Les cellules T mémoires (Tm) protègent l’organisme contre les réinfections de pathogènes qu’il a déjà combattu. Les Tm possèdent plusieurs propriétés en commun avec les cellules souches hématopoïétiques (CSH), notamment la capacité de se différencier, de s’auto-renouveler et de maintenir une population relativement constante au sein de l’organisme via des mécanismes homéostatiques. Il a été démontré que Hoxb4, un membre de la famille des facteurs de transcription Hox, était capable d’induire l’expansion des CSH in vivo et in vitro de façon rapide. Au vu de ces parallèles, nous avons posé l’hypothèse que la surexpression de Hoxb4 pourrait induire l’expansion de populations de Tm. Nous avons analysé les populations de Tm et lymphocytes T naïfs (Tn) dans les organes lymphoïdes de souris transgéniques surexprimant Hoxb4 et les avons comparées à des souris de type sauvage (wt). Alors que la fréquence des cellules T matures Hoxb4 diminuait avec l’âge, leur phénotype ainsi que leur viabilité demeuraient inchangés. Ensuite, nous avons procédé à des transplantations en compétition de Tm (CD4+CD44hi) Hoxb4 et wt chez des hôtes dépourvus de lymphocytes T (CD3-/-) dans le but d’évaluer leur contribution à la reconstitution du compartiment T après 2 mois. Au final, les Tm wt avait contribué un peu plus que les Tm Hoxb4 à la reconstitution (~60%). Des analyses fonctionnelles et phénotypiques ont montré que les Tm Hoxb4 possédaient une fonctionnalité normale, mais se distinguaient des Tm wt par la présence d’une faible population qui présentait un phénotype « mémoire central » (Tcm), conférant habituellement une longévité accrue. Les cellules des ganglions lymphatiques totaux des hôtes furent transplantées de façon sérielle chez trois générations de nouveaux hôtes. Le phénotype Tcm observés chez les Tm Hoxb4 était récapitulé chez les hôtes secondaires uniquement. Les ratios sont demeurés en faveur des Tm wt lors des deux transplantations suivantes, mais les Tm Hoxb4 ont commencé à montrer un avantage compétitif chez certains hôtes quaternaires. Une transplantation en compétition à court terme de Tm Hoxb4 et wt marqués avec un marqueur cytoplasmique ont démontré la présence chez les Tm Hoxb4 seulement d’une faible population CD62Lhi proliférant lentement. Ainsi, l’expansion préférentielle de Tcm CD4 par le biais d’une sélection ou d’une différenciation induite par la surexpression de Hoxb4 pourrait potentiellement leur permettre de maintenir un état de quiescence leur permettant de persister plus longtemps suite à des transplantations sérielles. / Memory T cells (Tm) protect the organism against reinfection from pathogens they’ve already encountered. Tm share characteristics with hematopoietic stem cells (HSC), such as the capacity to differentiate, self-renew and maintain a relatively constant population via homeostatic mechanisms. Hoxb4, a member of the Hox genes family of transcription factors, has been shown to expand HSCs rapidly in vivo and in vitro. Thus, drawing from these parallels we hypothesise that Hoxb4 overexpression could lead to expansion of Tm populations. Tm and naïve T cell (Tn) populations were analysed in the lymphoid organs of young and aged transgenic mice overexpressing Hoxb4 in comparison with wild type (wt) mice. While the frequencies of mature Hoxb4 T cells in lymphoid organs seemed to decline with age, the phenotype or the cell viability remained unaffected. Next, CD4+CD44hi Hoxb4 Tm were transferred into T cell deficient (CD3-/-) hosts in competition with wt CD4+CD44hi Tm and evaluated for their contribution to T cell reconstitution after 2 months. Engraftment of wt Tm in secondary lymphoid organs was slightly higher than Hoxb4 Tm (~60%). Functional assays and phenotypic analysis showed that Hoxb4 Tm exhibited normal functionality, but in contrast to wt Tm, a fraction of Hoxb4 Tm exhibited a more central memory (Tcm) phenotype, indicative of a longer lifespan. Total lymph nodes from hosts were serially re-transplanted for three generations. The Tcm phenotype of the Hoxb4 Tm present in the primary hosts was recapitulated in the secondary but not in the tertiary hosts. The ratios remained in favor of the wt Tm after two subsequent expansion rounds, but Hoxb4 Tm showed a competitive advantage over wt Tm in some quaternary hosts. Cell tracking of a short term transplantation of Hoxb4 and wt Tm in competition exposed a small population of CD62Lhi cells displaying slow proliferation in the Hoxb4 Tm only. Thus preferential CD4+ Tcm expansion by selection or differentiation could potentially allow Hoxb4 Tm to persist longer following serial transplantations due to a more quiescent state.

Our earlier work has shown that pre-B cells can be converted into macrophages by the transcription factor C/EBPα at very high frequencies and also that a clonal pre-B cell line with an inducible form of C/EBPα can be converted into macrophage-like cells. Using these systems we have performed a systematic analysis of the questions whether during transdifferentiation the cells retrodifferentiate to a precursor cell state and whether cell cycle is required for reprogramming. As for the first question, a transcriptome analysis of transdifferentiating cells showed that most genes are continuously up or downregulated, acquiring a macrophage phenotype within 5 days. In addition, we observed the transient reactivation of a subset of immature myeloid markers, as well as low levels of the progenitor markers Kit and Flt3 and a few lineage inappropriate genes. Importantly, we were unable to observe the re-expression of cell surface marker combinations that characterize hematopoietic stem and progenitor cells (HSPCs), including c-Kit and Flt3. This was the case even when C/EBPα was activated in pre-B cells under culture conditions that favor HSPC growth or when the transcription factor was activated in a time limited fashion. As for the second question, using the C11-inducible pre-B cell line, time-lapse experiments showed that a subpopulation of about 8% of the pre-B cells did not divide before acquiring macrophage properties, with the majority of cells dividing once and a few percent dividing twice. In agreement with these results we found that 8% of the induced cells did not incorporate BrdU during reprogramming. Importantly, the non-dividing cell subset expressed the highest levels of C/EBPα and was the fastest in acquiring a macrophage phenotype. Inhibition of DNA synthesis by aphidicolin led to an impairment of transdifferentiation in >70% of the cells, suggesting a requirement for traversing the cell cycle. However, sorting pre-B cells into G0/G1 and G2/M fractions followed by induction showed no significant differences in the reprogramming kinetics. Finally, we showed that knocking down p53 in the inducible pre-B cells does not alter their conversion into macrophages, suggesting that an acceleration of the cell cycle has no effect. Together, our findings show that the conversion of pre-B cells to macrophages does not involve overt retrodifferentiation and that high concentrations of C/EBPα bypass the cell cycle-dependency of immune cell transdifferentiation / Recientemente, nuestro grupo ha demostrado que las células pre-B se pueden reprogramar a macrófagos mediante la sobreexpresión del factor de transcripción C/EBP, con una eficiencia elevada. Así mismo, mediante la expresión de la forma inducible de C/EBP en una línea de células pre-B (C11), éstas también se puede convertir en células similares a macrófagos. Usando este sistema hemos estudiado si durante el proceso de trans-diferenciacion las células requieren volver a un estadio de célula precursora, y si el ciclo celular es necesario para este proceso. En cuanto a la primera cuestión, el análisis del transcriptoma de células trans-diferenciadas mostró que la expresión de la mayoría de los genes están regulados durante todo el proceso bien aumentando o disminuyendo, y que adquieren el fenotipo de macrófago a los 5 días después de iniciar el proceso. Así mismo, se observó la reactivación transitoria de un grupo de genes que codifican para marcadores de células mieloides inmaduras; también cabe destacar que observamos una disminución en la expresión de los genes expresados en células progenitoras Kit y Flt3, así como de genes de linajes impropios. Es importante destacar que nunca hemos llegado a observar la expresión de combinaciones de marcadores de superficie característicos de las células madre hematopoyéticas y las células progenitoras (HSPCs), incluyendo c-Kit y Flt3, mediante el análisis por citometría de flujo. Estos resultados se reprodujeron incluso cuando C/EBP se sobreexpresó en células pre-B que fueron cultivadas en condiciones que favorecen el crecimiento de las HSPC o cuando el factor de transcripción se activó de forma limitada en el tiempo. En cuanto a la segunda pregunta, usando la línea de células inducibles pre-B C11, el análisis mediante microscopia a diferentes tiempos después de la inducción de la reprogramación mostraron que una subpoblación de aproximadamente el 8% de las células pre-B no se dividen antes de adquirir las propiedades de macrófago, mientras que la mayoría de las células se dividen sólo una vez y un pequeño porcentaje dos veces antes de que se reprogramen totalmente a macrófagos. De acuerdo con estos resultados se encontró que un 8% de las células inducidas no incorporan BrdU durante la reprogramación. Es importante destacar que el subconjunto de células que no se dividen expresan los niveles más altos de C/EBP, con lo que cabe pensar que la adquisición del fenotipo de macrófago es más rápida en estas células. La inhibición de la síntesis de ADN por afidicolina bloqueó la transdiferenciación en mas de un 70% de las células, lo que sugiere que la correcta progresión del ciclo celular es un requisito para la transdiferenciación. Sin embargo, al separar la linea de células pre-B C11 en fracciones G0/G1 y G2/M seguido de la inducción, la cinética de la reprogramación no mostró diferencias significativas. Por último, también demostramos que la reducción en la expresión de p53 en las células pre-B inducibles no altera el proceso de conversión a macrófago, lo que sugiere que la aceleración del ciclo celular no tiene ningún efecto. En conjunto, nuestros resultados muestran que la conversión de células pre-B a macrófagos no requiere retro-diferenciación y que las células con una expresión mayor de C/EBP pueden llegar a prescindir de la dependencia del ciclo celular para la trans-diferenciación de las células inmunitarias.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 18, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 137-158.

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Les cellules souches hématopoïétiques (CSH) assurent la génération de toutes les lignées sanguines. Leur différenciation en cellules T matures se déroule dans un microenvironnement spécialisé, le thymus, orchestrée par des interactions complexes entre cytokines, chimiokines et cellules stromales. Les mutations bloquant la différenciation des cellules T ont un impact sur l'architecture du thymus, soulignant l’importance des interactions entre cellules T en développement et cellules stromales thymiques.Les déficits immunitaires combinés sévères sont généralement traités, avec succès, par transplantation de CSH allogénique par voie intraveineuse. Cependant, des complications peuvent survenir notamment en cas de greffe non compatible. Pour pallier à cela, la thérapie génique a été développée mais son efficacité et son innocuité restent à améliorer. Dans ce but, notre groupe a développé une approche par correction génique des progéniteurs T directement in vivo, via un vecteur lentiviral. Bien qu’efficace, là encore, l’efficacité de traitement reste insuffisante voire extrêmement limitée chez les macaques.Lors de ma thèse, j'ai donc évalué le potentiel de différents sérotypes de vecteur viraux adéno-associés (AAV) pour la transduction des thymocytes. L'administration IT de plusieurs sérotypes de AAV2 engendre une transduction des thymocytes >10 fois plus élevée que celle des vecteurs lentiviraux. Le sérotype AAV2/8 induit la transduction des thymocytes la plus efficace et les cellules transduites représentent jusqu'à 1% des cellules T périphériques d’une souris immunocompétente. En utilisant des souris immunodéficientes ZAP-70-/- comme paradigme, j'ai découvert que l'injection IT de l’AAV2/8-ZAP-70 entraîne une transduction et différentiation lymphocytaire T rapide, associée à la génération d’une medulla thymique. En effet, des cellules épithéliales thymiques de la médulla (mTEC) exprimant le régulateur auto-immun AIRE sont détectées en <2 semaines. Bien que cette reconstitution soit transitoire, les mTECs AIRE+ diminuant 10 semaines post-injection, les cellules T périphériques corrigées persistent >40 semaines et présentent environ 1 copie du vecteur AAV/cellule. Ces cellules T effectrices peuvent sécréter des niveaux élevés de cytokines et un nombre important de cellules T régulatrices est également généré. Ainsi, une seule vague de thymopoïèse à partir de progéniteurs transduits par l’AAV-ZAP-70, permet une restauration, rapide et transitoire de l'architecture thymique mais, à long terme de cellules T périphériques fonctionnelles.Pour évaluer les diverses populations de TEC régissant le développement et la sélection des cellules T, j'ai collaboré avec les groupes de P Jay/J Abramson/I Amit pour établir une cartographie de novo du compartiment stromal thymique. Nos analyses ont mis en évidence quatre populations majeures de mTEC (I-IV) avec des fonctions distinctes. Notamment, les mTEC-IV constituent une population unique présentant des similarités moléculaires et morphologiques avec les cellules tuft intestinales. Comme nous avions précédemment identifié la sécrétion d'IL-25 par les cellules tufts comme un régulateur des interactions entre compartiment épithélial et hématopoïétique dans l'intestin, nous avons évalué ce potentiel dans le thymus. Ainsi, des souris déficientes en cellules tuft intestinales présentent également une déficience spécifique en mTEC-IV et une homéostasie perturbée de diverses populations exprimant l'IL-25R dans le thymus. Notre recherche a donc permis d'identifier une nouvelle population de TEC tuft avec un rôle critique dans la formation de la niche immunitaire du thymus.L’ensemble de mes résultats montrent le potentiel thérapeutique de stratégie intrathymique de thérapie génique pour des patients ayant besoin d’une reconstitution rapide en cellules T et fournissent de nouvelles perspectives sur les populations stromales thymique et leur rôle dans l’équilibre de la niche immunitaire. / Hematopoietic stem cells (HSC) ensure the generation of all blood lineages. Their differentiation to mature T lymphocytes occurs in the specialized microenvironment of the thymus, orchestrated by complex interactions between cytokines, chemokines, and stromal cells. Mutations resulting in a block in T cell differentiation impact on the architecture of the thymus, pointing to the critical crosstalk between developing T cells and thymic stromal components.Genetic severe combined immunodeficiencies (SCID) are generally treated by the intravenous transplantation of healthy allogeneic HSCs. Although this therapy is often successful, complications can occur, especially for patients receiving non-histocompatible HSC transplants. To circumvent these problems , significant efforts have gone into developing gene therapy strategies but adverse events indicate the necessity of exploring other avenues. Our group hypothesized that in situ gene correction of T lymphoid progenitors in the thymus itself may overcome some of the drawbacks of ex vivo gene therapy. While intrathymic (IT) lentiviral vector administration corrected immunodeficient thymocyte precursors in mice, thymus transduction was inefficient and efficacy in macaques was limited.During my PhD, I assessed the in vivo potential of adeno-associated vectors (AAV) to transduce thymocyte precursors. Intrathymic administration of several different scAAV2 serotypes resulted in a >10-fold higher transduction of thymocytes (3-5%) as compared to lentiviral vectors. scAAV2/8 promoted the highest level of gene transfer and strikingly, transduced cells represented up to 1% of peripheral T lymphocytes in immunocompetent mice. Using ZAP-70-/- immunodeficient mice as a paradigm, I found that IT injection of an AAV2/8-ZAP-70 vector resulted in a rapid transduction and T cell differentiation, correlating with a dramatic generation of the thymus medulla. Indeed, medullary thymic epithelial cells (mTEC) expressing the AIRE autoimmune regulator were detected within <2 weeks. While this reconstitution was transient––AIRE+ mTECs decreased by 10 weeks post gene transfer––gene-corrected peripheral T cells, harboring approximately 1 AAV genome/ cell, persisted for >40 weeks. Effector T cells had the potential to secrete high levels of cytokines and significant numbers of gene-corrected regulatory T cells were also generated. Thus, a single wave of thymopoiesis, from intrathymic AAV-ZAP-70-transduced progenitors, allows for a rapid but transient restoration of the thymic architecture and long-term peripheral T cell function.To better assess the diverse TEC populations that orchestrate T cell development and selection, I collaborated with the groups of P. Jay/J. Abramson/I. Amit to combine single cell analysis and in-vivo fate-mapping to de novo characterize the entire stromal compartment of the thymus. Our analyses highlighted four major medullary TEC (mTEC I-IV) populations with distinct lineage regulator function and specifically, we found that mTEC-IV constitutes a highly divergent TEC subset that bears strong molecular and morphological characteristics to intestinal tuft cells. As we previously identified tuft cell secretion of IL-25 as a regulator of the crosstalk between the epithelial and hematopoietic compartments in the gut, we assessed the potential immune-modulatory function of mTEC-IV. Notably, mice deficient in intestinal tuft cells exhibited a specific depletion of mTEC-IV and a perturbed homeostasis of various IL25-R-expressing populations in the thymus. Taken together, our data identify a new tuft TEC population critical for shaping the thymus immune niche.In conclusion, the data generated during my PhD advance the therapeutic potential of intrathymic-based vector strategies for the treatment of patients requiring a rapid T cell reconstitution and provide new insights into thymic stromal subsets that are critical for shaping the thymus immune niche.

L’hématopoïèse se déroule dans un microenvironnement spécialisé appelé niche où les cellules souches hématopoïétiques (CSH) sont en contact étroit avec les cellules stromales mésenchymateuses. Cette interaction cellulaire associée à d’autres facteurs environnementaux, comme la présence des espèces réactives à l’oxygène, est cruciale pour la régulation des CSH normales, mais aussi leucémiques. Pour étudier ce microenvironnement, il est donc important de développer un modèle in vitro de niche humaine qui mime la physiologie in vivo. Nous avons choisi comme modèle deux lignées mésenchymateuses stromales humaines HS-27a et HS-5, très peu décrites dans la littérature. Le premier objectif a été de déterminer la qualité de cette niche tant du point de vue cellulaire, moléculaire que fonctionnel. Nos résultats montrent clairement que les cellules HS-27a participent à la formation d’une niche « quiescente » alors que les cellules HS-5 représentent une niche « proliférative ». Le deuxième objectif a été de créer une niche contrôlée pour le métabolisme oxydatif en régulant l’expression d’une protéine antioxydante, la glutathion peroxydase 3 ou GPx3. L’originalité de ce travail repose sur l’utilisation d’une méthode non virale de transfert de gène par le transposon piggyBac. Le plasmide porteur du gène d'intérêt a été apporté sous forme d’ADN et une source de transposase, enzyme catalysant la réaction d'intégration sous forme d’ARNm. Notre travail montre que GPx3 est un régulateur clé de l’homéostasie hématopoïétique favorisant le maintien des progéniteurs immatures. Pour la première fois, nous créons par ingénierie in vitro une niche hématopoïétique « calibrée » capable de mimer le microenvironnement normal et leucémique. Ce modèle permet non seulement d’identifier les acteurs clés de la régulation des cellules médullaires, mais aussi de développer des stratégies thérapeutiques ciblées. / Hematopoiesis occurs in a hypoxic microenvironment or niche in which hematopoietic stem cells (HSCs) are in close contact with mesenchymal stromal cells. Cellular interactions as well as microenvironmental factors such as reactive oxygen species are crucial for the maintenance of normal and leukemic HSCs. Developing an in vitro human culture system that closely mimcs marrow physiology is therefore essential to study the niche. Here, we present a model using two human stromal cell lines, HS-27a and HS-5. Previously poorly described in the literature, we have further characterized both of these cell lines. The first objective was to assess the quality of HS-27a and HS-5 niches by investigating their cellular, molecular and functional characteristics. Our results clearly show that HS-27a cells display features of a “quiescent” niche whereas HS-5 cells rather represent a “proliferative” niche. The second objective was to engineer a hematopoietic niche where the oxidative metabolism is optimized for the expression of an antioxidant protein, glutathione peroxidase 3 (GPx3). The originality of this work is the use of a non-viral gene transfer system by using the transposon piggyBac. This strategy was achieved by delivering a DNA plasmid carrying the gene of interest, and an mRNA source of transposase, the enzyme which catalyzes the transgene integration. Functionally, GPx3 was shown to be a key regulator for sustaining hematopoietic homeostasis by maintaining immature progenitor cells. For the first time, an original non-viral gene transfer has been used to create an in vitro hematopoietic niche that recapitulates the complexity of normal and leukemic microenvironment. This niche not only provides a platform to identify regulatory factors controlling medullary cells, but may also help in the development of targeted therapeutic strategies.

Introdução: Células-troco mesenquimais (CTM) vêm mostrando seus benefícios na doença do enxerto-versus-hospedeiro (DECH), observada no transplante de células tronco hematopoéticas (TCTH), existem três questões em aberto: (1) Expansão de CTM em meio de cultura suplementado com soro fetal bovino (SFB), pelo o risco de xenorreação; (2) Otimização de condições de cultura para a obtenção, em tempo hábil, de um numero que permita de 4 a 6 infusões de 2x106cells/kg do receptor; (3) Obter células do doador de medula óssea, evitando assim a utilização de um terceiro doador. Objetivos: Este estudo foi desenhado para comparar o lisado de plaquetas (LP) e o SFB na expansão de CTM, a densidade de plaqueamento das células e os dias entre cada passagem, e para investigar se as células nucleadas totais obtidas da bolsa e filtro do TCTH, podem ser utilizadas para expansão de CTM para utilização clínica. Métodos: Células residuais foram removidas do filtro e da bolsa utilizados para o TCTH, plaqueadas e depois da primeira passagem foram cultivadas em diferentes concentrações com SFB ou LP e observado o número de dias que levaram para chegar a 80% de confluência. Em seguida, as culturas com as mesmas densidades de plaqueamento foram suplementadas com LP ou SFB e depois de sete dias contou-se o número de células para analisar o quanto elas cresceram nesse período. Resultados: A proliferação de CTM, na presença de LP e SFB foi em média 11,88 e 2,5 vezes, respectivamente, num período de 7 dias. A concentração mais elevada de células usando LP demorou menos tempo para atingir a confluência, em comparação com os três inferiores. Este estudo sugere que o LP é a melhor escolha como suplemento para expandir CTM, e permite a proliferação de um número suficiente de CTM de doadores para uso clínico. / Introduction: Mesenchymal stromal cells (MSC) have shown their benefits in graft-versus-host disease (GVHD), with three unsettled matters:(1) MSCs expansion in medium with Fetal Calf Serum (FCS) and its risk of xenoreaction; (2) The number of cells indicated for therapy is 2x106cells/Kg with the need to optimize expansion, number and time wise; and (3) the utilization of third party donors. Aims: This study was designed to compare the platelet lysate (LP) and FCS on the expansion of MSC, the optimal cell plating density and days between each pass, and to investigate if donor total nucleated cells (TNC) obtained from the washouts of hematopoietic stem cell transplantation (HSCT) explants can be expanded to be used at clinical grade. Methods: TNC were removed, plated and after the first passage were cultivated in different concentrations with FCS or PL and the number of days reach 80% of confluence was observed. Next, cultures with the same plating density were fed either with PL or FCS and after seven days counted to analyze how much they have grown in that period. Results: The proliferation of mesenchymal stromal cells in the presence of PL and SFB was averaged 11.88 and 2.5 times, respectively, in a period of 7 days. The highest concentration of plating cells using PL, took less time to reach confluence as compared with the three lower ones. This study suggests that the PL is the best choice as a supplement to expand MSC, and allows the proliferation of a sufficient number of donors MSC at P2 for clinical use.

Objetivos. Avaliar o papel do transplante de células hematopoiéticas (TCH) e da interleucina 6 (IL – 6) sobre vários parâmetros de estresse oxidativo em pacientes com Adrenoleucodistrofia ligada ao cromossomo X (X-ALD). Métodos. A concentração de malondialdeído (MDA), o conteúdo de carbolinas e sulfidrilas e a concentração de ácido hexacosanóico (C26:0) foram quantificados no plasma de pacientes X-ALD antes e após serem submetidos ao TCH. E, a concentração de MDA, a formação de carbonilas e a concentração de IL-6 foram quantificados em plasma e o conteúdo de glutationa reduzida (GSH) foi quantificado em eritrócitos de pacientes X-ALD com fenótipos cerebral infantil (cALD) ou assintomáticos no momento diagnóstico. Resultados. Observamos um aumento significativo na concentração de MDA em plasma de pacientes X-ALD antes e após o TCH em comparação ao grupo controle e uma redução significativa nesses valores após o transplante em comparação aos anteriores ao procedimento. Verificamos uma redução significativa no conteúdo de sulfidrilas no plasma de pacientes X-ALD antes do TCH em comparação ao grupo controle e um aumento significativo desses níveis após o TCH. Não observamos diferenças significativas no conteúdo de carbonilas no plasma de X-ALD antes e após o TCH, em comparação aos controles, apesar de observarmos uma redução significativa nesta determinação nos pacientes após o transplante em relação a antes do TCH. Os pacientes X-ALD apresentam níveis plasmáticos de C26:0 significativamente aumentados antes do TCH em comparação aos controles e, após o TCH, as concentrações de C26:0 foram reduzidas. Observamos uma correlação negativa significativa entre a medida do conteúdo de sulfidrilas e os níveis plasmáticos de C26:0 de indivíduos X-ALD antes do TCH. Também evidenciamos elevados níveis de MDA e da formação de carbonilas no plasma de pacientes cALD e assintomáticos em comparação ao grupo controle. Ainda, observamos redução significativa do conteúdo de GSH nos dois grupos testados comparados aos controles. A quantificação de IL-6 foi significativamente maior nos pacientes cALD, o que não foi observado nos pacientes assintomáticos, apesar destes mostrarem uma tendência de aumento da concentração de IL-6. Conclusões. Os resultados obtidos a partir do plasma de pacientes X-ALD antes e após o TCH demonstram que esta terapia, quando bem indicada e bem sucedida, tem alta efetividade em reduzir a concentração plasmática de C26:0 e é eficaz em reduzir a peroxidação lipídica e o dano oxidativo às proteínas nos pacientes X-ALD. Ainda, é possível relacionar o acúmulo de C26:0 e o dano oxidativo na patogênese da X-ALD. Nossos dados permitem sugerir que a lipoperoxidação e o dano oxidativo às proteínas possam de alguma forma estar envolvidos na fisiopatologia da X-ALD. Além disso, podemos presumir que, nos pacientes X-ALD assintomáticos estudados, o dano oxidativo e os aspectos inflamatórios desempenham papéis importantes na evolução e nas futuras manifestações do fenótipo neuronal. Também podemos supor que a administração de antioxidantes deve ser considerada como uma terapia adjuvante potencial para os pacientes assintomáticos e sintomáticos afetados pela X-ALD, inclusive para aqueles submetidos ao TCH. / Objective. We aimed to evaluate the role of hematopoietic stem cell transplantation (HSCT) and interleukin 6 (IL – 6) on various parameters of oxidative stress in X-linked adrenoleukodystrophy (X-ALD) patients. Methods. Malondialdehyde (MDA), sulfhydryl, carbonyl and hexacosanoic acid (C26:0) levels were measured in plasma from X-ALD patients before and after HSCT. And, MDA, carbonyl and IL-6 levels were measured in plasma and reduced glutathione (GSH) content was measured in erythrocytes from X-ALD patients with different phenotype (asymptomatic and childhood cerebral (CCER patients) at diagnosis moment. Results. We observed increased levels of MDA in plasma from X-ALD before and after HSCT compared to control group, but there was a significant reduction in MDA values after transplantation compared to levels found before the procedure. We verified a significant decrease in sulfhydryl content in plasma of X-ALD patients before HSCT compared with the control group and we also verified a significant increase in the levels of sulfhydryl content after HSCT. No significant differences were observed in carbonyl content in plasma of X-ALD before and after HSCT, compared to controls. However, we observed a significant reduction of plasma carbonyl content from X-ALD patients after HSCT compared to before HSCT. X-ALD patients presented a significant increase of C26:0 plasma level before HSCT when compared to controls and an important reduction of C26:0 plasma concentration in X-ALD patients after HSCT when compared to before HSCT C26:0 levels. We observed an inverse significant correlation between sulfhydryl content and plasma C26:0 levels of X-ALD individuals before HSCT. We also evidenced high levels of MDA and carbonyl formation in plasma from CCER and asymptomatic patients compared to controls. Still, we observed a significant decrease of GSH content in both groups tested compared to controls. The quantification of IL-6 is significantly higher in CCER patients, which is not observed in asymptomatic patients, despite these patients show a tendency of increased concentration of IL-6. Conclusions. The results obtained from plasma of X-ALD patients before and after HSCT demonstrate that this therapy, when well indicated and successful, has high effectiveness in reducing C26:0 plasma and is effective in reducing lipid peroxidation and oxidative damage to proteins in X-ALD patients. Still, it is possible to relate the accumulation of C26:0 and oxidative damage in the pathogenesis of X-ALD. Our data also suggest that lipid peroxidation and protein damage may somehow be involved in the pathophysiology of X-ALD. Moreover, we can assume that in our asymptomatic X-ALD patients, oxidative damage and inflammatory issues seem to play an important role in the evolution and future manifestations of neuronal phenotype. We can also assume that the administration of antioxidants should be considered as a potential adjuvant therapy for asymptomatic and symptomatic patients affected by X-ALD, including those that are submitted to HSCT.