Orientadores: Cármino Antonio de Souza, Afonso Celso Vigorito / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T14:24:56Z (GMT). No. of bitstreams: 1 Colella_MarcosPaulo_M.pdf: 4664560 bytes, checksum: ce76d7ca7d69ede5a86dc9fe14e7bbca (MD5) Previous issue date: 2014 / Resumo: O Transplante de Células Tronco Hematopoiéticas (TCTH) Alogênico representa uma possibilidade de cura para pacientes portadores de doenças hematológicas malignas e benignas. Porém, como qualquer modalidade de tratamento, apresenta efeitos adversos que podem ser graves, inclusive causando a morte. Com o intuito de se avaliar a influência que as comorbidades teriam na mortalidade não relacionada à recidiva (MNRR), foi criada uma ferramenta, o Índice de Comorbidade específico do Transplante de Células Tronco Hematopoiéticas (Hematopoietic Cell Transplant Specific Comorbidity Index - HCT-CI). Nossos objetivos, portanto, foram validar o HCT-CI na população de pacientes submetidos a TCTH Alogênico em nossa instituição, no período de 1993 a 2010, e avaliar outros fatores de riscos envolvidos na MNRR e na Sobrevida Global (SG). Os prontuários de 457 pacientes foram revistos e as informações referentes às comorbidades contidas no HCT-CI foram registradas. A maioria dos pacientes (59%) recebeu o índice 0, seguido de 30% de pacientes com índice de 1-2 e 11% com índice ? 3. Na análise univariada, os pacientes com HCT-CI igual a zero, comparados aos com HCT-CI ?1, apresentaram uma MNRR de 33% vs. 45% (p=0.01) e SG de 53% vs. 35% (p=0.001); nos pacientes que ao transplante apresentavam doença de baixo risco, comparados aos com doença de alto risco, a MNRR foi de 30% vs. 50% (p<0.0001) e SG de 57% vs. 27% (p<0.0001); o tipo de enxerto (medula óssea vs. sangue periférico) apresentou MNRR de 29% vs. 49% (p<0.0001) e SG de 56% vs. 34% (p<0.0001). A análise multivariada confirmou a influência do HCT-CI na MNRR e na SG, do risco da doença sobre a SG e do tipo de enxerto na MNRR. O tipo de condicionamento (baixa dose vs. alta dose) não teve influência na MNRR e SG, tanto na análise univariada quanto na multivariada. Quanto o grupo foi estratificado pelo HCT-CI (0 e ?1) o risco da doença ao transplante e o tipo de enxerto tiveram influência na MNRR e na SG, na análise univariada e multivariada, tanto nos pacientes com HCT-CI 0 e ?1. Não houve influência do tipo de condicionamento. O HCT-CI foi validado na nossa população de pacientes submetidos ao TCTH alogênico e identificamos outros fatores de risco que tiveram influência na MNRR e na SG. O HCT-CI, portanto, deve ser utilizado como guia no planejamento da estratégia terapêutica em pacientes portadores de comorbidades / Abstract: Allogeneic hematopoietic stem cell transplant is an important modality of treatment for patients bearing malignant and benign hematologic diseases, representing a chance of cure. As every treatment, it has a treatment-related mortality, possibly influenced by comorbidities. To better evaluate the influence of comorbidities on Non-Relapse Mortality (NRM) and Overall Survival (OS), the Hematopoietic Cell Transplant Specific Comorbidity Index (HCT-CI) was developed. Our objective was to apply the HCT-CI and to find risk factors for NRM and OS in patients who underwent an allogeneic hematopoietic stem cell transplant in our institution, between 1993 and 2010. Medical charts from 457 patients were reviewed. Most patients (59%) were classified in score 0, followed by 30% of cases with score 1-2 and 11% score 3-7. In a univariate analysis, patients with comorbidity score 0, compared with score ?1 had a NRM of 33% vs. 45% (p=0.01) and OS at 5 years of 53% vs. 35% (p=0.001); patients with low risk disease at transplant, compared with high risk disease, had a NRM of 30% vs. 50% (p<0.0001) and OS of 57% vs. 27% (p<0.0001); graft source (bone marrow vs. peripheral blood) had a NRM of 29% vs. 49% (p<0.0001) and OS 56% vs. 34% (p<0.0001). The multivariate analysis confirmed the influence of HCT-CI score on NRM and OS, disease risk at transplant on OS and graft source on NRM. The conditioning type (low dose vs. high dose) did not influence the NRM and OS in both univariate and multivariate analysis. When stratified by comorbidity (0 and ?1), disease status at transplant and graft source influenced NRM and OS in univariate and multivariate analysis, either in the group of patients with HCT-CI 0 and ?1. The conditioning type had no impact. Based on our data, we were able to validate de HCT-CI in our institution and to identify other risk factors with influence on NRM and OS. The HCT-CI, therefore, could be used to guide the treatment strategy of patients with comorbidities / Mestrado / Clinica Medica / Mestre em Clinica Medica

Introdução: O transplante de células‐tronco hematopoéticas (TCTH) é um procedimento complexo, que envolve diferentes fatores e condições biopsicossociais. O acompanhamento dos dados desses pacientes é fundamental para a obtenção de informações que possam auxiliar a gestão, aperfeiçoar a assistência prestada e subsidiar novas pesquisas sobre o assunto. Objetivos: desenvolver um modelo de banco de dados (BD) de pacientes submetidos a TCTH, contemplando as principais variáveis de interesse na área. Métodos: Trata‐se de um estudo aplicado, onde utilizou‐se a metodologia de desenvolvimento de um BD relacional, seguindo três etapas principais (modelo conceitual, modelo relacional, modelo físico). O modelo físico proposto foi desenvolvido na plataforma Research Electronic Data Capture (REDCap). Um teste piloto foi realizado com dados de três pacientes submetidos a TCTH no Hospital Moinhos de Vento no ano de 2016/2017, a fim de avaliar a utilização das ferramentas e sua aplicabilidade. Resultados: Foram desenvolvidos nove formulários no REDCap: dados sociodemográficos; dados diagnósticos; histórico, dados clínicos prévios; avaliação prétransplante; procedimento; acompanhamento pós‐imediato; acompanhamento pós‐tardio; reinternações; óbito. Adicionalmente foram desenvolvidos três modelos de relatórios, com as variáveis contidas nos formulários para auxiliar na exportação de dados para as instituições envolvidas com o TCTH. Após o teste piloto foram realizados pequenos ajustes na nomenclatura de algumas variáveis e exclusão de outras devido à complexidade na sua obtenção. Conclusão: Espera‐se que com a sua utilização, o modelo de BD proposto possa servir como subsídio para qualificar a assistência prestada ao paciente, auxiliar a gestão e facilitar futuras pesquisas na área. / Introduction: hematopoietic stem cell transplantation (HSCT) is a complex procedure involving different biopsychosocial factors and conditions. Monitoring the data of these patients is fundamental for obtaining information that can help the management, improve the assistance provided and subsidize new research on the subject. Objectives: to develop a database model (DB) of patients submitted to HSCT, considering the main variables of interest in the area. Methods: it is an applied study, where the methodology of development of a relational DB was used, following three main steps (conceptual model, relational model, physical model). The proposed physical model was developed in the research electronic data capture (Redcap) platform. A pilot test was performed with data from three patients submitted to HSCT at Moinhos de Vento Hospital in 2016, in order to evaluate the use of the tools and their applicability. Results: nine forms were developed in redcap: demographic data; diagnostic data; previous clinical data; pre‐transplant evaluation; procedure; post‐immediate follow‐up; post‐late follow‐up; readmissions; death. In addition, three reporting models were developed, with the variables contained in the forms to assist in the export of data to the institutions involved with the TCTH. After the pilot test small adjustments were made in the nomenclature of some variables and others were excluded due to the complexity in obtaining them. Conclusion: it is hoped that with its use, the proposed BD model can serve as a subsidy to qualify the care provided to the patient, assist the management and facilitate research in the area.

(English) Using flow cytometry we analyzed absolute and relative proportions of hematopoietic stem and progenitors cells (HSPC) populations including hematopoietic stem cells (HSC), multipotent progenitors (MPP), multilymphoid progenitors (MLP) and pro B cells from bone marrow of patients with mature B cell malignancies and in healthy controls. We found lower absolute and relative numbers of MLP and higher relative numbers of HSC were observed in patients when compared to age-matched controls irrespective of bone marrow (BM) involvement. On the other hand significantly decreased absolute numbers of MPP were observed only in patients who had their BM infiltrated by disease. We also confirmed published data showing increasing absolute and relative percentages of MLP with increasing age, decreasing relative percentages of HSC with increasing age, and decreasing absolute and relative pro B cell frequencies with increasing age in healthy subjects. While decreased absolute and relative pro B cell numbers were also found in patient samples as age increased, no significant correlations were detected in patients HSC, MPP or MLP populations. Age-related sub-analysis of PTs samples demonstrated that most of the disease associated changes in HSPC frequencies were observable more prominently in the elderly (>45...

Hematopoietic stem cell transplantation (HSCT) is used to treat patient with hematological diseases such as leukemia and genetic conditions such as sickle cell anemia. After HSCT the patients are supervised for signs of relapse of disease or rejection of transplanted cells. This is done by using chimerism analysis. At the department of clinical genetics at Akademiska sjukhuset fragment analysis of short tandem repeats is used for chimerism analysis, which is to be replaced by a Next generation sequencing (NGS) based method called Devyser chimerism, which includes an IVDR labelled kit. The aim of this project was to validate the new method for chimerism analysis. DNA samples from twelve HSCT patients and their donors were analyzed with Devyser chimerism and the results were compared to the results from the current method. The sensitivity of the new method was tested by analysis of artificial chimerism samples from blood donors. The results from the comparison showed a good correlation between methods (R2 = 0,9864) and the sensitivity of the method was confirmed to be 0,1% mixed chimerism. There was some difficulty in identifying enough informative markers for re-transplanted patients two had separate donors. This is a known problem for chimerism analysis in general and not a specific problem to the new method and will not be a hindrance for the implementation of Devyser chimerism at the clinical laboratory.

Background: Multiple sclerosis (MS) is a neurological, autoimmune disease which mainly affects people in the age of 20 to 40. The disease course is unpredictable affecting each patient differently, leading to progressiveand irreversible degradation of the central nervous system. There is no treatment that cures this disease, however, there are treatments that either slows down the disease course or prevents progressive disabilities. A treatment called autologous hematopoietic stem cell transplantation (AHSCT) is thought to reset the immune system and induce a new, more tolerant one, thus haltering the disease course. However, the knowledge about the effects causing the improvement seen in patients treated with AHSCT is limited. Methods: To investigate the effect of AHSCT in MS patients, serum lipidomics data from 16 patients was collected at ten timepoints. The lipidomics data was collected for both positively and negatively charged molecules separately as well as within a single experiment called polarity switching, using mass spectrometry. Since the standard method requires two separate experiments to analyze both positively and negatively charged lipids it requires twice the time and resources compared to polarity switching. Results: Comparing the two mass spectrometry protocols showed that the coefficient of variation (CV) was slightly higher for polarity switching compared to the standard method. Nevertheless, the difference was not significant and both methods had in general a good CV, indicating low technical variation. In addition, this thesis showed that polarity switching has a slightly higher percentage of lipids with zero carryover compared to the standard method. The results also indicated that the expression levels of differentially expressed lipids follow two distinct patterns throughout the AHSCT treatment. The largest intensity variation arises after stem cellreinfusion and the lipid intensities are back to nearly initial levels atthe three month follow-up. Finally, many lipids were found to be associated with the change in c-protein levels as well as erythrocyte, leukocyte, and thrombocyte levels that occurred during treatment. Conclusions: This master thesis showed that polarity switching is a good alternative to the standard method, saving both time and resources without losing too much in specificity. In addition, this thesis has shown that differentially expressed lipids follow two distinct expression patterns through the treatment. The lipids levels for both differentially expressed lipids and lipids associated with clinical data were nearly back to baseline levels three months after AHSCT. Hence, AHSCT has a major but short-lasting impact on the lipid levels in peripheral blood.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21018号 / 医博第4364号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 柳田 素子, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le transfert adoptif de lymphocytes T virus-spécifiques ou ‘’Chimeric Antigen Receptors’’ (CAR) s’est avéré efficace pour le traitement de plusieurs types d’infections virales et certains cancers à la suite d’une greffe de cellules souches hématopoïétiques. Cependant, l’immunosuppression administrée pour la prévention du rejet de greffe et de la maladie du greffon contre l’hôte limite l’efficacité et la persistance à long terme des réponses médiées par ces lymphocytes. L’agent immunosuppresseur Tacrolimus (FK506) est parmi les plus utilisés, et fonctionne en liant la protéine FK506-Binding protein (FKBP12) afin d’exercer ses effets immunosuppresseurs sur les lymphocytes T. Dans le but de fournir une protection anti-virale, mais également anti-lymphoprolifératif des lymphocytes B et permettre la poursuite de cette immunosuppression préventive, nous avons pour objectif de générer des lymphocytes CAR-T et virus-spécifiques résistants à l’action du FK506 par l’invalidation du FKBP12. En utilisant la méthode d’édition génique basée sur les CRISPR ciblés par la nucléase Cas9, nous avons pu invalider le gène du FKBP12 sur des lymphocytes T stimulés par CD3-CD28. L’efficacité du knockout a été validée par Western Blot et TIDE sequencing. Le knock-out du gène du FKBP12 a conféré un maintien de la croissance cellulaire et des fonctions effectrices telles que la synthèse de cytokines IL-2, TNFα et IFN γ en présence de Tacrolimus comparativement aux cellules contrôles. Par la même méthode, nous avons pu invalider le gène du FKBP12 sur des lymphocytes T multivirus-spécifiques dont l’efficacité a été validée par cytométrie en flux. Les fonctions effectrices ont également été maintenues en présence de tacrolimus et ont été évaluées par ELISpot. Enfin, des lignées de lymphocytes T multivirus spécifiques dont le gène du FKBP12 a été invalidé ont été transduites avec un vecteur lentiviral dans le but d’exprimer un CAR CD19 dont l’expression a été validée par cytométrie en flux et la réactivité maintenue en présence de tacrolimus. En conclusion, ces résultats nous ont permis de démontrer la faisabilité de génération de lymphocytes T « triple fonction » anti-tumorales, anti-virales et résistantes au tacrolimus. L’application de cette approche semble prometteuse dans un contexte d’une immunothérapie adoptive anti-virale et anti-tumorale post-transplantation de moelle osseuse. / Adoptive transfer of virus-specific T lymphocytes or CAR-T cells has been shown to be effective for the treatment of several types of viral infections and certain cancers following hematopoietic cell transplantation. However, immunosuppression administered for the prevention of transplant rejection and graft-versus-host disease limits the efficacy and long-term persistence of responses mediated by these lymphocytes. The widely used immunosuppressive agent Tacrolimus (FK506) requires FK506-Binding protein (FKBP12) to exert its immunosuppressive effects on T cells. We undertook to engineer a multifunctional T-cell therapy to both optimally prevent viral reactivation and relapse of B-cell malignancies post-transplant in the context of immunosuppression. The objective of our work is to generate tacrolimus resistant, multivirus-specific T-cell lines expressing an anti-CD19 CAR. Using the gene editing method based on Clustered Regular interspaced short palidromic repeats (CRISPR) targeted by the CRISPR-associated protein 9 (Cas9) nuclease, we were able to invalidate the FKBP12 gene on activated T cells (confirmed by TIDE sequencing and western blotting). Invalidation of FKBP12 conferred maintenance of cell growth and effector functions such as the synthesis of cytokines IL-2, TNFα and IFNγ in the presence of Tacrolimus. Using the same method, we were able to delete the FKBP12 gene in virus-specific T lymphocytes. Effector functions were also maintained in the presence of tacrolimus. Finally, we integrated an anti-CD19 CAR by lentiviral transduction into FKBP12-edited multi-virus T-cell lines, and the efficiency of transduction was determined by flow cytometry. The cells maintained their viral reactivity in the presence of tacrolimus. In conclusion, we were able to confirm the feasibility of generation of ‘’triple function’’ T cells (anti-viral, anti-tumoral and tacrolimus resistant). Multifunctional T-cell product manufacturing is a promising approach to optimize post-transplant T-cell immunity against opportunistic pathogens and underlying malignancies.

Indiana University-Purdue University Indianapolis (IUPUI) / The innate and adaptive immune responses are critical for host survival. The TNFα/NF-κB signaling pathway is a major regulator of the immune response. The TNFα/NF-κB signaling pathway has also been proposed to play a role in the regulation of hematopoiesis. In the TNFα signaling pathway, full induction of NF-κB (specifically the p65 subunit) dependent transcription is regulated by a co-activator SIMPL. The biological significance of SIMPL in TNFα dependent responses is poorly understood. To study SIMPL in vitro and in vivo in mammalian cells, a knockdown system utilizing shRNA (short hairpin RNA) was used. Analysis of hematopoietic progenitor cells infected with a retrovirus encoding the SIMPL shRNA was used to study the role of SIMPL in hematopoiesis. The ability of progenitor cells lacking SIMPL to grow and differentiate was not compromised. In contrast in the progenitors cells lacking SIMPL, TNFα mediated inhibition of colony formation was significantly enhanced. These growth inhibitory effects of SIMPL were not due to an increase in apoptosis. The enhanced inhibitory affects were specific for TNFα and not found in other common hematopoietic inhibitors (TGF-β1 and IFNγ). Results of this work reveal that SIMPL is a component of the hematopoiesis that is required for TNFα dependent effects upon myeloid progenitors.

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