Clinical application of natural killer (NK) cells against leukemia is an area of intense investigation. In human leukocyte antigen-mismatched allogeneic hematopoietic stem cell transplantations (HSCT), alloreactive NK cells exert powerful anti-leukemic activity in preventing relapse in the absence of graft-versus-host disease, particularly in acute myeloid leukemia patients. Adoptive transfer of donor NK cells post-HSCT or in non-transplant scenarios may be superior to the currently widely used unmanipulated donor lymphocyte infusion. This concept could be further improved through transfusion of activated NK cells. Significant progress has been made in good manufacturing practice (GMP)-compliant large-scale production of stimulated effectors. However, inherent limitations remain. These include differing yields and compositions of the end-product due to donor variability and inefficient means for cryopreservation. Moreover, the impact of the various novel activation strategies on NK cell biology and in vivo behavior are barely understood. In contrast, reproduction of the thirdparty NK-92 drug from a cryostored GMP-compliant master cell bank is straightforward and efficient. Safety for the application of this highly cytotoxic cell line was demonstrated in first clinical trials. This novel ‘off-theshelf’ product could become a treatment option for a broad patient population. For specific tumor targeting chimeric-antigen-receptor-engineered NK-92 cells have been designed.

Die vorliegende medizinische Dissertation untersucht nicht-invasive bildgebende Verfahren wie die konventionelle Sonographie, die Acoustic radiation force impulse (ARFI)-Elastographie sowie die Transiente Elastographie (TE) zur Detektion von Komplikationen in der Frühphase nach allogener Stammzelltransplantation. Dem kurativen Therapieansatz der Stammzelltransplantation steht ein hohes Komplikationspotential gegenüber. Besonders hepatobiliär treten Graft-versus-host Erkrankungen (GvHD) sowie Gefäßkomplikationen (VOD) auf. Der bisherige diagnostische Goldstandard, die Leberbiopsie, ist als invasives Verfahren mit einer hohen Intra- und Inter-Untersucher-Variabilität sowie der geringen Repräsentativität als Screeningmethode ungeeignet. Die Elastographieverfahren ARFI und TE als nicht-invasive Alternativen ermitteln die Lebergewebesteifigkeit als Surrogatparameter fibrotischer Veränderungen und wurden bereits in zahlreichen Studien als geeignete Diagnoseverfahren für Leberfibrose und -zirrhose unterschiedlicher Ätiologie definiert. Ziel dieser prospektiven Pilotstudie war die Evaluation der genannten Methoden zur Detektion von Frühkomplikationen nach allogener Stammzelltransplantation. Die Ergebnisse der Studie zeigen, dass sowohl die konventionelle Sonographie als auch die Transiente Elastographie pathologische Organveränderungen vor allem des hepatobiliären Systems detektieren können. Allerdings erscheinen diese Veränderungen unspezifisch. Es bestehen keine signifikanten Unterschiede zwischen Patienten mit und ohne Komplikationen. Anders bei der ARFI-Elastographie. Hier zeigten die Messwerte im linken Leberlappen signifikant höhere Werte bei Patienten mit Komplikationen. Zusammenfassend ist die ARFI-Elastographie zur Prädiktion möglicher Komplikationen nach allogener Stammzelltransplantation geeignet, sollte allerdings mit anderen diagnostischen Verfahren ergänzt werden.:III. Inhaltsverzeichnis I. Vorbemerkungen 2 II. Bibliographische Beschreibung 3 III. Inhaltsverzeichnis 4 IV. Abkürzungsverzeichnis 5 1. Einleitung 1.1. Hämatopoetische Stammzelltransplantation 6 2. Komplikationen nach allogener Stammzelltransplantation und deren Diagnostik 2.1. Akute Komplikationen 8 2.2. Akute und chronische Graft-versus-host Erkrankung 9 2.3. Hepatobiliäre Komplikationen 12 2.3.1. Veno-occlusive disease 12 2.3.2. Drug-induced liver injury 14 2.3.3. Problematik der Diagnostik 16 3. Risikostratifizierung bei Stammzelltransplantation 16 3.1. Karnovsky Index und Eastern Cooperative Oncology Group Index 16 3.2. Hematopoietic cell transplantation comorbidity index 17 3.3. Leberspezifisches Risikoassessment 18 4. Nicht-invasive Leberdiagnostik 19 4.1. Konventionelle Sonographie 19 4.2. Elastographie 20 4.2.1. Transiente Elastographie 21 4.2.2. Acoustic radiation force impulse imaging 22 5. Prospektive Studie: Sonographische Evaluation von Komplikationen in der Frühphase nach allogener Stammzelltransplantation 23 5.1. Methodik 24 5.2. Eigener Arbeitsanteil 24 6. Publikationsmanuskript 25 7. Zusammenfassung 32 8. Literaturverzeichnis 36 9. Selbständigkeitserklärung 44 10. Lebenslauf 45 11. Danksagung 46

Analysis of donor chimerism has become a routine method for the documentation of engraftment after allogeneic hematopoietic stem cell transplantation (HSCT). In recent years several groups have also focused on the application of this technique for the detection of relapsing disease after allogeneic HSCT. This review addresses technical issues (sensitivity, specificity) and discusses the advantages and limitations of methods currently used for chimerism analysis and their usefulness for the detection of MRD. In addition, the potential impact of novel procedures, e.g. subset chimerism or real-time PCR-based procedures, is discussed. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

GM1 gangliosidosis is an autosomal recessive lysosomal storage disease, caused by a deficiency in the enzyme β-galactosidase. The disease affects the CNS, liver, kidney, heart and skeletal system, leading to severe neurodegeneration and death. We propose to treat this disorder using ex vivo hematopoietic stem cell therapy. The effectiveness of this therapy requires the recruitment of transduced donor cells to the CNS. This is only found to occur after mice are conditioned with total body irradiation, due to the increase in CNS cytokine production and blood brain barrier permeability that occurs. As the use of total body irradiation in pediatric patients has been linked to future developmental problems, this myeloablation approach is often avoided in younger patients in favor of a conditioning regimen using the chemotherapy drugs, busulfan and cyclophosphamide. Whether donor cells can enter the CNS when a busulfan and cyclophosphamide conditioning regimen is used has not been determined. In this study we plan to quantify the cytokine and blood-brain barrier permeability increases necessary for donor cells to be recruited to the CNS after total body irradiation. We will then investigate whether busulfan and cyclophosphamide conditioning and/or the chronic neuroinflammation present in GM1 mice can produce similar conditions and facilitate the recruitment of donor hematopoietic stem cells to the CNS. Finally we will assess whether ex vivo hematopoietic stem cell gene therapy is still an effective therapy when busulfan and cyclophosphamide are used for myeloablative conditioning.

The HLA-DRB3/4/5 loci are closely linked to the HLA-DRB1 gene. Mismatches in these loci occur with a frequency of about 8%–12% in otherwise 10/10 HLA-matched transplant pairs. There is preliminary evidence that these disparities may associate with increased acute graft-versus-host disease (GvHD) rates. The aim of this study was to analyze a large cohort of German patients and their donors for HLA-DRB3/4/5 compatibility and to correlate the HLA-DRB3/4/5 matching status with the outcome of unrelated hematopoietic stem cell transplantation (uHSCT). To this end, 3,410 patients and their respective donors were HLA-DRB3/4/5 and HLA-DPB1 typed by amplicon-based nextgeneration sequencing (NGS). All patients included received their first allogeneic transplant for malignant hematologic diseases between 2000 and 2014. Mismatches in the antigen recognition domain (ARD) of HLA-DRB3/4/5 genes were correlated with clinical outcome. HLA-DRB3/4/5 incompatibility was seen in 12.5% (n = 296) and 17.8% (n = 185) of the 10/10 and 9/10 HLA-matched cases, respectively. HLA-DRB3/4/5 mismatches in the ARD associated with a worse overall survival (OS), as shown in univariate (5-year OS: 46.1% vs. 39.8%, log-rank p = 0.038) and multivariate analyses [hazard ratio (HR) 1.25, 95% CI 1.02–1.54, p = 0.034] in the otherwise 10/10 HLAmatched subgroup. The worse outcome was mainly driven by a significantly higher nonrelapse mortality (HR 1.35, 95% CI 1.05–1.73, p = 0.017). In the 9/10 HLA-matched cases, the effect was not statistically significant. Our study results suggest that mismatches within the ARD of HLA-DRB3/4/5 genes significantly impact the outcome of otherwise fully matched uHSCT and support their consideration upon donor selection in the future.

De nos jours, la greffe de cellules souches hématopoïétiques (GCSH) de type allogénique est le traitement standard pour de nombreuses hémopathies malignes, et ce, en dépit des taux élevés de complications liées à la transplantation, telles que les infections, les rechutes et la maladie du greffon contre l’hôte (GVH), associées à cette procédure. Depuis les deux dernières décennies, plusieurs stratégies visant à contrôler la maladie du GVH ont émergé et incluent la déplétion partielle et la manipulation ex vivo des cellules T du greffon. En revanche, ces stratégies peuvent entrainer de sévères déficits immunitaires post-greffe. En comparaison avec les autres sources de cellules souches, une faible incidence de la maladie du GVH et de rechute est observée chez les patients recevant une greffe de sang de cordon ombilical (SC). En contrepartie, ce type de greffe engendre des retards dans la restauration immunitaire, rendant ainsi les patients plus susceptibles aux agents pathogènes. À l’inverse, une augmentation plus importante de la production de cellules T naïves, d’émigrants thymiques récents et de l’abondance des clonotypes des cellules T fut détectée chez les patients ayant reçu une GCSH dérivée du sang d’un seul cordon traité avec UM171, une molécule utilisée pour l'amplification ex vivo des CSHs dérivés de SC, relativement aux patients ayant reçu une GCSH standard. Dans ce mémoire, nous essaierons de comprendre le mécanisme moléculaire sous-jacent à la restauration immunitaire chez les patients transplantés avec un greffon de SC traité avec UM171, nous tenterons de déceler des progéniteurs lymphoïdes au sein des cellules de SC traitées avec UM171 et nous essaierons d’identifier un marqueur de surface qui pourrait être utilisé pour enrichir les progéniteurs lymphoïdes. Nos résultats démontrent la présence de deux "clusters" lymphoïdes, PCL et LMPP, qui expriment potentiellement CD10, et sont plus nombreux dans les échantillons de SC CD34+ traités avec UM171 comparativement aux contrôles. En utilisant les marqueurs de surface CD10 et CD45RA, nous avons pu identifier deux populations CD34+ (CD10medCD45RAmed/lo et CD10hiCD45RAhi) qui sont multipliées dans les cultures traitées avec UM171 relativement aux contrôles. En outre, nous démontrons également que seules les cellules CD45RA- peuvent générer les deux populations CD10+ in vitro, ce qui suggère que les cellules CD10medCD45RAmed/lo définissent une population plus primitive que les cellules CD10hiCD45RAhi. De surcroit, nous proposons que la population CD10medCD45RAmed/lo pourrait contenir des LMPPs qui se différencieront en PCLs inclus dans la population CD10hiCD45RAhi. D'un point de vue fonctionnel, nous observons un développement et une maturation des lymphocytes T nettement plus rapide pour la population CD10+CD34+ relativement à la population CD34+ totale à 3 et 6 semaines, ce qui suggère que CD10 pourrait être utilisé comme marqueur de progéniteurs lymphoïdes. Par ailleurs, nous détectons également un pourcentage plus élevé de cellules ayant un phénotype ILC dans la population CD10+CD34+ comparativement à la population CD34+ totale, ce qui suggère que la population CD10+CD34+ peut potentiellement contenir des progéniteurs lymphoïdes capables de produire différents types de cellules immunitaires. D’autre part, la population CD45RA+ produit plus rapidement des lymphocytes T contrairement à la population CD45RA-. En conséquence, nous proposons que la population CD45RA- contient des cellules primitives (ex. CSH) qui à leur tour génèrent des progéniteurs multipotents contenus dans la population CD10+CD34+ (ex. LMPP) et qui éventuellement produiront des progéniteurs à potentiel plus restreint inclus dans la population CD45RA+ (ex. CLP). En somme, ce mémoire identifie pour la première fois une population CD10+CD34+ présente dans le greffon traité avec UM171 avec un potentiel T et produisant potentiellement des ILCs. L’un des problèmes majeurs liés à la GCSH dérivée du SC est une restauration immunitaire retardée; par conséquent, optimiser l’infusion de cellules CD34- en augmentant le nombre de progéniteurs lymphoïdes pourrait considérablement aider à stimuler la restauration immunitaire chez les patients recevant une GCSH dérivée de SC. / Currently, allogeneic hematopoietic stem cell transplantation is a standard treatment for many hematological malignancies, although high rates of transplant-related complications such as infections, relapse, and GVHD has constrained the implementation of HSCT. Strategies have emerged to manage GVHD and include partial T-cell depletion and ex vivo manipulation of donor T cells, albeit they have adverse effects on post-transplantation immune recovery and can cause profound immunodeficiency. In comparison to other stem cell sources, a lower incidence of chronic GVHD and relapse is reported in patient receiving cord blood (CB) transplant, although they also display an enhanced susceptibility to pathogens caused by an ineffective T-cell reconstitution. In contrast, we reported a greater increase in naïve T cells production, recent thymic emigrants and T cell clonotype from 3 months to 6 and 12 months in patient transplanted with a single CB graft expanded with UM171, a small molecule used for the ex vivo expansion of CB HSCs, as compared to counterpart patients receiving unmanipulated CB graft. In this master research, we aimed to understand the molecular mechanism underlying T-cell reconstitution in patients transplanted with UM171- expanded CB graft. We tried to identify lymphoid progenitors within the highly heterogeneous CD34+ CB cells treated with UM171 using Cite-Seq, find a surface marker that can be used to enrich for early lymphoid progenitors using flow cytometry, and assess their lineage potential using artificial thymic organoids. Our results highlight the presence of two lymphoid clusters, CLP and LMPP, expressing CD10. The LMPP cluster is about 6 fold expanded in UM171-treated cells as compared to uncultured and DMSO-treated cells. Using the marker CD10 and CD45RA we identify two CD34+ populations (CD10medCD45RAmed/lo and CD10hiCD45RAhi) that are significantly expanded in CD34+ CB cells cultured in the presence of UM171 in contrast to uncultured CD34+ CB cells and DMSO-supplemented cultures. Furthermore, we demonstrate that only the CD45RA-negative cells can give rise to both CD10-positive subsets in vitro, suggesting that CD10medCD45RAmed/lo cells define a subset with a more primitive phenotype than CD10hiCD45RAhi. We propose that the CD10medCD45RAmed/lo subset could contain LMPPs that will commit to CLPs contained within the CD10hiCD45RAhi subset. Functionally, we compared T-cell development and maturation of the CD10+CD34+ subset (including CD10medCD45RAmed/lo and CD10hiCD45RAhi) to the CD45RA+CD34+, CD45RA-CD34+ and total CD34+ subsets and denote a faster T cell development and maturation at 3 and 6 weeks within the CD10-positive subset in contrast to the total CD34+ subset, suggesting that CD10 can be used as a marker of lymphoid precursors in UM171 cultures. We also observe a higher percentage of ILC-like cells within the CD10-positive subset as compared to total CD34+ cells suggesting that the CD10- positive subset potentially contains lymphoid precursors with multilineage capacity. Faster T cell development is observed for the CD45RA+ subset whereas CD45RA- cells display the slowest T cell development, hence we propose that the CD45RA- subset contains primitive cells (e.g. HSC) that segregate into lineage-biased multipotent progenitors enriched in the CD10-positive subset (e.g. LMPP) that will give rise to lineage-restricted precursors that are CD45RA+ (e.g. CLP). In sum, our findings represent the first identification of a CD10-positive population within the UM171- expanded graft that has T potential and could potentially produce ILCs. Delayed immune reconstitution is a major obstacle impeding the implementation of CB HSCT; therefore optimizing infusion of CD3+ donor cells by enriching for lymphoid precursors could help boost T-cell reconstitution following allogeneic HSCT.

The HLA-DRB3/4/5 loci are closely linked to the HLA-DRB1 gene. Mismatches in these loci occur with a frequency of about 8%–12% in otherwise 10/10 HLA-matched transplant pairs. There is preliminary evidence that these disparities may associate with increased acute graft-versus-host disease (GvHD) rates. The aim of this study was to analyze a large cohort of German patients and their donors for HLA-DRB3/4/5 compatibility and to correlate the HLA-DRB3/4/5 matching status with the outcome of unrelated hematopoietic stem cell transplantation (uHSCT). To this end, 3,410 patients and their respective donors were HLA-DRB3/4/5 and HLA-DPB1 typed by amplicon-based nextgeneration sequencing (NGS). All patients included received their first allogeneic transplant for malignant hematologic diseases between 2000 and 2014. Mismatches in the antigen recognition domain (ARD) of HLA-DRB3/4/5 genes were correlated with clinical outcome. HLA-DRB3/4/5 incompatibility was seen in 12.5% (n = 296) and 17.8% (n = 185) of the 10/10 and 9/10 HLA-matched cases, respectively. HLA-DRB3/4/5 mismatches in the ARD associated with a worse overall survival (OS), as shown in univariate (5-year OS: 46.1% vs. 39.8%, log-rank p = 0.038) and multivariate analyses [hazard ratio (HR) 1.25, 95% CI 1.02–1.54, p = 0.034] in the otherwise 10/10 HLAmatched subgroup. The worse outcome was mainly driven by a significantly higher nonrelapse mortality (HR 1.35, 95% CI 1.05–1.73, p = 0.017). In the 9/10 HLA-matched cases, the effect was not statistically significant. Our study results suggest that mismatches within the ARD of HLA-DRB3/4/5 genes significantly impact the outcome of otherwise fully matched uHSCT and support their consideration upon donor selection in the future.

Functional impairment of the bone marrow (BM) niche has been suggested as a major reason for prolonged cytopenia and secondary graft failure after allogeneic hematopoietic cell transplantation (alloHCT). Because mesenchymal stromal cells (MSCs) serve as multipotent progenitors for several niche components in the BM, they might play a key role in this process. We used collagenase digested trephine biopsies to directly quantify MSCs in 73 patients before (n = 18) and/or after alloHCT (n = 65). For the first time, we demonstrate that acute graft-versus-host disease (aGvHD, n = 39) is associated with a significant decrease in MSC numbers. MSC reduction can be observed even before the clinical onset of aGvHD (n = 10). Assessing MSCs instantly after biopsy collection revealed phenotypic and functional differences depending on the occurrence of aGvHD. These differences vanished during ex vivo expansion. The MSC endotypes observed revealed an enhanced population of donor-derived classical dendritic cells type 1 and alloreactive T cells as the causing agent for compartmental inflammation and MSC damage before clinical onset of aGvHD was ascertained. In conclusion, MSCs endotypes may constitute a predisposing conductor of alloreactivity after alloHCT preceding the clinical diagnosis of aGvHD.

Purpose: We determined the indication, outcome and risk factors of single and multiple hematopoietic stem cell transplantation(s) (HSCT) in children and adolescents mostly with advanced disease. Methods: Forty-one out of 483 patients (8.5%; median age 9 years) diagnosed at the University of Leipzig with haematological and oncological diseases required HSCT from 1999 to 2011. Results: Patients had overall survival (OS) of 63±10% and 63±16%, event-free survival (EFS) of 57±10% and 42±16%, relapse incidence (RI) of 39±10% and 44±18% and non-relapse mor-tality (NRM) of 4±4% and 13±9% at 10-years after one or more HSCT for allogeneic and autologous HSCT, respectively. One patient in complete remission (CR)1 and five with advanced disease received two HSCT. Four of the six patients maintained/achieved CR for a median of 13 months. Three died of progression and one of NRM. Two patients had a third HSCT and one survived in CR +231 days after HSCT. Risk factors for OS and EFS were disease stage at HSCT and EBMT risk-score. Center (paediatric or JACIE accredited paediatric/adult) was not a determinant for survival. Conclusion: Paediatric single and multiple HSCT are important curative approaches for high-risk malignant diseases with low NRM. Efforts to reduce high RI remain the major aim.

O diabetes mellitus tipo 1 (DM-1) e a esclerose múltipla (EM) são doenças auto-imunes órgão-específicas, inflamatórias, mediadas por células T e B auto-reativas e caracterizadas pela destruição seletiva de células b pancreáticas produtoras de insulina e do sistema nervoso central, respectivamente. Acredita-se que a desregulação da expressão de genes reguladores da maquinaria apoptótica possa contribuir para o desenvolvimento da auto-imunidade, visto que algumas dessas moléculas participam nos processos de tolerância central e periférica de linfócitos auto-reativos. O objetivo deste projeto foi analisar a expressão de moléculas reguladoras das vias intrínseca, extrínseca e da Família de proteínas inibidoras da apoptose (IAP) em 33 indivíduos saudáveis, 15 pacientes com DM-1 e 18 com EM submetidos à terapia de imunossupressão em altas doses seguida do transplante autólogo de células-tronco hematopoéticas (IAD/TACTH). As células mononucleares (CMN) foram isoladas do sangue periférico dos controles e de pacientes nos períodos pré-mobilização (pré-mob), pré-condicionamento (pré-cond), D+180, D+360, D+540 e D+720 pós-transplante. As CMN foram utilizadas para extração de RNA, síntese de cDNA, quantificação da expressão por PCR em tempo real dos genes a1, bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bid, bik, bim, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 e cIAP-2 e protéica de Bcl-2, Bcl-xL, Bak, Bim e c-FLIPL por western-blotting. Os resultados de expressão gênica foram representados por unidades relativas de expressão em medianas nas diferentes amostras. Os pacientes com DM-1 apresentaram diminuição da expressão dos genes anti-apoptóticos bcl-2 (mediana: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) e cIAP-1 (1,24; p=0,003) nas CMN dos pacientes no período pré-mob em relação aos indivíduos saudáveis (medianas: bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), enquanto a expressão de cIAP-2 (60,8; p=0,0005) estava aumentada em relação aos controles (23,3). Foi observada redução significativa na expressão dos genes pró-apoptóticos bad (0,002; p<0,0001), bax (0,01; p=0,002) e fasL (1,66; p=0,001) no período pré-mob comparada aos controles sadios (bad: 0,23; bax: 2,79; fasL: 3,56). Os níveis de RNAm de bid (0,10; p=0,001) e bok (0,72; p=0,006) estavam elevados no pré-mob em relação ao grupo controle (bid: 0,004; bok: 0,31). As moléculas bcl-2, bcl-w, bcl-xL, mcl-1, bad, bax, bok, fasL e cIAP-1 atingiram níveis de RNAm similares aos controles após o TACTH. Foi verificado que a expressão de bcl-w, cIAP-1 e noxa estava maior nos pacientes com DM-1 em remissão quando comparados àqueles em recaída. A diminuição da expressão de a1, bcl-2 e bcl-w e o aumento de fas e noxa correlacionaram-se às porcentagens de hemoglobina glicosilada, concentração de auto-anticorpos GAD65, e aos níveis séricos de peptídeo-C após o transplante. Os pacientes com EM mostraram uma expressão reduzida dos genes anti-apoptóticos bcl-w (0,11; p=0,02) e cIAP-1 (1,87; p=0,04) no pré-mob comparada aos valores dos controles (bcl-w: 0,27; cIAP-1: 7,75) e maior expressão dos genes a1 (90,8; p=0,001) e cIAP-2 (58,8; p=0,009) em relação aos controles (a1: 12,7; cIAP-2: 22,3). As moléculas pró-apoptóticas bad (0,007; p=0,01) e bax (0,0007; p=0,004) mostraram menor expressão nas CMN no período pré-mob do que nos controles (bad: 0,27; bax: 1,24). Os genes bid (20,7; p=0,004), bik (0,84; p=0,02) e bok (1,77; p=0,0001) possuíam maior expressão no período pré-mob em relação aos indivíduos sadios (bid: 2,64; bik: 0,33; bok: 0,26). Não foram observadas diferenças significativas na expressão das moléculas da via extrínseca da apoptose nos pacientes com EM (p>0,05) nos períodos avaliados. Os valores de expressão de bcl-w, bak, bax, bik, bok e cIAP-1 atingiram níveis semelhantes aos controles após o transplante. A expressão dos genes bcl-2, cIAP-1, bad e bax estava maior nos pacientes em remissão da EM quando comparados àqueles em progressão neurológica. O aumento da expressão dos genes pró-apoptóticos bax, bak e bimEL correlacionou-se inversamente aos valores de EDSS dos pacientes com EM após o TACTH. Os resultados de expressão protéica foram equivalentes aos de expressão gênica nas duas doenças, com exceção dos dados das proteínas Bcl-2 e Bim. Em conjunto, os resultados demonstraram a desregulação da expressão de várias moléculas anti- e pró-apoptóticas nas CMN dos pacientes com DM-1 e EM. Esses achados sugerem a associação de alterações nos processos de apoptose celular com o surgimento e persistência de células auto-reativas no DM-1 e EM. Os dados indicam que essas alterações, principalmente a diminuição da expressão de moléculas pró-apoptóticas, como bak e bax, possam contribuir para a patogênese do DM-1 e EM. Além disso, a terapia de IAD/TACTH foi capaz de modular a expressão da maioria dos genes anormalmente expressos nas CMN dos pacientes com DM-1 e EM, já que esses atingiram níveis de expressão similares ao grupo controle após o transplante. Esta normalização da expressão de vários genes analisados correlacionou-se com a remissão clínica da doença na maioria dos pacientes / Type 1 diabetes mellitus (T1DM) and multiple sclerosis (MS) are inflammatory, organ-specific autoimmune diseases characterized by selective destruction of insulin-producing pancreatic -cells and central nervous system, respectively, by autoreactive B and T cells. Deregulation of apoptotic machinery is supposed to contribute to self-tolerance breakdown and autoimmune diseases pathogenesis, since apoptotic molecules have an important role in B and T lymphocytes central and peripheral tolerance mechanisms. The aim of this study was to evaluate the expression of pro and anti-apoptotic molecules from intrinsic and extrinsic apoptotic pathways and IAP Family members in 33 healthy individuals, 15 T1DM and 18 MS patients submitted to high-dose immunossupression therapy followed by autologous hematopoietic stem cell transplantation (HDI/AHSCT). Peripheral blood mononuclear cells (PBMC) were isolated from controls and patients at pre-mobilization (pre-mob), pre-conditioning (pre-cond), D+180, D+360, D+540 and D+720 post-transplantation. PBMC were used for RNA extraction, cDNA synthesis, gene quantification of a1, bcl-2, bcl-w, bcl-xL, bad, bak, bax, bid, bik, bimEL, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 and cIAP-2 by Real Time PCR and Bcl-2, Bcl-xL, Bak, BimEL and c-FLIPL proteins detection by western-blotting. Results are expressed as median of relative expression units. Results from T1DM patients indicated that antiapoptotic molecules bcl-2 (median: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) and cIAP-1 (1,24; p=0,003) were downregulated at pre-mob compared with healthy controls (medians bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), while cIAP-2 (60,8; p=0,0005) gene expression was upregulated compared to healthy controls (23,3). We observed a significant decrease in proapoptotic bad (0,002; p<0,0001), bax (0,01; p=0,002) and fasL (1,66; p=0,001) genes expression in patients PBMC at pre-mob period compared to healthy subjects (bad: 0,23; bax: 2,79; fasL: 3,56). mRNA levels of bid (0.10; p=0.001) and bok (0.72; p=0.006) were elevated at pre-mob period when compared to control group (bid: 0.004; bok: 0.31). The bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bok, fasL and cIAP-1 mRNA levels reached controls levels after HDI/AHSCT. We observed that bcl-w, cIAP-1 and noxa gene expression were increased in T1DM patients in remission when compared to relapsed patients. The decreased antiapoptotic gene expression and increased in proapoptotic molecules correlated with decreased glicosilated hemoglobin percentages (Hb A1C) and anti-GAD65 antibodies and increased peptide-C levels. Results from MS patients showed decreased bcl-w (0,11; p=0,02) and cIAP-1 gene expression (1,87; p=0,04) in patients PBMC at pre-mob period compared to healthy controls (bcl-w: 0,27; cIAP-1: 7,75) and increased expression of a1 (90,8; p=0,001) and cIAP-2 (58,8; p=0,009) compared to controls (a1: 12,7; cIAP-2: 22,3). Proapoptotic molecules bad (0.007; p=0.01) and bax (0.0007; p=0.004) showed decreased gene expression at pre-mob compared to control group (bad: 0.27; bax: 1.24). bid (20.7; p=0.004), bik (0.84; p=0.01) and bok genes (1.77; p=0.0001) showed increased expression at pre-mob compared to healthy controls (bid: 2.64; bik: 0.33; bok: 0.26). Significant differences were not observed in the expression of the extrinsic pathway genes in pre-mob and healthy controls samples (p>0.05). bcl-w, bak, bax, bik, bok and cIAP-1 expression values reached healthy control values after transplantation. We observed that bcl-2, cIAP-1, bad and bax gene expression was increased in MS patients in disease remission when compared to patients with neurologic progression. Significant correlation of increased proapoptotic genes expression with decreased EDSS values in MS patients after HDI/AHSCT was observed. Results of protein quantification of apoptotic molecules in PBMC of T1DM and MS patients were similar to the gene expression results of these molecules, except for Bcl-2 and Bim proteins. Taken together, these data indicate a deregulated expression of anti- and proapoptotic genes in T1DM and MS patients PBMC. These data suggest an association of deregulated apoptosis with emergence and maintenance of autoreactive lymphocytes in analyzed patients. Based on these results, we suggest that this altered gene expression profile, mainly the decreased proapoptotic genes expression, as bak and bax, may contribute to T1DM and MS pathogenesis. Furthermore, we showed that the HDI/AHSCT therapy was able to modulate and normalize the expression of most genes abnormally expressed in T1DM and MS patients at pre-transplant period. Many analyzed genes achieved expression levels similar to healthy controls. The normalization of the expression of many evaluated genes correlated to disease remission in the majority of the patients.