH reaction in the skin explant model. Furthermore, this work has revealed that despite Treg impairing in vitro GvL mechanisms at a cellular level there was no association observed between increased Treg levels and the incidence of relapse in a small clinical cohort of HSCT patients. In conclusion this study has provided further insight into the mechanisms by which Treg are able to modulate GvHD. This would inform future clinical trials using Treg as a therapeutic alternative to current GvHD treatment and prophylaxis.

Abstract The investigation of the web of relationships between the different elements of the immune system has proven instrumental to better understand this complex biological system. This is particularly true in the case of the interactions between B and T lymphocytes, both during cellular development and at the stage of cellular effectors functions. The understanding of the B–T cells interdependency and the possibility to manipulate this relationship may be directly applicable to situations where immunity is deficient, as is the case of cancer or immune suppression after radio and chemotherapy. The work presented here started with the development of a novel and accurate tool to directly assess the diversity of the cellular repertoire (Chapter III). Contractions of T cell receptor diversity have been related with a deficient immune status. This method uses gene chips platforms where nucleic acids coding for lymphocyte receptors are hybridized and is based on the fact that the frequency of hybridization of nucleic acids to the oligonucleotides on a gene chip varies in direct proportion to diversity. Subsequently, and using this new method and other techniques of cell quantification I examined, in an animal model, the role that polyclonal B cells and immunoglobulin exert upon T cell development in the thymus, specifically on the acquisition of a broader repertoire diversity by the T cell receptors (Chapter IV and V). The hypothesis tested was if the presence of more diverse peptides in the thymus, namely polyclonal immunoglobulin, would induce the generation of more diverse T cells precursors. The results obtained demonstrated that the diversity of the T cell compartment is increased by the presence of polyclonal immunoglobulin. Polyclonal immunoglobulin, and particularly the Fab fragments of the molecule, represent the most diverse self-molecules in the body and its peptides are presented by antigen presenting cells to precursor T cells in the thymus during its development. This probably contributes significantly to the generation of receptor diversity. Furthermore, we also demonstrated that a more diverse repertoire of T lymphocytes is associated with a more effective and robust T cell immune function in vivo, as mice with a more diverse T cell receptors reject minor histocompatiblility discordant skin grafts faster than mice with a shrunken T cell receptor repertoire (Chapter V). We believe that a broader T cell receptor diversity allows a more efficient recognition and rejection of a higher range of external and internal aggressions. In this work it is demonstrated that a reduction of TCR diversity by thymectomy in wild type mice significantly increased survival of H-Y incompatible skin grafts, indicating decrease on T cell function. In addiction reconstitution of T-cell diversity in mice with a decreased T cell repertoire diversity with immunoglobulin Fab fragments, lead to a increase on TCR diversity and to a significantly decreased survival of the skin grafts (Chapter V). These results strongly suggest that increases on T cell repertoire diversity contribute to improvement of T cell function. Our results may have important implications on therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post myeloablative treatments. Based on the previous results, we tested the clinical hypothesis that patients with haematological malignancies subjected to stem cell transplantation who recovered a robust immune system would have a better survival compared to patients who did not recover such a robust immune system. This study was undertaken by the examination of the progression and overall survival of 42 patients with mantle cell non-Hodgkin lymphoma receiving autologous hematopoietic stem cell transplantation (Chapter VI). The results obtained show that patients who recovered higher numbers of lymphocytes soon after autologous transplantation had a statistically significantly longer progression free and overall survivals. These results demonstrate the positive impact that a more robust immune system reconstitution after stem cell transplantation may have upon the survival of patients with haematological malignancies. In a similar clinical research framework, this dissertation also includes the study of the impact of recovering normal serum levels of polyclonal immunoglobulin on the survival of patients with another B cell haematological malignancy, multiple myeloma, after autologous stem cell transplantation (Chapter VII). The relapse free survival of the 110 patients with multiple myeloma analysed was associated with their ability to recover normal serum levels of the polyclonal compartment of immunoglobulin. These results suggest again the important effect of polyclonal immunoglobulin for the (re)generation of the immune competence. We also studied the impact of a robust immunity for the response to treatment with the antibody anti CD20, rituximab, in patients with non- Hodgkin’s lymphoma (NHL) (Chapter VIII). Patients with higher absolute counts of CD4+ T lymphocytes respond better (in terms of longer progression free survival) to rituximab compared to patients with lower number of CD4+ T lymphocytes. These observations highlight again the fact that a competent immune system is required for the clinical benefit of rituximab therapy in NHL patients. In conclusion, the work presented in this dissertation demonstrates, for the first time, that diverse B cells and polyclonal immunoglobulin promote T cell diversification in the thymus and improve T lymphocyte function. Also, it shows that in the setting of immune reconstitution, as after autologous stem cell transplantation for mantle cell lymphoma and in the setting of immune therapy for NHL, the absolute lymphocyte counts are an independent factor predicting progression free and overall survival. These results can have an important application in the clinical practice since the majority of the current treatments for cancer are immunosuppressive and implicate a subsequent immune recovery. Also, the effects of a number of antineoplastic treatments, including biological agents, depend on the immune system activity. In this way, studies similar to the ones presented here, where methods to improve the immune reconstitution are examined, may prove to be instrumental for a better understanding of the immune system and to guide more efficient treatment options and the design of future clinical trials. Resumo O estudo da rede de inter-relações entre os diversos elementos do sistema immune tem-se mostrado um instrumento essencial para uma melhor compreensão deste complexo sistema biológico. Tal é particularmente verdade no caso das interacções entre os linfócitos B e T, quer durante o desenvolvimento celular, quer ao nível das funções celulares efectoras. A compreensão da interdependência entre linfócitos B e T e a possibilidade de manipular esta relação pode ser directamente aplicável a situações em que a imunidade está deficiente, como é o caso das doenças neoplásicas ou da imunossupressão após radio ou quimioterapia. O trabalho apresentado nesta dissertação iniciou-se com o desenvolvimento de um novo método laboratorial para medir directamente a diversidade do reportório celular (Capítulo III). Reduções da diversidade do reportório dos receptores de células T têm sido relacionadas com um estado de imunodeficiência. O método desenvolvido utiliza “gene chips”, aos quais hibridizam os ácidos nucleicos codificantes das cadeias proteicas dos receptores linfocitários. A diversidade é calculada com base na frequência de hibridização do ácido nucleico da amostra aos oligonucleótidos presentes no “gene chip”. De seguida, e utilizando este novo método e outras técnicas de quantificação celular examinei, num modelo animal, o papel que as células policlonais B e a imunoglobulina exercem sobre o desenvolvimento linfocitário T no timo, especificamente na aquisição de um reportório diverso de receptores T (Capítulos IV e V). Testei, então, a hipótese de que a presença no timo de péptidos mais diversos, como a imunoglobulna policlonal, induzisse a génese de precursores T mais diversos. Demonstrámos que a diversidade do compartimento T é aumentado pela presença de imunoglobulina policlonal. A imunoglobulina policlonal, e particularmente os fragmentos Fab desta molécula, representam as moléculas autólogas mais diversas presentes nos organismos vertebrados. Estes péptidos são apresentados por células apresentadoras de antigénio às células precursoras T no timo, durante o desenvolvimento celular T. Tal, provavelmente, contribui para a génese da diversidade dos receptores. Também demonstrámos que a presença de um reportório mais diverso de linfócitos T se associa a um incremento da função imunológica T in vivo. Uma diversidade de receptores T mais extensa parece permitir um reconhecimento e rejeição mais eficientes de um maior número de agressores internos e externos. Demonstrámos que ratinhos com receptores de células T (RCT) com maior diversidade rejeitam transplantes cutâneos discordantes para antigénios minor de histocompatibilidade mais rapidamente do que ratinhos com um menor reportório T (Capítulo V). Por outro lado, uma redução da diversidade do RCT, causada por timectomia de ratinhos de estirpes selvagens, mostrou aumentar significativamente a sobrevivência de transplantes cutâneos incompatíveis para o antigénio H-Y (antigénio minor de histocompatibilidade), indicando uma diminuição da função linfocitária T. Além disso, a reconstituição da diversidade dos linfócitos T em ratinhos com uma diversidade de reportório T diminuída, induzida pela administração de fragmentos Fab de imunoglobulina, conduz a um aumento da diversidade dos RCT e a uma diminuição significativa da sobrevivência dos enxertos cutâneos (Capítulo V). Estes resultados sugerem que o aumento do reportório de células T contribui para uma melhoria das funções celulares T e poderão ter implicações importantes na terapêutica e reconstitutição imunológica em contexto de SIDA, neoplasias, autoimunidade e após tratamentos mieloablativos. Baseado nos resultados anteriores, decidimos testar a hipótese clínica de que doentes com neoplasias hematológicas sujeitos a transplantação de precursores hematopoiéticos e com recuperação imunológica precoce após transplante teriam uma sobrevivência mais longa do que doentes que não recuperassem tão bem a sua imunidade. Analisámos a sobrevivência global e sobrevivência sem doença de 42 doentes com linfoma não Hodgkin de células do manto sujeitos a transplante autólogo de precursores hematopoiéticos (Capítulo VI). Os resultados obtidos mostraram que os doentes que recuperaram contagens mais elevadas de linfócitos imediatamente após o transplante autólogo, apresentaram uma sobrevivência global e sem progressão mais longa do que doentes que não recuperaram contagens linfocitárias tão precocemente. Estes resultados demonstram o efeito positivo de uma reconstitutição imunológica robusta após transplante de presursores hematopoiéticos, sobre a sobrevivência de doentes com neoplasias hematológicas. Do mesmo modo, estudámos o efeito que a recuperação de níveis séricos normais de imunoglobulina policlonal tem na sobrevivência de doentes com outras neoplasias hematológicas de linfócitos B, como o mieloma múltiplo,após transplante autólogo de precursos hematopoiéticos (Capítulo VII). A sobrevivência livre de doença dos 110 doentes com mieloma múltiplo analizados está associada com a sua capacidade de recuperar níveis séricos normais do compartmento policlonal de imunoglobulina. Estes resultados pioneiros indicam a importância da imunoglobulina policlonal para a génese de competência imunológica. Também estudámos o impacto de um sistema imunitário eficiente sobre a resposta ao tratamento com o anticorpo anti CD20, ituximab, em doentes com linfoma não Hodgkin (LNH) (Capítulo VIII). Os resultados mostram que doentes com valores mais elevados de linfócitos T CD4+ respondem melhor (em termos de maior sobrevida livre de doença) ao rituximab, do que doentes com valores mais baixos. Estas observações ilustram a necessidade de um sistema imunitário competente para o benefício clínico da terapêutica com rituximab em doentes com LNH. Em conclusão, o trabalho apresentado nesta dissertação demonstra que as células B e a imunoglobulina policlonal promovem a diversidade das células T no timo e melhoram a função linfocitária T periférica. Concomitantemente, também demonstrámos que, no contexto de reconstituição imune, por exemplo, após transplante autólogo de precursores hematopoiéticos em doentes com linfomas de células do manto, o número absoluto de linfócitos é uma factor independente da sobrevivência. Os resultados demonstram, também, a importância dos valores de linfocitos T na resposta ao tratamento com rituximab no caso de doentes com LNH. O mesmo princípio se prova pelo facto de que doentes com mieloma múltiplo sujeitos a transplante autólogo de precursores hematopoiéticos que recuperam valores normais séricos de imunoglobulinas policlonais, terem melhores taxas de resposta em comparação com doentes que não recuperam valores normais de imunoglobulinas policlonais. Estes resultados podem ter importantes aplicações na prática clínica dado que a maioria dos tratamentos de doenças neoplásicas implica imunossupressão e, subsequente, recuperação imunológica. Estes estudos podem ser um instrumento fundamental para uma melhor compreensão do sistema imune e guiar uma escolha mais eficiente de opções terapêuticas bem como contribuir para a concepção de futuros estudos clínicos.

京都大学
新制・課程博士
博士(医学)
甲第23419号
医博第4764号
新制||医||1052(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 滝田 順子, 教授 杉田 昌彦, 教授 朝長 啓造
学位規則第4条第1項該当
Doctor of Medical Science
Kyoto University
DFAM

Haematopoietic stem cell transplantation (HSCT) is an effective therapy for many malignant and non‐malignant diseases. However, adverse events such as infections, Graft‐versus‐Host Disease (GvHD) and relapse limit the wider use of HSCT. The identification of factors that predict the outcome of the transplant can be a vital tool to offer patients better options for treatment. Since there is a significant immunological contribution to the outcome of the transplant, it is of interest to ask to what extent the immune status of the patient prior to transplant might influence their subsequent recovery. After transplantation, the reconstitution of the T cell compartment in the patient relies on both expansion of existing T cells and the production of new ones via the thymus; in older patients this latter process is compromised by age‐dependent thymic involution. Improvements in thymus function leading to improvement in immune reconstitution after an HSCT may provide significant benefits, potentially reducing mortality from both infections and GvHD. In one study described in this thesis, an improvement in T cell reconstitution, in particular of the CD4 compartment, could be demonstrated after the administration of an LHRH agonist before transplantation from an allogeneic donor. Such an effect could not be demonstrated in an autologous setting. This may reflect a differential requirement for thymus function in the two transplant settings. In additional studies, various immune parameters including T cell subsets and cytokine profiles from the patient, that could affect the transplant outcome were analysed. The findings indicate that high levels of regulatory T cells (Tregs) as well as high levels of regulatory cytokines in patients pretransplant are factors that predict relapse after transplantation. It is likely that these act by suppressing anti‐tumour responses in the patient. These findings may provide a useful tool to stratify the patients into high and low risk categories prior to transplantation.

Severe injury may result in disconnection between the peripheral and central nervous system. Regeneration of the central portion of sensory neurons into the spinal cord is notoriously poor in adult mammals, with low regenerative drive and an unpermissive central environment, most likely resulting in persistent loss of sensory function. A variety of strategies have been addressedto augment regeneration, including application of growth promoting factors, counteraction of inhibitory molecules, and provision of growth permissive substrates. Stem cells have been investigated in these contexts, as well as for the possibility of providing new neurons to act as a relay between the periphery and spinal cord. Here we have investigated different sources of neural stem cells for their ability to form neurons and glia after transplantation to the periphery; to project axons into the spinal cord; and to assist regeneration of surviving sensory neurons. These have been performed at two locations: the "dorsal root ganglion cavity", and the transitional zone following dorsal root avulsion. Neurons and glia were generated form mouse boundary cap neural crest stem cells and embryonic stem cell derived ventral spinal cord progenitors, and in addition to this, regeneration of sensory fibers was observed after transplantation of human fetal spinal cord derived progenitors and human embryonic stem cell derived ventral spinal cord progenitors. Further, delivery of neurotrophic factor mimetics via mesoporous silica nanoparticles proved a valuable tool for stem cell survival and differentiation. While technological advances make in vivo differentiation a realistic goal, our findings indicate that so far assisting regeneration of host sensory fibers to reconnect with the spinal cord by transplantation of stem cells is a more reliable strategy.

Spinal root avulsion leads to paralysis and loss of sensory function. Surgical methods can improve motor function and ameliorate pain but sensory recovery in adults is poor. Previous studies have shown that cell transplantation or treatment with trophic factors can improve functional outcome in rodents after dorsal root transection or crush. Here, a dorsal root injury model, more similar to human avulsion injuries, was used. The aims of this thesis were to investigate the behaviour of different stem cells following transplantation to avulsed dorsal roots and asses their potential to serve as possible regenerative therapy. In paper I, different murine stem cell types were transplanted to avulsed dorsal roots in rats. Murine embryonic stem cells remained outside the spinal cord and were surrounded by glutamatergic terminals. Boundary cap neural crest stem cells (bNCSC) formed elongated bands outside the spinal cord and migrated to the spinal cord as single cells. In paper II, transplanted bNCSC were further characterized. bNCSC remaining outside the spinal cord expressed glial markers and were associated with different types of sensory fibres. bNCSC that migrated into the injured spinal cord expressed different neuronal markers. In paper III, effects of bNCSC transplantation on local vasculature and glial scar formation were studied. bNCSC increase angiogenesis in a non dose response manner and participate in boundary glial scar formation. In paper IV, bNCSC spinal migration was analysed using two different injury models - dorsal root transection and dorsal root avulsion. In addition, bNCSC capacity to support sensory regeneration was assessed and the results suggest that bNCSC do not support robust regeneration of avulsed afferents. In paper V, an in vitro stem cell model system was used to assess the possibility of using artificial nanomaterials to deliver differentiation factors. Cells treated with either soluble factors or particle-delivered factors showed similar differentiation patterns. Stem cell transplantation offers several opportunities following dorsal root avulsion, including cell replacement and regenerative support. By elucidating the mechanisms by which stem cells can assist regeneration of avulsed afferents will allow for more targeted or combinatorial approaches, including growth factor treatment.

Retinal pigment epithelium (RPE) cells perform a variety of roles that are principally directed at maintaining retinal function and homeostasis and their loss causes conditions such as age-related macular degeneration (AMD) and Stargardt disease (STGD). Regeneration of the loss or dystrophic RPE cells with stem cell-derived RPE may provide a potential therapeutic option. STGD is the commonest form of juvenile-onset, inherited macular degeneration. In order to determine the safety and efficacy of embryonic stem (ES) cell-derived RPE transplantation for the treatment of STGD, twelve subjects with STGD received a suspension of hES-derived RPE cells in escalating dose cohorts. Following the intervention, areas of sub retinal pigmentation were noted in all participants, suggestive of engraftment and survival. Transplanted hES-derived RPE cells were often observed overlying regions of atrophic Bruch's membrane (BrM). There was no evidence of tumorigenicity, immune adverse events or other serious safety concerns related to the transplanted cells. Furthermore, there was no significant change in visual function in the study eye of any participant. In order to improve the efficacy of ES-derived RPE transplantation, I developed an improved rodent model of retinal degeneration that features focal regions of atrophic BrM. I used a diode laser to selectively ablate RPE and observed focal regions of RPE atrophy with corresponding changes in choroidal vasculature that resemble those observed in retinal degenerative disease. Moreover, transplantation of human ES- and human induced pluripotent stem cell (iPS)-derived RPE resulted in re-population and restoration of RPE morphology post ablation. Although transplanted ES-derived RPE survived well on healthy BrM, attachment and survival of RPE is compromised on BrM that exhibits AMD or senescent changes. A potential strategy to promote survival and engraftment where there is damaged BrM is to deliver ES-derived RPE on a carrier substrate. I used a bespoke electrospun scaffold consisting of poly(e-caprolactone) and seeded this with either hESC or hiPSC-derived RPE. Transplantation of ES-derived RPE resulted in a functional monolayer of RPE with correct orientation and polarity on a biodegradable, porous and biocompatible membrane. These studies support further work in large animal models using ES-derived RPE scaffolds to restore the RPE in the presence of a compromised BrM.

Allogeneic stem cell transplantation is associated with a powerful T cell-mediated ‘graft-versus-leukaemia’ (GvL) effect and also ‘graft-versus-host disease’ (GvHD). Developing therapies to improve survival relies on greater understanding of these responses in order to enhance GvL and suppress GvHD. To gain an increased understanding of the mechanisms of GvHD I measured frequencies of Th1, Th17 and Treg subsets in the blood of 32 GvHD patients (during and outside of disease episodes) and in 21 patients who did not suffer GvHD. No associations between T cell subset frequencies or serum cytokine concentrations and incidence of GvHD were evident. My GvL work addressed whether T cell responses to cancer/testis antigens (CTAg) could be detected in a cohort of 41 patients who had undergone allogeneic stem cell transplantation for the management of acute myeloid leukaemia and multiple myeloma. CTAg-specific CD8+ T cell immune responses were observed within peripheral blood of five patients, with an average magnitude of 0.045% of the CD8+ T cell repertoire. T cell immunity was focussed against peptides derived from MAGE proteins and was increased within the bone marrow. These immune responses are likely to contribute to tumour eradication following transplantation and represent a potential novel mechanism for GvL.

One strategy to restore vision in retinitis pigmentosa and related retinal degenerations is by cell replacement. Typically, patients lose vision when the outer retinal photoreceptor layer is lost, and so the therapeutic ideal would be to restore vision at this stage of disease. It is not currently known if a degenerate retina lacking the outer nuclear layer of photoreceptor cells would allow the survival, maturation and reconnection of replacement photoreceptors, as prior studies used hosts with a pre-existing outer nuclear layer at the time of treatment. Here, using a murine model of severe human retinitis pigmentosa at a stage when no host rod cells remain, transplanted rod precursors are shown to reform an anatomically distinct and appropriately polarised outer nuclear layer. A trilaminar nuclear organisation is returned to the rd1 hosts that had only two retinal layers before treatment. The newly introduced rod precursor cells were able to resume their developmental programme in the degenerate host niche to become mature rods with light- sensitive outer segments, and reconnected with host neurons downstream. Visual function, assayed in the same animals before and after transplantation, was restored in animals with zero rod function at baseline. These observations suggest that a cell therapy approach may reconstitute a light-sensitive cell layer de novo and hence repair a structurally damaged visual circuit. Rather than placing discrete photoreceptors amongst pre-existing host outer retinal cells, total photoreceptor layer reconstruction may provide a clinically relevant model to investigate cell-based strategies for retinal repair.

Haemopoietic recovery after high dose chemotherapy (HOC) in the treatment of haematological disease may be slow and/or incomplete. This is generally attributed to progressive haemopoietic stem cell failure, however we hypothesize that HOC induced defective haemopoiesis may be in part due to poor stromal function. Although chemotherapy is known to damage mature bone marrow stromal cells in-vitro, the extent to which marrow mesenchymal stem cells (MSC) are damaged by HOC in-vivo and in-vitro is unknown. To firstly address this question the physical characteristics and functional properties of marrow MSC derived from patients who have received chemotherapeutic treatment for various haematological diseases were investigated. Subsequently a suitable in-vitro treatment culture model was developed and the effects of chemotherapy exposure in-vitro using cell culture and proteomic techniques were shown. Results of this study demonstrate proliferative and phenotypic changes to patient MSC caused by HOC regimens. In contrast, the differentiation capacity, and ability to form functional marrow stroma after exposure to HOC in-vivo was equal to that of patient MSC studied prior to HOC. Chemotherapeutic exposure to MSC cultures in-vitro have confirmed changes seen in-vivo, with abnormalities evident in MSC proliferation, differentiation and also in their ability to support haemopoietic stem cell migration and repopulation after treatment. A reduced C044 expression on MSC, after exposure to cyclophosphamide, was also observed and the importance of this molecule shown through con-focal microscopy demonstrating its interaction with C034+ haemopoietic stem cells. Using proteomic techniques differences in protein expression by MSC, relating to changes in their function after chemotherapy exposure, were also observed after treatment with cyclophosphamide or melphalan. Finally the role of keratinocyte growth factor as a potential cyto-protective agent to MSC against damage caused by chemotherapy exposure was tested. Results indicated there was significant evidence to indicate that KGF was able to preserve reductions in C044 expression levels after MSC exposure. It was concluded that marrow MSC sustain prolonged injury due to recurrent courses of HOC in-vivo and in-vitro. However, the clinical importance of the chemotherapy induced defects we have observed must be determined through the initiation of prospective randomized trials of the effects of MSC co-transplantation on haemopoietic recovery in the setting of HOC with and without haemopoietic stem cell rescue.

Trouver de nouvelles sources de foie pour les transplantations est un vrai enjeu. Les obstacles principaux à la transplantation d'organes sont la pénurie de greffons et les lourds traitements immunosuppresseurs à vie. La possibilité de reprogrammer les cellules du patient et les cultiver donne accès à une quantité virtuellement illimitée de cellules a transplanter, éliminant de ce fait le besoin de donneurs d'organesMon doctorat se divise en deux parties : la première consiste à faire des cellules hépatiques en programmant des cellules souches humaines. La deuxième consiste à transplanter ces dernières dans des foies de souris ayant des insuffisances hépatiques aigues et chroniques pour étudier la régénération du foie. Les nouveautés de ce projet sont le fait d'étudier la première phase d'intégration post transplantation des cellules car cette phase est critique dans la régénération. De plus je comparerai la régénération du foie par les cellules transplantées dans une insuffisance hépatique aigue avec une insuffisance hépatique chronique. Une semaine après transplantation, le foie de ces souris contenait les cellules greffées qui se sont implantées et celles-ci se comportaient comme des cellules de foie bona fide, produisant une protéine, l'albumine humaine. Encore mieux : l'analyse des fonctions du foie a relevé un retour à une fonction normale en 3 jours avec transplantation contre 7 jours sans cellules transplantées. Ces résultats sont très encourageants quant à la possibilité d'utiliser des cellules dérivées de cellules souches à des fins thérapeutiques
Finding new sources for liver transplants is a real issue. The main obstacles to organ transplantation are the shortage of grafts and heavy immunosuppressive treatment for life. The ability to reprogram the patient's cells and grow them gives access to a virtually unlimited amount of transplanted cells, thereby eliminating the need for an organ donor. My doctorate is in two parts: the first is to make liver cells by programming of human stem cells. The second is to transplant those cells in the livers of mice with acute and chronic liver failure to study liver regeneration. The novel parts of this project are to study the first phase of post-integration cell transplantation because this phase is critical in regeneration. Also I would compare liver regeneration by transplanted cells in acute liver failure with chronic liver failure. One week after transplantation, the liver of these mice contained the grafted cells and which are located if they behaved as bona fide liver cells producing a protein, human albumin. Even better: analysis of liver function noted a return to normal function in three days with seven days without transplantation against transplanted cells. These results are very encouraging when the possibility of using cells derived from stem cells for therapeutic purposes

Spermatogonial stem cells (SSC) self-renew and differentiate into spermatozoa throughout the life of the male. SSC transplantation is a potential method for the propagation of genetically important males. These cells have been isolated in different mammalian species using specific cell surface markers, but not in felines. The goal of this study was to explore a relevant strategy for conservation of endangered felids by characterizing domestic cat (Felis catus) SSCs and assessing their ability for self-renewal after transplantation. Firstly, SSC and pluripotent surface markers, identified in non-feline species, were tested for expression in mixed germ cells from adults by immunocytochemistry and flow cytometry, with immunohistochemical confirmation of expression in prepubertal and adult testis tissue. Secondly, subpopulations were purified through fluorescence-activated cell sorting using spermatogonia-specific markers and molecularly characterized to ascertain levels of pluripotent transcription factors expressed in cat embryos. Thirdly, subpopulations of mixed germ cells and purified spermatogonial cells were transplanted to prepubertal cats to determine: 1) if SSCs capable of colonization were present, and 2) the value of using adolescent domestic cats without depletion of endogenous germ cells as recipients. Fourthly, various culture conditions were evaluated to identify proteins and factors required to maintain proliferation of cat SSCs. Lastly, adult lion testis tissue was characterized with the same surface markers, and mixed germ cells were transplanted to cat testes to evaluate the cat as a suitable host for lion SSC colonization and differentiation. Pluripotent surface markers were more reliable than the common SSC surface markers for isolating cat SSCs. Varying expression levels of pluripotent transcription factors between the different purified cell populations identified spermatogonial subpopulations. Cell purification was not necessary to colonize recipient testes, and transplantation validated the use of prepubertal males as recipients without depletion of endogenous cells. Unlike spermatogonia within mixed germ cells, purified spermatogonia were not maintained under various culture conditions; therefore, SSC culture conditions must be optimized. Similarities in the expression patterns of surface markers in lion and cat spermatogonia were revealed, and colonization of lion SSCs in cat testes provided further evidence of the domestic cat’s relevance as a model for exotic felid SSC transplantation.

Doctor of Philosophy (PhD)
The BCNU + O6benzylguanine (O6BG) driven selective enrichment strategy was first established for enhanced transplantation of hematopoietic stem cells. This study describes a novel application of this BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation. Furthermore, this study addresses the three main limitations observed in previously reported skeletal muscle stem cell transplantation strategies. Limitation of ineffective donor cells which lack the ability for successful engraftment was overcome by using a heterogeneous population of donor cells which are present during a normal skeletal muscle regeneration response. The limitation of donor cell death upon transplantation as a result of competition from the endogenous stem cells of the host muscles was overcome by elimination of host muscle stem cells with BCNU + O6BG treatment. Efficiency of elimination of host muscle stem cells was further demonstrated by the complete inhibition of a regeneration response up to 3 months in injured, BCNU + O6BG treated muscles. The limitation of localised engraftment as a result of intramuscular injection of donor cells was also addressed. The transplanted donor cells demonstrated the ability to migrate via systemic circulation. This characteristic of the donor cells would allow the transplantation of cells via intraarterial or intravenous delivery which would overcome the limitation of localised engraftment. Finally, application of the BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation demonstrated enhanced engraftment. This is the first reported attempt of enhanced stem cell transplantation in a solid tissue achieved upon application of the BCNU + O6BG driven selective enrichment strategy. This study provides the basis for application of the BCNU + O6BG driven selective enrichment strategy in other tissues where stem cell transplantation is considered.

In 51 (33 adult-18 paediatric) allogeneic hematopoietic stem cell transplant recipients we aimed to evaluate: i) the incidence of EBV infection and potential risk factors; ii) the suitability of whole blood (WB) as clinical specimen to monitor the risk of patients to develop EBV-related post-transplant lymphoproliferative disorder (EBV-PTLD); iii) the clinical utility of combined virological-immunological monitoring; iv) the management of infection and the incidence of EBV-PTLD. Quantitative real-time PCR assay was performed on WB samples for all patients. EBV-DNA quantification also in peripheral blood mononuclear cells (PBMCs) samples was adopted for actively EBV infected patients. Immunological monitoring of infection was performed by Enzyme-linked ImmunoSPOT assay evaluating the EBV-specific cell-mediated immunity (CMI). The incidence of EBV infection was 51% and the frequency of EBV-PTLD was 3.9%. Reduced-intensity conditioning (RIC) in combination with in vivo T-cell depletion was associated with higher frequencies of infection (P=0.036). A significant correlation (P<0.001) between EBV-DNA levels in WB and PBMC samples was obtained in adult (r=0.787) and paediatric (r=0.976) patients. A similar kinetics of EBV-DNA in blood compartments was observed. Clinically, both specimen types appeared to be equally informative. The lack of EBV-specific CMI during/after active infection was associated with a higher median peak level of EBV-DNA in WB (P=0.013) and a greater severity of infection. The 54.5% of the patients without EBV-specific CMI needed anti-CD20 therapy and the 27.2% developed EBV-related complications, including a lethal PTLD. All patients with EBV-specific CMI controlled EBV replication and were asymptomatic. WB proved to be a suitable clinical specimen to monitor the risk of patients to develop EBV-related complications. RIC combined with in vivo T-cell depletion is a risk factor for the development of infection. EBV-specific CMI is a critical determinant in controlling the infection and consequently the EBV-related complications. Combined virological-immunological monitoring could improve the management of infection.

The doctoral thesis aimed to evaluate in a comparative way the benefit chimerism assessment in isolated cell populations (ICP) versus conventional monitoring in whole blood cells (WBC). The study was initiated in Pediatric Bone Marrow Transplant Service of Charité Children’s Hospital (Humboldt University, Berlin). Children suffered from: acute lymphoblastic leukemia (ALL), Fanconi anemia (FA) and adrenoleukodystrophy (ALD) were included into the study. Thereafter the doctoral thesis was accomplished at the Vilnius University where evaluation of the own experience of chimerism analysis in pediatric patients was performed. Chimerism was prospectively monitored in WBC and three cell subsets: CD3, CD19 and CD34. Cell populations were extracted from peripheral blood using immunomagnetic beads. Following polymorphisms of short tandem repeats between donor and recipient were compared. The analysis reveled that following myeloablative conditioning in patients with ALL and ALD analysis of ICP revealed persistent autologous hemopoiesis despite stable donor chimerism in WBC. In contrast after reduced intensity conditioning 45.5% of FA recipients had no evidence of autologous signals either in WBC, or in ICP. FA were found to develop stable long-lasting mixed chimerism associated with better overall survival. development of MC was related to the infusion of bone marrow but not to peripheral blood stem cells. Chimerism in ICP did no affected transplant outcome in any disease group.
Moklsiniame darbe tirtas chimerizmas vaikams po alogeninės kraujodaros kamieninių ląstelių transplantacijos. Tyrimas pradėtas Charité klinikose (Humboldto universitetas, Berlynas), kur chimerizmo tyrinėjimams pasirinkti recipientai, sergantys ūmia limfoblastine leukemija (ŪLL), Fanconi anemija (FA) ir adrenoleukodistrofija (ALD). Sergant šiomis ligomis nuoseklus chimerizmo stebėjimas ALP atliktas pirmą kartą. Darbas tęstas Vilniaus universitete, kuomet pirmą kartą buvo atlikti chimerizmo tyrinėjimai Lietuvoje transplantuotiems vaikams. Donoro ir recipiento ląstelėms atskirti naudoti polimorfiniai DNR žymenys, kurie tirti periferinio kraujo leukocituose (PKL) ir atskirose ląstelių populiacijose (ALP): CD3, CD19 ir CD34. Ląstelių frakcijoms atskirti naudotos imunomagnetinės dalelės. Atlikus chimerizmo analizę nutatyta, kad esant visiškam donoro chimerimui PKL, 74,4% ŪLL ir 66,7% ALD recipientų po mieloabliacinio kondicionavimo ląstelių populiacijose išlieka autologinė, aptinkama iki kelerių metų po transplantacijos. Po sumažinto intensyvumo kondicionavimo 45,5% FA recipientų autologinės kraujodaros pėdsakų nerandama nei PKL, nei ALP. FA ligoniams vystosi stabilus ilgalaikis mišrus chimerizmas, kuris susijęs su su ilgesniu išgyvenamumu po transplantacijos. Nustatėme, kad sergantiems FA galimybė išsivystyti mišriam chimerizmui yra 20 kartų didesnė perpylus kaulų čiulpus, nei periferinio kraujo kamienines ląsteles.