Academic literature on the topic 'Cellules pro-B'

O-linked β-N-acetylglucosamine (O-GlcNAc) modification regulates the activity of hundreds of nucleocytoplasmic proteins involved in a wide variety of cellular processes, such as gene expression, signaling, and cell growth; however, the mechanism underlying the regulation of B cell development and function by O-GlcNAcylation remains largely unknown. Here, we demonstrate that changes in cellular O-GlcNAc levels significantly affected the growth of pre-B cells, which rapidly proliferate to allow expansion of functional clones that express successfully rearranged heavy chains at the pro-B stage during early B cell development. In our study, the overall O-GlcNAc levels in these proliferative pre-B cells, which are linked to the glucose uptake rate, were highly induced when compared with those in pro-B cells. Thus, pharmacologically, genetically, or nutritionally, inhibition of O-GlcNAcylation in pre-B cells markedly downregulated c-Myc expression, resulting in cell cycle arrest via blockade of cyclin expression. Importantly, the population of B cells after the pro-B cell stage in mouse bone marrow was severely impaired by the administration of an O-GlcNAc inhibitor. These results strongly suggest that O-GlcNAcylation-dependent expression of c-Myc represents a new regulatory component of pre-B cell proliferation, as well as a potential therapeutic target for the treatment of pre-B cell-derived leukemia.

The most important feature of humoral immunity is the adaptation of the diversity of newly generated B cell receptors, that is, the antigen receptor repertoire, to the body’s own and foreign structures. This includes the transient propagation of B progenitor cells and B cells, which possess receptors that are positively selected via anabolic signalling pathways under highly competitive conditions. The metabolic regulation of early B-cell development thus has important consequences for the expansion of normal or malignant pre-B cell clones. In addition, cellular senescence programs based on the expression of B cell identity factors, such as Pax5, act to prevent excessive proliferation and cellular deviation. Here, we review the basic mechanisms underlying the regulation of glycolysis and oxidative phosphorylation during early B cell development in bone marrow. We focus on the regulation of glycolysis and mitochondrial oxidative phosphorylation at the transition from non-transformed pro- to pre-B cells and discuss some ongoing issues. We introduce Swiprosin-2/EFhd1 as a potential regulator of glycolysis in pro-B cells that has also been linked to Ca2+-mediated mitoflashes. Mitoflashes are bioenergetic mitochondrial events that control mitochondrial metabolism and signalling in both healthy and disease states. We discuss how Ca2+ fluctuations in pro- and pre-B cells may translate into mitoflashes in early B cells and speculate about the consequences of these changes.

Abstract In the bone marrow (BM), stromal cells constitute a supportive tissue indispensable for the generation of pro-B/pre-BI, pre-BII, and immature B lymphocytes. IL-7–producing stromal cells constitute a cellular niche for pro-B/pre-BI cells, but no specific stromal cell microenvironment was identified for pre-BII cells expressing a functional pre-B cell receptor (pre-BCR). However expression of the pre-BCR represents a crucial checkpoint during B-cell development. We recently demonstrated that the stromal cell derived-galectin1 (GAL1) is a ligand for the pre-BCR, involved in the proliferation and differentiation of normal mouse pre-BII cells. Here we show that nonhematopoietic osteoblasts and reticular cells in the BM express GAL1. We observed that pre-BII cells, unlike the other B-cell subsets, were specifically localized in close contact with GAL1+ reticular cells. We also determined that IL-7+ and GAL1+ cells represent 2 distinct mesenchymal populations with different BM localization. These results demonstrate the existence of a pre-BII specific stromal cell niche and indicate that early B cells move from IL-7+ to GAL1+ supportive BM niches during their development.

Abstract Abstract 3532 B cell acute lymphoblastic leukemia (B-ALL) arises by transformation of progenitor (pre-B) cells. Cure rates in adults remain low and treatment is complicated by microenvironment-mediated resistance to cytotoxic drugs, indicating an urgent need for development of new, more targeted treatment approaches. Spleen tyrosine kinase (Syk), a B cell receptor (BCR)-associated tyrosine kinase, recently was identified as a novel therapeutic target in mature B cell malignancies, such as chronic lymphocytic leukemia (CLL). Besides its role in BCR signaling in mature B cells, Syk also plays an important role in maintenance and expansion of immature B cells. Syk-deficient mice display a severe defect of B lymphopoiesis, with a block at the pro-B to pre-B transition, consistent with a key role for Syk in pre-BCR signaling. We therefore hypothesized that pre-B ALL cells which express pre-BCRs might be more sensitive to Syk inhibition than other ALL subtypes, which either do not have functional pre-BCRs (pro-B ALL) or display modifying genetic lesions (Ph+/BCR-ABL+ B-ALL). PRT060318, a highly specific, ATP-competitive, small molecular inhibitor of Syk, has preclinical activity in CLL and DLBCL models (Hoellenriegel J et al., Leukemia 26:1576–83, 2012). Syk specificity was confirmed by cellular and non-cellular kinase inhibition assays. We performed metabolic assays using the tetrazolium dye XTT to screen B-ALL cell lines (RS4;11, REH, TOM-1, Nalm-20, Nalm-21, Nalm-6, 697, Kasumi-2, KOPN-8, RCH-ACV, SMS-SB) for responses to PRT060318. ALL cells were incubated with increasing concentrations of PRT060318 (100 nM – 10 μM) for 72 hours. Based on the XTT assays, we determined half maximal inhibitory concentration (IC50), and separated B-ALL cells into responsive (IC50 < 4.5 μM) and non-responsive (IC50 > 4.5 μM) groups. Interestingly, the responsive group consisted only of pre-B ALL cells (CD10+, TdT+, cytoIgμ+), whereas the resistant group comprised only ALL cell lines with a pro-B cell phenotype (CD10+/−, TdT+, cytoIgμ-). All BCR-ABL positive cell lines tested exhibit the pro-B cell phenotype and, similar to their BCR-ABL negative counterparts, were resistant to PRT060318. The Figure depicts XTT assay results for two PRT060318-sensitive pre-B (grey lines) and two resistant pro-B (black lines) B-ALL cell lines. Western Blot analysis demonstrated SYK protein expression in all B-ALL cell lines, excluding lack of target protein expression as possible cause for different response rates between pre-B and pro-B ALL cells. Cell proliferation assays revealed a significant, dose-dependent inhibition of pre-B ALL cell proliferation by PRT060318. Concordantly, dose depended S phase reduction was detected in all PRT060318-sensitive cell lines. In apoptosis assays, in which sensitive pre-B ALL lines were incubated with increasing concentrations of PRT060318 (up to 5 μM) we did not find any significant induction of apoptosis, suggesting anti-proliferative, rather than cytotoxic effects as a main mechanism of action of PRT060318. Preliminary data of primary ALL patient samples, cultured with KUSA-H1 marrow stroma cells in the presence or absence of PRT060318 demonstrate induction of apoptosis in 3 out of 10 samples, other assays are ongoing. In conclusion, we provide evidence that the Syk inhibitor PRT060318 thwarts pre-B ALL cell proliferation, providing a first rationale for clinical testing of PRT060318 in selected patients with B cell ALL. Disclosures: Coffey: Portola Pharmaceuticals Inc.: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment. Pandey:Portola Pharmaceuticals Inc.: Employment.

Abstract The initial B-cell repertoire is generated by combinatorial immunoglobulin V(D)J gene segment rearrangements that occur in a preferential sequence. Because cellular proliferation occurs during the course of these rearrangement events, it has been proposed that intraclonal diversification occurs during this phase of B-cell development. An opportunity to examine this hypothesis directly was provided by the identification of a human acute lymphoblastic leukemic cell line that undergoes spontaneous differentiation from pro-B cell to the pre-B and B-cell stages with concomitant changes in the gene expression profile that normally occur during B-cell differentiation. After confirming the clonality of the progressively differentiating cells, an analysis of immunoglobulin genes and transcripts indicated that pro-B cell members marked by the same DJ rearrangement generated daughter B cells with multiple VH and VL gene segment rearrangements. These findings validate the principle of intraclonal V(D)J diversification during B-cell generation and define a manipulable model of human B-cell differentiation.

Objectif : La présente étude a pour but d’examiner la réponse à un schéma chimiothérapeutique administré à des enfants atteints de LAL à cellules pré-B à risque élevé selon le protocole COG. Méthode : L’étude transversale porte sur 55 enfants traités selon le protocole du groupe d’oncologie pédiatrique (mieux connu sous le nom de Children’s Oncology Group ou COG), de septembre 2010 à février 2015, et évalue les résultats du schéma chimiothérapeutique. Résultats : Durant la première semaine suivant le traitement, le taux de rétablissement complet a été de 76,4 %. Les taux de survie après trois ans et cinq ans étaient respectivement de 85,5 % et de 81 %. Le taux de rechute après le premier épisode de rémission a été de 20 % et le taux de mortalité consécutif à cette rechute a été de 50 %. Trente pour cent de l’ensemble des décès ont eu lieu durant la période d’induction. Dans tous les cas, une septicémie en est la cause. Conclusion : Les résultats indiquent que le taux de survie a augmenté. Il est donc possible d’améliorer le taux de survie en optant pour le protocole COG et en contrôlant les infections chez les patients, et ce, sans égard au groupe de risque.

We determined the temporal and spatial distribution of surfactant protein B (pro-SP-B) and C (pro-SP-C) mRNAs and proteins by immunohistochemistry and in situ hybridization in fetal, neonatal, and adult human lung. Pro-SP-B and SP-B mRNA were detected in bronchi and bronchioles by 15 weeks' gestation. After 25 weeks, pro-SP-B, active SP-B peptide, and SP-B mRNA were co-localized in bronchiolo-alveolar portal cells and in Type II epithelial cells. In adult lung, pro-SP-B and SP-B mRNA were detected primarily in non-ciliated bronchiolar epithelial cells and in Type II cells in the alveolus. Pro-SP-C and SP-C mRNA were detected in cells lining terminal airways from 15 weeks' gestation and thereafter. After 25 weeks, SP-C mRNA and precursor protein were detected in epithelial cells of the bronchiolo-alveolar portals and in Type II cells, where expression increased with advancing gestational age. Distinct cellular patterns of staining for pro-SP-B compared with SP-B active peptide support the concept that its proteolytic processing or cellular routing may be influenced by cell type and/or cell differentiation. SP-B and SP-C are expressed primarily in distal conducting and terminal airway epithelium of human fetal lung well in advance of surfactant lipid synthesis or physiologic requirements to produce pulmonary surfactant at the time of birth.

Abstract Early B lymphopoiesis in mammals is induced within the bone marrow (BM) microenvironment, but which cells constitute this niche is not known. Previous studies had shown that osteoblasts (OBs) support hematopoietic stem cell (HSC) proliferation and myeloid differentiation. We now find that purified primary murine OBs also support the differentiation of primitive hematopoietic stem cells through lymphoid commitment and subsequent differentiation to all stages of B-cell precursors and mature B cells. Lin−Sca-1+Rag-2− BM cell differentiation to B cells requires their attachment to OBs in vitro, and this developmental process is mediated via VCAM-1, SDF-1, and IL-7 signaling induced by parathyroid hormone (PTH). Addition of cytokines produced by nonosteoblastic stromal cells (c-Kit ligand, IL-6, and IL-3) shifted the cultures toward myelopoiesis. Confirming the role of OBs in B lymphopoiesis, we found that selective elimination of osteoblasts in Col2.3Δ-TK transgenic mice severely depleted pre-pro-B and pro-B cells from BM, preceding any decline in HSCs. Taken together, these results demonstrate that osteoblasts are both necessary and sufficient for murine B-cell commitment and maturation, and thereby constitute the cellular homolog of the avian bursa of Fabricius.

Abstract The expression of Pax5 commits common lymphoid progenitor cells to B-lymphoid lineage differentiation. Little is known of possible variations in the levels of Pax5 expression and their influences on hematopoietic development. We have developed a retroviral transduction system that allows for the study of possible intermediate stages of this commitment by controlling the levels of Pax5 expressed in Pax5-deficient progenitors in vitro and in vivo. Retroviral transduction of Pax5-deficient pro-/pre-B cell lines with a doxycycline-inducible (TetON) form of the human Pax5 (huPax5) gene yielded cell clones that could be induced to different levels of huPax5 expression. Clones inducible to high levels developed B220+/CD19+/IgM+ B cells, while clones with low levels differentiated to B220+/CD19−/CD11b+/Gr-1− B-lymphoid/myeloid biphenotypic cells in vitro and in vivo. Microarray analyses of genes expressed at these lower levels of huPax5 identified C/ebpα, C/ebpδ, Pu.1, Csf1r, Csf2r, and Gata-3 as myeloid-related genes selectively expressed in the pro-/pre-B cells that can develop under myeloid/lymphoid conditions to biphenotypic cells. Therefore, reduced expression of huPax5 during the induction of early lymphoid progenitors to B-lineage–committed cells can fix this cellular development at a stage that has previously been seen during embryonic development and in acute lymphoblastic lymphoma–like biphenotypic acute leukemias.

Le développement des cellules B est conduit par l'action combinée de facteurs de transcription, de facteurs solubles et d'interactions cellulaires, qui régulent leur engagement et différenciation. La signalisation par l'IL-7R et le pré-BCR régule la prolifération et la survie des cellules B précoces, en même temps qu'elle coordonne l'expression et le réarrangement des Ig. Un des facteurs de transcription impliqués dans le développement des cellules B est Etsl, un facteur fortement exprimé dans les lignées lymphoïdes, où il contrôle la différenciation des cellules B, T et NK. Son inactivation altère le développement des cellules B, en particulier dans la différenciation des cellules pro-B à pré-B. La dérégulation du processus de différenciation des cellules B est à l'origine des leucémies. La Leucémie Aiguë Lymphoblastique (LAL) est une prolifération clonale qui se développe à partir d'une cellule lymphoïde bloquée à un stade précoce de différenciation. Il existe plusieurs formes de LAL ayant des altérations génétiques diverses, parmi elles la translocation t(9,22), qui induit l'expression de la protéine oncogénique BCR-ABLI et est associée à un mauvais pronostic. BCR-ABL I active constitutivement STAT5, et contribue à leucémogenèse à travers la voie de signalisation JAK/STAT. L'objectif de cette thèse est d'étudier la fonction du facteur de transcription Etsl dans le développement des cellules B précoces. Nos résultats montrent une nouvelle fonction de Etsl dans la régulation de l'activité de STAT5 dans le développement des cellules B et dans le contrôle du programme de transcription en aval de BCR-ABLI pendant les étapes initiales de leucémogenèse dans les cellules pré-B
B-cell development is driven through combined actions of transcription factors, soluble factors and cellular interactions, which regulate the commitment and differentiation of B tells. Signaling through IL-7R and pre-BCR regulates the transcription of genes involved in proliferation and survival of early B tells, while it coordinates the expression and rearrangement of Ig genes. One of the transcription factors involved on B tell development is Etsl, which is a member of the ETS family of transcription factors and it is highly expressed in the lymphoid lineages where it controls the differentiation of B, T and NK tells. Its inactivation impairs B tell development, particularly during the differentiation of pro-B into pre-B tells. Deregulation of the process of B tells development causes leukemia. Acute lymphoblastic leukemia (ALL) is a clonai proliferation that develop from a lymphoid tell blocked at an early stage of differentiation. This can be due to diverse genetic alterations, among them the t(9,22) translocation induces the expression of an oncogenic fusion protein, called BCR-ABL1, which is associated with poor prognosis. BCR-ABL1 activates constitutively STAT5 and thus likely contributes to leukemogenesis by signaling via the JAK/STAT pathway. The aim of this thesis is to study the role and function of Ets I transcription factor in early B tell development. These findings demonstrate a nove] function for the Etsl transcription factor in the regulation of STAT5 activity during early B-tell development and in the control of the transcription program downstream BCR-ABL1 during the initial steps of leukemogenesis in pre-B tells

La tuberculose causée par Mycobacterium tuberculosis est la plus mortelle des maladies infectieuses. Les mycobactéries sont des pathogènes intra-cellulaires qui ont développé des mécanismes d’évasion spécifiques pour survivre au sein de leur hôte. Une réponse immune anti-mycobatérienne efficace met en jeu l’activation des cellules T CD4+ Th1 ainsi que la génération d’IFN- microbicide. Cependant, des patients ayant une tuberculose active présentent une réponse immune de type Th2 durant les stades les plus tardifs de l’infection. L’association de ce pathogène avec le VIH chez les patients atteints du SIDA et l’émergence de souches extrêmement résistantes aux traitements actuelles font de la tuberculose une « urgence mondiale ». Ainsi, découvrir les interactions moléculaires des motifs antigéniques associés aux mycobactéries pathogènes (« PAMPs ») avec le système immunitaire de l’hôte est important pour comprendre la pathogenèse de la tuberculose et pour permettre une immunointervention thérapeutique efficace. La mise en place d’une réponse immune contre M. Tuberulosis met en jeu l’interaction entre les systèmes immunitaires innés et adaptatifs. Les cellules de l’immunité innée comme les cellules dendritiques (CD) sont impliquées dans la reconnaissance du pathogène et des antigènes qui en dérives, dans la présentation de peptides antigéniques aux cellules T et dans la sécrétion d’un large panel de cytokines qui permettent la différenciation et l’expansion des cellules T CD4+ et T CD8+. Les cellules T CD4+ Th fournissent l’aide nécessaire aux lymphocytes B pour la production des anticorps. Outre la production d’anticorps, les cellules B peuvent aussi agir comme cellules présentatrices de l’antigène et produire différentes cytokines et chimiokines immunorégulatrices qui modulent la réponse immune et l’inflammation. Durant ma thèse, j’ai étudié l’interaction moléculaire des « PAMPs » de M. Tuberculosis avec des CD et des cellules B humaines et j’ai examiné in vivo les réponses immunes dirigées contre les « PAMPs » en association avec différentes molécules adjuvantes. Dans un premier temps, j’ai étudié la régulation des fonctions des CD humaines par un antigène mycobactérien de surface mannosylé : le mannose lipoarabinomannan (ManLAM). Le ManLAM est un composant principal de la membrane cellulaire des mycobactéries et est impliqué dans l’immunopathogenèse de la tuberculose. J’ai démontré que le ManLAM induisait la maturation des CD vers une réponse immune de type Th2 et la production de chimiokines impliquées dans la migration des cellules T Th2 et T régulatrice (Treg). De plus, les cellules B ont été retrouvées sur le site de réactions granulomateuses chez l’homme et la souris atteints de la tuberculose. Cependant le rôle des cellules B dans la réponse immune contre la tuberculose reste inconnu. A cet effet, j’ai étudié l’interaction du ManLAM avec des cellules B humaines issues de donneurs sains. J’ai démontré que le ManLAM stimulait la prolifération des cellules B avec une forte production d’IL-8 et d’IL-6. Mes résultats montrent de même que la prolifération des cellules B stimulées par le ManLAM avait pour origine l’interaction entre le ManLAM et le TLR-2. Ainsi, les cellules B éduquées par le ManLAM propagent la réponse immune de type Th2. L’ensemble de mes résultats démontrent que le ManLAM module la fonction des CD et des cellules B afin d’échapper à la réponse cellulaire T efficace contre M. Tuberculosis. PE, PPE sont de nouvelles familles de protéines qui ont été identifiées après que le génome de M. Tuberculosis ait été entièrement séquencé. Ces classes de protéines sont propres aux mycobactéries et la plupart d’entre elles sont des protéines de surface qui ont la capacité d’interagir avec les cellules du système immunitaire. J’ai étudié l’interaction de PE_PGRS 62 (Rv3812) avec les CD humaines et j’ai montré que PE_PGRS 62 agissait comme un ligant de TLR-2 et induisait la maturation des CD vers des réponses de type Th1. De plus, j’ai démontré que PE_PGRS 62 générait de fortes réponses immunitaires in vivo dans différents modèles expérimentaux. L’ensemble de ces résultats montre que PE_PGRS 62 met en place une réponse cellulaire de type Th1 en présence ou non d’antigènes immunorégulateurs tels que le ManLAM et la protéine gp120 du VIH. Ceci permet d’envisager l’utilisation de PE_PGRS 62 comme vaccin candidat pour combattre la tuberculose.

Les cellules Natural Killer (NK) représentent une population de cellules innées lymphoïdes aux fonctions anti-infectieuses et antitumorales. Les leucémies aiguës lymphoblastiques pré-B (LAL pré-B) constituent le cancer de l’enfant le plus fréquent et ont été décrites comme résistantes à la cytotoxicité médiée par les NK bien que les bases moléculaires demeurent inconnues.L’objectif de ces travaux a été de caractériser cette résistance. En développant un essai de cytotoxicité par cytométrie en flux et en utilisant des cellules effectrices activées in vitro, nous avons établi la sensibilité retardée des LAL pré-B à la cytotoxicité NK : initialement résistantes après 4h d’incubation, elles sont fortement tuées après 25h.Cette cytotoxicité est contact-dépendante mais ni la voie de l’exocytose des granules cytotoxiques ni celle des récepteurs de mort n’y contribuent. La mort cellulaire des cibles est de profil apoptotique mais indépendante des caspases ; la signalisation mitochondriale l’amplifie partiellement. Interférer avec les dérivés de l’oxygène par un antioxydant diminue la cytotoxicité. Nous montrons que les cellules NK de patients atteints de granulomatose septique chronique liée à l’X présentent un défaut de cette nouvelle cytotoxicité. Nous démontrons l’expression par les NK des composants clefs d’une NADPH oxydase distincte du complexe utilisé par les phagocytes. Nos travaux établissent l’existence d’une voie de cytotoxicité non conventionnelle et en définissent les principaux prérequis moléculaires
Natural Killer (NK) cells are innate lymphoid cells with anti-infectious and anti-tumoral activities. Among neoplasia, pre-B acute lymphoblastic leukemias (pre-B ALL) represent the most common form of cancer in childhood and were shown to be resistant to NK cell mediated cytotoxicity although the mechanisms explaining this phenomenon are incompletely understood.In the present work, we investigated the relative immune resistance of pediatric pre-B ALL targets to activated NK cells. We developed a flow cytometry based cytotoxicity assay to assess the NK activity and the involvement of long term cytotoxic pathways. Although pre-B ALL blasts were strongly resistant at 4h, we found a considerable delayed NK killing at 25h.Further investigations revealed that cell contact was mandatory for efficient killing but also that neither the granule exocytosis nor the death receptor pathway were involved. Target cell death was caspase independent but mitochondria signaling amplified it. We then showed that NK cells from patients with X-linked chronic granulomatous disease could not kill efficiently ALL blasts and that NK cells expressed key components of a NADPH oxidase complex that was distinct from the phagocyte type. Our work reveals an uncharacterized effector pathway among cytotoxic lymphocytes and establishes key molecular requirements for this unconventional pathway

Le stade pre-B représente un point de contrôle crucial du développement des lymphocytes B dans la moelle osseuse. A ce stade, il y aura formation d'un récepteur intermédiaire nommé pre-BCR. Le pre-BCR est constitué de deux chaines lourdes Igµ et de deux pseudo-chaines légères (SLCs). Chaque SLC est constituée des protéines λ5 et VpreB, qui possèdent des régions « uniques » à leur extrémité N et C-terminale, respectivement. Ces régions uniques sont cruciales pour les fonctions du récepteur. La première partie de mes travaux sur l'étude du domaine λ5-UR nous a permis de proposer un modèle original d'assemblage du pre-BCR et d'apporter les bases structurales du rôle de chaperonne intramoléculaire de λ5-UR. L'activation du récepteur est permis par la formation d'une synapse immunologique. Des interactions entre la galectine-1 et λ5-UR vont permettre la formation d'un treillis d'interactions. L'étude structurale du complexe GAL1/λ5-UR, réalisée dans la seconde partie de ma thèse, a permis de déterminer la structure du complexe. L'interaction GAL1/λ5-UR engendre une modification d'affinité de GAL1 pour le lactose. Ce résultat suggère que l'interaction entre le pre-BCR et la galectine-1 peut influencer l'équilibre des interactions au niveau de la lattice en modulant l'affinité de la galectine-1 pour certains glycans. Dans la troisième partie de mon travail de thèse, des approches de glycomique fonctionnelle et structurale nous a permis l'élaboration d'un mécanisme de formation-dissolution de la synapse pre-B basés sur une modification d'affinité de GAL1 pour certains carbohydrates en présence de λ5-UR
The pre-B stage represents a critical checkpoint in the development of B cells in the bone marrow. At this stage , there will be formation of a receptor intermediate called pre-BCR . The pre -BCR is composed of two heavy chains Igμ and two surrogate light chains ( SLCs ) . Each SLC consists of two proteins: λ5 and VpreB , which have "unique region" to their N-terminus and C -terminus, respectively. These unique regions are crucial for the functions of the receptor. The first part of my work on the domain λ5-UR has allowed us to propose an original model for assembling the pre -BCR and provide the structural basis of the role of intramolecular chaperone of λ5-UR. Receptor activation is allowed by the formation of an immunological synapse. Interactions between galectin-1 and λ5-UR will allow the formation of a lattice interactions. The structural study of complex GAL1/λ5-UR , conducted in the second part of my thesis, has allowed to determine the structure of the complex. These interactions GAL1/λ5-UR generate a modification of affinity of GAL1 for lactose. This result suggests that the interaction between the pre-BCR and galectin-1 may affect the balance of interactions at the lattice by modulating the affinity for galectin-1 for some glycans. In the third part of my thesis, approaches to structural and functional glycomics has allowed us to develop a mechanism of formation-dissolution of the synapse pre-B based on a modified affinity of GAL1 for certain carbohydrates in presence of λ5-UR

Les cellules gliales sont essentielles au fonctionnement du système nerveux. Dans la rétine, les cellules gliales de Müller assurent à la fois l’homéostasie du tissu et la protection des neurones, notamment celle des cellules ganglionnaires de la rétine (CGRs). L’hypothèse principale de la thèse est que les cellules de Müller joueraient un rôle primordial dans la survie neuronale tant au plan de la signalisation des neurotrophines/proneurotrophines par suite d’une blessure que lors des mécanismes d’excitotoxicité. Contrairement au brain-derived neurotrophic factor (BDNF), le nerve growth factor (NGF) n’est pas en mesure d’induire la survie des CGRs après une section du nerf optique. Le premier objectif de la thèse a donc été de localiser les récepteurs p75NTR et TrkA du NGF dans la rétine adulte et d’établir leur fonction respective en utilisant des ligands peptidomimétiques agonistes ou antagonistes spécifiques pour chacun des récepteurs. Nos résultats ont démontré que TrkA est surexprimé par les CGRs après l’axotomie, tandis que p75NTR est spécifiquement exprimé par les cellules de Müller. Alors que NGF n’est pas en mesure d’induire la survie des CGRs, l’activation spécifique de TrkA par des ligands peptidomimétique est nettement neuroprotectrice. De façon surprenante, le blocage sélectif de p75NTR ou l’absence de celui-ci protège les CGRs de la mort induite par l’axotomie. De plus, la combinaison de NGF avec l’antagoniste de p75NTR agit de façon synergique sur la survie des CGRS. Ces résultats révèlent un nouveau mécanisme par lequel le récepteur p75NTR exprimé par les cellules gliales de Müller peut grandement influencer la survie neuronale. Ensuite, nous avons voulu déterminer l’effet des proneurotrophines dans la rétine adulte. Nous avons démontré que l’injection de proNGF induit la mort des CGRs chez le rat et la souris par un mécanisme dépendant de p75NTR. L’expression de p75NTR étant exclusive aux cellules de Müller, nous avons testé l’hypothèse que le proNGF active une signalisation cellulaire non-autonome qui aboutit à la mort des CGRs. En suivant cette idée, nous avons montré que le proNGF induit une forte expression du tumor necrosis factor α (TNFα) dans les cellules de Müller et que l’inhibition du TNF bloque la mort neuronale induite par le proNGF. L’utilisation de souris knock-out pour la protéine p75NTR, son co-récepteur sortiline, ou la protéine adaptatrice NRAGE a permis de montrer que la production de TNF par les cellules gliales était dépendante de ces protéines. Le proNGF semble activer une signalisation cellulaire non-autonome qui cause la mort des neurones de façon dépendante du TNF in vivo. L’hypothèse centrale de l’excitotoxicité est que la stimulation excessive des récepteurs du glutamate sensibles au N-Methyl-D-Aspartate (NMDA) est dommageable pour les neurones et contribue à plusieurs maladies neurodégénératives. Les cellules gliales sont soupçonnées de contribuer à la mort neuronale par excitotoxicité, mais leur rôle précis est encore méconnu. Le dernier objectif de ma thèse était d’établir le rôle des cellules de Müller dans cette mort neuronale. Nos résultats ont démontré que l’injection de NMDA induit une activation du nuclear factor κB (NF-κB) dans les cellules de Müller, mais pas dans les CGRs, et que l’utilisation d’inhibiteurs du NF-κB empêche la mort des CGRs. De plus, nous avons montré que les cellules de Müller en réaction à l’activation du NF-κB produisent la protéine TNFα laquelle semble être directement impliquée dans la mort des CGRs par excitotoxicité. Cette mort cellulaire se produit principalement par l’augmentation à la surface des neurones des récepteurs AMPA perméables au Ca2+, un phénomène dépendant du TNFα. Ces donnés révèlent un nouveau mécanisme cellululaire non-autonome par lequel les cellules gliales peuvent exacerber la mort neuronale lors de la mise en jeu de mécanismes excitotoxiques.
Glial cells are essential for the functioning of the nervous system. In the retina, the Müller glial cells ensure the homeostasis of this tissue as well as the protection of neurons including the retinal ganglion cells (RGCs). The main hypothesis of this thesis is that Müller cells play a predominant role in neuronal survival both at the levels of neurotrophin/proneurotrophin signaling following injury and excitotoxic mechanisms. Unlike the brain-derived neurotrophic factor (BDNF), the nerve growth factor (NGF) is unable to induce RGCs survival following optic nerve transection. The first objective of the thesis was therefore to describe the expression of the two receptors of NGF, p75NTR and TrkA, in the adult retina and to address their functional role by using peptidomimetic agonistic or antagonistic ligands specific for each receptor. Our results showed that TrkA is overexpressed by RGCs following axotomy, whereas p75NTR is specifically expressed by Müller cells. While NGF by itself does not promote RGC survival, selective activation of TrkA receptors using peptidomimetic ligands is markedly neuroprotective. Surprisingly, selective blockers of p75NTR, or the absence of p75NTR, protect RGCs from axotomy-induced death. Moreover, combination of NGF or TrkA agonists with p75NTR antagonists functions synergistically to enhance RGC survival. These results reveal a new mechanism by which p75NTR expression by Müller glia may profoundly influence neuronal survival. Next, we wanted to address the effect of proneurotrophins in the adult retina. We showed that injection of proNGF induces the death of RGCs in rats and mice by a p75NTR-dependent signaling mechanism. Expression of p75NTR in the adult retina being confined to Müller glial cells, we tested the hypothesis that proNGF activates a non-cell autonomous signaling pathway to induce RGC death. Consistent with this notion, we showed that proNGF induced a robust expression of tumor necrosis factor α (TNFα) in Müller cells, and that genetic or biochemical ablation of TNFα blocked proNGF-induced death of retinal neurons. Mice rendered null for p75NTR, its co-receptor sortilin, or the adaptor protein NRAGE were defective in proNGF-induced glial TNFα production and did not undergo proNGF-induced retinal ganglion cell death. We concluded that proNGF activates a non-cell autonomous signaling pathway that causes TNFα-dependent death of retinal neurons in vivo. The central hypothesis of excitotoxicity is that excessive stimulation of neuronal N-Methyl-D-Aspartate (NMDA)-sensitive glutamate receptors is harmful to neurons and contributes to a variety of neurological disorders. Glial cells have been proposed to participate in excitotoxic neuronal loss, but their precise role is poorly defined. In this in vivo study, we showed that NMDA induces a strong NF-κB activation in Müller glia, but not in retinal neurons. Intriguingly, NMDA-induced death of retinal neurons was effectively blocked by inhibitors of NF-κB activity. We demonstrated that TNFα protein produced in Müller glial cells via an NMDA-induced NF-κB dependent pathway plays a crucial role in the excitotoxic loss of retinal neurons. This cell loss occurs mainly through a TNFα-dependent increase in Ca2+-permeable AMPA receptors on susceptible neurons. Thus, our data reveal a novel non-cell-autonomous mechanism by which glial cells can profoundly exacerbate neuronal death following excitotoxic injury.

博士
國立成功大學
基礎醫學研究所
95
Chronic hepatitis B virus (HBV) infection is a major global cause of hepatocellular carcinoma (HCC). Hepatocytes expressing the HBV large surface antigen (LHBs) pre-S2 mutant, which is partially deleted in the pre-S2 region on HBV surface gene, exhibited nodule formation, clonal expansion and growth advantage. The majority of HBV-related HCC patients harbored pre-S2 mutant LHBs, indicating that pre-S2 mutant LHBsis highly associated with hepatocellular carcinogenesis. It was found that pre-S2 mutant LHBs accumulates in endoplasmic reticulum (ER) inducing ER stress and oxidative DNA damage. In this study, we aim to explore the molecular mechanisms regulated by pre-S2 mutant LHBs and seek potential prophylaxis and therapeutic approaches for pre-S2 mutant LHBs-associated HCC. We found that the oxidative stress and DNA damages induced by pre-S2 mutant LHBs were dependent on ER stress, as the ER stress inhibitors vomitoxin or TMB8 dramatically decreased such oxidative stress. By yeast two-hybrid screening assays, the pre-S2 mutant LHBs was found to directly interact with the Jun activation domain-binding protein 1 (JAB1), a subunit of COP9 signalosome. JAB1 has been shown to interact with p27Kip1 cyclin-dependent kinase inhibitor and target it to cytosolic 26S proteasome for degradation. We found that JAB1 and pre-S2 mutant LHBs enhanced the nuclear translocation of JAB1 and trigger p27Kip1 degradation and Cdk2 activation, resulting in inactivation of the tumor suppressor retinoblastoma (RB), a downstream molecule regulated by Cdk2. In transgenic mice carrying the pre-S2 mutant LHBs, p27Kip1 degradation, Cdk2 activation and RB inactivation were also seen, indicating the pre-S2 mutant LHBs triggers cell cycle progression in vivo and in vitro. These results suggest that cell cycle progression caused by RB inactivation might contribute to HCC associated with pre-S2 mutant LHBs. In the last part of our study, we seek potential targeted prophylaxis and therapeutic approaches for HCC induced by pre-S2 mutant LHBs. Vitamin D3 (VitD3) and the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) have been found to enhance thioredoxin binding protein 2 (TBP2) expression and JAB1-TBP2 interactions, resulting in blockage of JAB1-p27Kip1 interaction and stabilization of p27Kip1. We found VitD3 and the HDAC inhibitors SAHA, trichostatin A and sodium butyrate enhanced p27Kip1 stabilization in the cells expressing the pre-S2 mutant LHBs. We also found that the oxidative DNA damages induced by pre-S2 mutant LHBs were dramatically decreased after treatments with vitD3 and HDAC inhibitors, indicating that HDAC inhibitors are effective targeted drugs to repress p27Kip1 degradation and oxidative stress caused by the pre-S2 mutant LHBs. The HDAC inhibitors might provide a novel therapeutic approach for the pre-S2 mutant LHBs-induced hepatocellular carcinogenesis.

abstract: The Philadelphia chromosome in humans, is on oncogenic translocation between chromosomes 9 and 22 that gives rise to the fusion protein BCR-Abl. This protein is constitutively active resulting in rapid and uncontrolled cell growth in affected cells. The BCR-Abl protein is the hallmark feature of chronic myeloid leukemia (CML) and is seen in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cases. Currently, the first line of treatment is the Abl specific inhibitor Imatinib. Some patients will, however, develop resistance to Imatinib. Research has shown how transformation of progenitor B cells with v-Abl, an oncogene expressed by the Abelson murine leukemia virus, causes rapid proliferation, prevents further differentiation and produces a potentially malignant transformation. We have used progenitor B cells transformed with a temperature-sensitive form of the v-Abl protein that allows us to inactivate or re-activate v-Abl by shifting the incubation temperature. We are trying to use this line as a model to study both the progression from pre-malignancy to malignancy in CML and Imatinib resistance in Ph+ ALL and CML. These progenitor B cells, once v-Abl is reactivated, in most cases, will not return to their natural cell cycle. In this they resemble Ph+ ALL and CML under Imatinib treatment. With some manipulation these cells can break this prolonged G1 arrested phenotype and become a malignant cell line and resistant to Imatinib treatment. Cellular senescence can be a complicated process requiring inter-play between a variety of players. It serves as an alternate option to apoptosis, in that the cell loses proliferative potential, but does not die. Treatment with some cancer therapeutics will induce senescence in some cancers. Such is the case with Imatinib treatment of CML and Ph+ ALL. By using the S9 cell line we have been able to explore the possible routes for breaking of prolonged G1 arrest in these Ph+ leukemias. We inhibited the DNA damage sensor protein ataxia telangiectasia mutated (ATM) and found that prolonged G1 arrest in our S9 cells was broken. While previous research has suggested that the DNA damage sensor protein ataxia-telangiectasia mutated (ATM) has little impact in CML, our research indicates that ATM may play a role in either senescence induction or release.
Dissertation/Thesis
M.S. Microbiology 2011

Les extrémités des chromosomes contiennent des répétitions de séquences d’ADN appelées télomères qui empêchent l’activation inopportune de la réponse aux dommages de l'ADN afin de préserver l'intégrité génomique. Les télomères raccourcissent à chaque cycle de réplication d’ADN et la télomérase a pour fonction de contrebalancer cette érosion en allongeant les télomères. Les cellules somatiques n’expriment pas la télomérase, donc leur durée de vie est normalement limitée par ce raccourcissement progressif des télomères qui conduit à l'activation de la voie p53 entraînant un arrêt de la croissance cellulaire. En revanche, les cellules cancéreuses acquièrent l'immortalité cellulaire principalement en réactivant la télomérase ou en utilisant des méthodes alternatives d'allongement des télomères basées sur la recombinaison d’ADN. Auparavant, dans notre laboratoire, un criblage CRISPR à l'échelle du génome a été réalisé dans la lignée cellulaire pré-B NALM-6 traitée avec la molécule BIBR1532, un inhibiteur de la télomérase. Ces résultats suggéraient que cinq sous-unités du complexe de remodelage de la chromatine INO80, lorsque supprimées indépendamment, réduisaient la prolifération des cellules ayant un raccourcissement des télomères induit par le BIBR1532. Mon objectif était d'étudier cette interaction génétique afin de comprendre les processus biologiques impliqués dans cette létalité synthétique. Après l'élimination des gènes codant à la fois pour la sous-unité enzymatique de la télomérase humaine (hTERT) ainsi que les sous-unités spécifiques du complexe INO80 humain, nous avons constaté que les cellules double-négatives avaient une capacité proliférative réduite, ce qui démontre que l’interaction génétique mesurée par criblage CRISPR est bel et bien spécifique. Étant donné le rôle du facteur de transcription p53 dans la réponse cellulaire au raccourcissement télomérique, nous avons exploré l’importance de cette voie de signalisation pour l’interaction entre le complexe INO80 humain et la télomérase. Après l’activation de p53 avec un traitement avec la molécule nutlin-3a, les niveaux d'expression de plusieurs cibles de p53 tels que MDM2 et CDKN1A ont augmenté dans les cellules ayant une délétion du gène NFRKB, codant pour une sous-unité du complexe INO80 humain. Les cellules ayant une délétion du gène UCHL5, codant pour le partenaire d’interaction de NFRKB, ont également montré une augmentation de l’expression de MDM2 lorsque traitées avec nutlin-3a. Enfin, la perte de télomérase (hTERT) modifie les niveaux d'expression des composants de la 2 voie p53 CDKN1A, BAX et MDM2. En conclusion, la suppression des gènes codant pour des sous-unités du complexe INO80 telles que NFRKB ou UCHL5 est nuisible aux cellules ayant une délétion de la télomérase. Le complexe INO80 humain peut être impliqué dans l'inhibition de la voie p53, en réponse à l'activation de p53 soit par des télomères courts ou avec un traitement avec nutlin-3a. Des recherches plus approfondies sur cette interaction génétique pourraient mener au développement de nouvelles thérapies combinatoires afin d’inhiber la croissance des cellules cancéreuses.
The ends of chromosomes contain telomeric repeats that prevent the DNA damage response from being activated in order to preserve genomic integrity. Telomerase functions to alleviate incomplete DNA replication at telomeres, and to repair those telomeres damaged by various means including oxidative damage. The lifespan of telomerase negative somatic cells is normally restricted by gradual telomere shortening which can lead to the activation of the p53 pathway resulting in cellular growth arrest. Cancer cells often elongate their telomeres in order to acquire cellular immortality predominantly by reactivating telomerase or by using recombination-based, alternative telomere lengthening methods. Previously in our lab, a genome-wide CRISPR screen was conducted in the pre-B cell line NALM-6 treated with a small molecule inhibitor of telomerase, BIBR1532. These previous results suggested that five subunits of the INO80 chromatin-remodeling complex, when independently deleted, reduced cellular proliferation in cells with BIBR1532 induced telomere shortening. My goal was to investigate this genetic interaction in order to understand the biological processes implicated in this synthetic lethal relationship. After the knockout of the genes encoding both the enzymatic subunit of human telomerase (hTERT) and specific subunits of the human INO80 complex, I found that the proliferative capacity of NALM-6 cells was reduced. This result indicates the genetic interaction identified by CRISPR screening is in fact specific. In addition, after p53 stimulation with nutlin-3a treatment, expression levels of the p53 pathway component MDM2 were altered after the knockout of the genes encoding specific subunits of the human INO80 complex, NFRKB and UCHL5, individually. CDKN1A expression was also altered after nutlin-3a treatment and NFRKB knockout. Finally, the loss of telomerase (hTERT) alters the expression levels of the p53 pathway components CDKN1A, BAX and MDM2. In conclusion, the deletion of the genes encoding specific subunits of the INO80 complex, including NFRKB and UCHL5, is harmful to cells after hTERT knockout. The human INO80 complex may be involved in inhibiting the p53 pathway, in response to p53 activation by short telomeres or nutlin-3a treatment. Further investigation into this synthetic lethal relationship may shed light on new combinatorial therapeutics in cancer.

Oral, small-molecule signalling pathway inhibitors, including ones that inhibit the JAK and other pathways, are currently in development for the treatment of rheumatoid arthritis (RA). Many of the pro-inflammatory cytokines implicated in the pathogenesis of RA utilize cell signalling that involves the JAK-STAT pathways and therefore inhibition of JAK-STAT signalling, by targeting multiple RA-associated cytokine pathways, has the potential to simultaneously reduce inflammation, cellular activation, and proliferation of key immune cells. Spleen tyrosine kinase (SyK) is a cytoplasmic tyrosine kinase that is an important mediator of immunoreceptor signalling in mast cells, macrophages, neutrophils, and B cells. Interruption of SyK signalling should interrupt production of tumour necrosis factor (TNF) and metalloproteinase and therefore affect RA disease activity. Tofacitinib, approved in many countries for the treatment of RA, is an orally administered small-molecule inhibitor that targets the intracellular Janus kinase 3 and 1 (JAK1/3) molecules to a greater extent than JAK2; there are other JAK inhibitors in development which are purported to be more specific for JAK3 (Vertex 509), specific for JAK1/2 (baricitinib) or more specific for JAK1 (Galapagos and INCYTE) where clinical data has been reported. Tofacitinib has been investigated in multiple clinical trials which have investigated its efficacy (clinical, functional, and radiographic) and safety in patients who have failed disease-modifying anti-inflammatory drugs (DMARDs) as monotherapy or in combination with DMARDs, compared to an inhibitor of tumour necrosis factor alpha (TNFα‎‎) and in patients who have failed TNFα‎‎ inhibitors. Vertex 509 has been investigated as monotherapy or in combination with MTX in DMARD failures while baricitinib, GLPG0634 (Galapagos), and INCB039110 (Incyte) have been investigated in phase 1 and 2 clinical trials in combination with MTX. Each of these medications has demonstrated efficacy; their safety profile has been shown to be generally similar although with some differences from each other and some differences from most of the currently approved biological agents. Fostamatinib disodium is an orally available inhibitor of SyK which was investigated in multiple phase 3 clinical trials in RA but was found to be generally ineffective with significant safety signals. This chapter discusses what is currently known and understood about the efficacy and safety of these oral, small-molecule DMARDs.

Background: The role of B cells in multiple sclerosis (MS) is increasingly recognized. B cells undergo compartmentalized redistribution in blood and cerebrospinal fluid (CSF) during active MS, whereby memory B cells accumulate in the CSF. While B-cell trafficking across the blood– brain barrier has been intensely investigated, cellular diapedesis through the blood–CSF barrier (BCSFB) is incompletely understood. Objectives: To investigate how B cells interact with the choroid plexus to transmigrate into the CSF, we isolated circulating B cells from healthy donors (HC) and MS patients, utilized an inverted cell culture filter system of human choroid plexus papilloma (HIBCPP) cells to determine transmigration rates of B-cell subsets, immunofluorescence, and electron microscopy to analyze migration routes, and qRT-PCR to determine cytokines/chemokines mediating B-cell diapedesis. We also screened the transcriptome of intrathecal B cells from MS patients. Results: We found that spontaneous transmigration of HC- and MS-derived B cells was scant yet increased significantly in response to B-cell specific chemokines CXCL-12/CXCL-13, was further boosted upon pre-activation and occurred via paracellular and transcellular pathways. Migrating cells exhibited upregulation of several genes involved in B-cell activation/migration and enhanced expression of chemokine receptors CXCR4/CXCR5 and were predominantly of isotype class switched memory phenotype. This antigen-experienced migratory subset displayed more pronounced chemotactic activities in MS than in HC and was retrieved in intrathecal B cells from patients with active MS. Trafficking of class-switched memory B cells was downscaled in a small cohort of natalizumab-exposed MS patients and the proportions of these phenotypes were reduced in peripheral blood yet were enriched intrathecally in patients who experienced recurrence of disease activity after withdrawal of natalizumab. Conclusion: Our findings highlight the relevance of the BCSFB as an important gate for the entry of potentially harmful activated B cells into the CSF.

Achieving cell adhesion, growth and homeostasis on an underlying biomaterial surface may be a desirable feature in implant device design and tissue engineering. Insight has been gained from numerous cell patterning strategies where spatial cues and physical constraints have been shown to regulate the structure and function of cells. Despite significant advances in modifying substrates for cellular attachment, migration and proliferation, the achievement of confluent and aligned growth of functional endothelial cells on cardiovascular blood-contacting implants under physiologically significant wall shear stress has proven difficult. Recently we have reported on a method that enhances cellular adhesion under flow conditions on synthetic polymer surfaces, without reliance on pro-adhesive protein biomaterials, which are often thrombogenic. In this method we utilize electron beam lithography and size-dependent self-assembly to fabricate line arrays of nanowells allowing entrapment and retention of charged nanoparticles, covalently conjugated with a RGD adhesive ligand, GRGDSPK. This approach is an additive strategy of combining substrata topographic alteration, electrostatic charge and biochemical ligands, all uniquely incorporated as an ensemble of charged, ligand-bearing nanoparticles entrapped in arrays of nanowells. However, the modulation of endothelial cell physiologic mechanisms as a result of ensemble surface exposure remains to be characterized. In this report, we extend our studies and probe cell physiologic mechanisms altered as a result of nanofeatured surface exposure. We first examined the functional intactness or normalcy of endothelial cells adherent to the nanofeatured ensemble surface utilizing standard immunostaining and flow cytometry methods. We found β1-integrin expression dominated quiescent adherent endothelial cells while αVβ3-integrins expression was more common in migratory cells. Endothelial cells were noted to express high levels of PECAM-1 over time when exposed to nanofeatured surface and RGD peptides. For understanding the contribution of the nanofeatured surface (entrapped RGD conjugated nanoparticles) to cell adhesion, cytochalasin B was used to alter cell spreading. Confocal microscopy illustrated the uptake of nanoparticles in endothelial cells on composite surfaces, as well as the inhibition of this endocytosis by cytochalasin B. After prohibiting the cells from engulfing nanoparticles, we found an 80% reduction in cell adhesion; suggesting that an endocytic mechanism might play a role in maintaining cell adhesion. Nanofeatured ensemble surfaces appear to be good substrates for achieving a high level of EC adhesion, with maintained growth and stability.