Thèses sur le sujet « Bone marrow precursors »

Introduction: Atherosclerosis and its complications are a major cause of death and disability and it remains a major challenge to develop new therapies for patients with irreversible end organ damage and ongoing ischaemia. The discovery of adult stem and progenitor cells with the ability to regenerate adult tissues holds great promise. Bone marrow is the source of both endothelial progenitor cells (EPCs) and multi-potent adult progenitor cells (MAPCs). MAPCs are rare pluripotent bone marrow derived cells with the theoretical potential to differentiate into tissues of all three germ cell layers, including endothelium. These cells may have the potential to facilitate cardiac repair. The aim of this thesis was to further characterise bone marrow derived endothelial progenitor cells including multi potent adult progenitor cells and assess their angiogenic potential and mechanisms of action in animal models of cardiovascular disease. Findings: EPCs were isolated from humans and mice and their phenotype, markers and function determined, including gene tracking experiments in mice utilising the Cre/Lox system. It was not possible to isolate cells with the same phenotype as MAPCs from rodent bone marrow. However, cells with pluri-potent properties, named rat multi-potent progenitor cells (rMPCs), were isolated from rat bone marrow. These cells had the ability to up regulate tissue specific antigens from all 3 germ cell lineages and in addition secreted multiple cytokines related to angiogenesis and inflammation. To investigate the in vivo properties of rMPCs a rat hind limb model of ischaemia was established and syngeneic rMPCs were transplanted into the ischaemic hind limbs. rMPCs engrafted selectively into the adventitia of arterioles of ischaemic muscles. However, engrafted cells did not differentiate into an endothelial or smooth muscle phenotype. Cytokine analysis of muscles 5 days after rMPC injection revealed raised levels of cytokines, including chemokines MCP1 and SDR. Limb perfusion, measured by microspheres, increased after rMPC injection. In addition a novel MRI based assessment of ischaemic muscles revealed a significant normalisation of MRI signal after rMPC transplantation. However, there was no improvement in limb function assessed by treadmill running distance 4 weeks after cell injection. These findings suggest that transplantation of rMPCs into ischaemic muscles may modulate local inflammatory and angiogenic responses through paracrine mechanisms. Conclusion: Despite the potential for stem and progenitor cells to be used for the treatment of chronic cardiac ischaemia the biology of stem cells is still relatively poorly understood, as is the mechanism of action of cells after transplantation. As set out in the aims, the work in this thesis adds further to our understanding of both EPCs and BM derived pluri-potent stem cells. In addition it provides insight into the hind limb ischaemia model and the mechanism of action of cell therapy after transplantation into ischaemic muscle.

The alteration of insulin-like growth factor I (IGF-I) and its binding proteins (IGFBP) and their effects on proliferation and differentiation of murine bone marrow-derived macrophage (BMM) precursors were investigated. Bone marrow cells exposed to 20% L929-fibroblast conditioned medium (LCM) were cultured in serum-free medium for 24 h. Western ligand blotting (WLB) analysis detected four bands in all samples. 41-kDa and 30-kDa bands were detected after 12 h and remained constant during BMM differentiation. The 28-kDa and 25-kDa proteins were almost undetectable until day 2, but accumulated significantly from day 3 to day 7. Immunoblotting analysis verified these two bands as IGFBP-4. Northern blotting analyses detected both IGFBP-4 and IGFBP-3 mRNA in the cells. The 41-kDa protein was postulated to be IGFBP-3 in a glycosylated form. The identity of the 30-kDa band is not known. Northern blotting analysis showed that IGF-I mRNA level increased in a time-dependent manner until day 3, and decreased thereafter during BMM differentiation. The effect of IGF-I and its analogs on cell proliferation was studied by ($ sp3$H) thymidine incorporation. IGF-I and its analogs enhanced cell proliferation of freshly isolated bone marrow cells. Both IGF-I and long R$ sp3$ IGF-I, but not des(1-3)IGF-I, continued to exert a stimulating effect on day 1, although to a lesser extent. The effect of IGF-I and its analogs on BMM differentiation was studied by checking morphology, non-specific esterase-1 (NSE-1) activity, and mannose receptor expression. No significant differences in morphology and NSE-1 activity were observed among the treatment groups. There was no difference of mannose receptor expression on day 4 between the IGF-I group and the control cells, whereas long R${ sp3}$ and des(1-3)IGF-I increased the receptor number by 260% and 228% respectively, with less increased K$ rm sb{d}$ values. (Abstract shortened by UMI.)

Chronic myeloid leukaemia (CML) is caused by the BCR-ABL hybrid gene. The molecular mechanisms leading from chronic phase (CP) to blast crisis (BC) are not understood. However, both the presence and the levels of BCR-ABL seem to be important for CML progression. BCR-ABL is under the transcriptional control of BCR promoter. Here we focused on the gene expression control of BCR and BCR-ABL upon myeloid differentiation in healthy donors (HDs), CP and BC patients. As previously reported, BCR-ABL is downregulated during myeloid maturation in CP patients. A similar pattern was detected for BCR (but not for ABL) in CP-CML and in HD, thus suggesting that the two genes may be under a similar transcriptional control. In BC this mechanism is similarly impaired for both BCR-ABL and BCR. These data indicate the presence of an ‘in trans’ deregulated transcription of both BCR and BCR-ABL promoters, associated with CML progression. The results of the luciferase assay indicate that the region comprised between 420 and 900 bp from the coding ATG site is required to achieve a basal transcription level. Previous studies suggest that a putative SP1 binding site could have a role in the basal promoter activity. In fact, an almost complete absence of transcriptional activity was measured in delta1041 and delta1271 constructs, lacking both the main transcription start site and the putative SP1 binding region. We hypothesize that SP1 could be responsible for the basal promoter activity, present in the delta541 and in longer constructs. The ChIP assay confirmed the SP1-binding to the BCR promoter. The presence of 10 additional putative protein binding sites (PBSs), along the BCR promoter is also known from previous works. Six of these putative PBSs are localized in the region between −1443 to −1202 bp, which appears to be critical from in silico studies. In fact, only in presence of a 221 bp region upstream from delta241, a strong luciferase signal could be detected, suggesting that the promoter region between −1443 and −1202 bp from the coding ATG is indeed critical to achieve the highest level of expression.

The in situ microenvironmental organization of early precursor B cells in mouse bone marrow has been studied using three experimental models: (1) mutant mice with severe combined immunodeficiency (SCID), which develop pro-B cells but no pre-B and B cells; (2) SCID/myc transgenic mice having expanded pro-B cell populations, but no pre-B and B cells; (3) x-irradiated C3H/HeJ mice during early stages in the regeneration of pro-B cells in bone marrow seeded from a shielded marrow site. The in vivo localization of B220$ sp+$ cells was revealed by the binding of i.v. injected $ sp{125}$I-mAb 14.8 detected by light and electron microscope radioautography of femoral marrow sections. Many B220$ sp+$ pro-B cells were located in peripheral regions of SCID and SCID/myc bone marrow, often in clusters, associated with an electron dense extracellular matrix and with the processes of stromal reticular cells. Many B220$ sp+$ cells were associated with macrophages which contained numerous ingested bodies. Macrophage associations were more numerous in SCID/myc than in SCID mice, especially in the peripheral marrow regions. 3-5 day post-irradiation endocolonizing marrow contained increasing numbers of B220$ sp+$ cells in subosteal and peripheral regions, situated both within sinusoids and extravascularly, associated with stromal reticular cell processes and often close to nerve fibers. The results demonstrate that early B220$ sp+$ precursors begin to differentiate in peripheral marrow regions and develop intimate associations with reticular cells and macrophages. These findings suggest that through these associations, the bone marrow reticular cells promote the early development of the B cell lineage, while the bone marrow macrophages play a role in the elimination of aberrant precursor B cells.

The localisation of B lymphocyte precursor cells in mouse bone marrow and their associations with stromal reticular cells and macrophages have been investigated by in vivo radioimmunolabeling combined with light and electron microscope radioautography. Many early B lineage cells expressing the B220 glycoprotein prior to the expression of surface immunoglobulin and those regenerating after sub-lethal $ gamma$-irradiation, were located in bone-associated regions of femoral marrow. Labeled B220$ sp*$ lymphoid cells of undifferentiated morphology were closely associated with complex cytoplasmic processes of stromal reticular cells. The binding of a monoclonal antibody raised against a B lymphocyte-supportive stromal cell line (mAb KMI6), was highly restricted to the interface between stromal cells and undifferentiated lymphoid cells. The VCAM-1 specific mAb M/K-2, labeled electron-dense stromal cells that contacted lymphoid cells and a variety of other hemopoietic cell lineages. Within the bone marrow of normal mice there was evidence of death of B220$ sp+$ B lineage cells by apoptosis and their removal by macrophages. Cell loss, apoptosis and macrophage deletion of B precursor cells were greatly enhanced in E$ mu$-myc transgenic mice. The results reveal a complex in vivo microenvironmental organisation of B lymphocytopoiesis characterised by intimate interactions with stromal reticular cells and macrophages which regulate the development of precursor B cells and determine the ultimate output of B lymphocytes from the bone marrow.

Les progéniteurs hématopoïétiques présentent la faculté de détecter des signaux infectieux et inflammatoires. Leur éducation précoce par de tels signaux au sein de la moelle osseuse avant leur sortie vers la périphérie peut influencer l'évolution des réponses immunes. Alors que les cellules dendritiques plasmacytoïdes matures peuvent soit aggraver soit améliorer des maladies auto-immunes ou allogéniques, nous avons exploré la possibilité que de tels signaux innés confèrent des propriétés immunorégulatrices à des précurseurs médullaires des pDC. Nous avons caractérisé une population médullaire émergeant après interaction avec un agoniste du Toll-like receptor-9, l'oligonucléotide CpG, qui présente le phénotype c-kit+Sca-1+B220intPDCA-1+, est engagée dans la voie des cellules dendritiques plasmacytoïdes (pDC) et l'avons dénommée CpG-pre-pDC. Nous avons évalué le potentiel immunorégulateur des CpG-prepDCs en opérant leur transfert adoptif dans deux types de pathologies murines : l'Encéphalite Auto-immune Expérimentales (EAE), un modèle de sclérose en plaques qui est une maladie auto-immune, ainsi que la maladie du greffon contre l'hôte (GVHD) qui est une réponse allogénique. Il s'est avéré que le transfert d'un nombre assez faible de précurseurs (80 000 en EAE et 200 000 en GVHD) est capable de limiter la maladie à différents temps cliniques. De façon intéressante, les CpG-pre-pDC migrent spécifiquement à la moelle épinière dans l'EAE et à la rate dans la GVHD où leur descendance conserve un phénotype de pDC encore relativement immature. Dans le cadre de l'EAE, la descendance des précurseurs injectés produit essentiellement de l'IL-27 et du TGFß et plus modestement du GM-CSF. Au niveau du système nerveux central inflammé, elles font dévier la réponse immunitaire des lymphocytes T CD4+ infiltrants d'un profil pro-inflammatoire (IFNy+ GM-CSF+ IL-17+) vers un profil anti-inflammatoire (TGFß+, IL-27+, IL-17-, GM-CSFlo). L'utilisation de précurseurs déficients dans chacune de ces deux cytokines a permis de démontrer que TGFß et IL-27 interviennent séquentiellement dans la protection conférée par les CpG-prepDCs, le TGFß à des temps précoces et l'IL-27 aux phases tardives de la maladie. Des mécanismes semblables interviennent dans la protection envers la GVHD conférée par les CpG-prepDCs, car leur descendance est toujours capable de produire du TGFß mais cette fois en association avec l'IL-12, une autre cytokine de la même famille que l'IL-27. Par ailleurs, ces cellules sont capables de diminuer la production d'IL-17 tant par les lymphocytes T CD4+ que par les CD8+. Une thérapie cellulaire avec l'équivalent humain des CpG-pre-pDCs pourrait constituer un nouvel outil thérapeutique pour le traitement à la fois de la sclérose en plaques réfractaire et de la maladie du greffon contre l'hôte soit injectés seuls soit en enrichissement des greffes de cellules souches hématopoïétiques qui sont pratiquées dans ces deux maladies
Hematopoietic progenitors can sense innate signals. Their early education by such signals within the bone marrow, prior to their egress, may have considerable impact on the outcome of immune responses. While mature plasmacytoid dendritic cells (pDC) are known to either aggravate or ameliorate disease both auto-immune and allogeneic, it remains unknown whether immune regulatory function can be stably imprinted at the precursor stage in the pDC lineage onwards. We herein investigated whether activation with the oligonucleotide CpG, a Toll-like receptor-9 agonist, confers to bone marrow pDC precursors (CpG-prepDCs) characterized by the c-kit+Sca-1+B220intPDCA-1+ phenotype the capacity to protect against two kinds of murine immune pathologies: Experimental Autoimmune Encephalomyelitis (EAE), a model of multiple sclerosis which is an autoimmune disease and graft versus host disease (GVHD), an allogeneic response. We demonstrate that the adoptive transfer of relatively low number of CpG-pre-pDCs (80.000 in EAE and 200.000 in GVHD) was able to clinically reduce both diseases. Interestingly, CpG-pre-pDCs migrated to the spinal cord in EAE and to the spleen in GVHD where their progeny retained a relatively immature pDC phenotype. In EAE, the progeny of CpG-pre-pDCs massively produces IL-27 and TGFß and moderately GM-CSF. In the inflamed central nervous system, the progeny switches the immune response of infiltrating CD4+ T cells from pro-inflammatory (IFNy+ GM-CSF+ IL-17+) to anti-inflammatory (TGFß+, IL-27+, IL-17-, GM-CSFlo). The key role of TGFß and IL-27 was assessed using precursors incapacitated for the production of each of those cytokines. These experiments demonstrated that the two soluble factors acted sequentially: TGFß ensures early phases of the immunomodulation mediated by the CpG-pre-pDC while IL-27 is required for later protection. In GVHD, the mechanisms of protection are different yet similar in some ways. As for EAE, the progeny of CpG-pre-pDCs is still able to produce TGFß but this time in combination with IL-12, another cytokine from the IL-27 family. Additionally, those cells were able to reduce the IL-17 production by both pathogenic CD4+ and CD8+ T cells. The human equivalent of CpG-pre-pDC could be a new therapeutic tool in patients with multiple sclerosis or graft versus host disease either per se or enriched in the hematopoietic stem cell transfer already implemented to treat those two immune conditions

Myelin is essential for neuronal survival and maintenance of normal functions of the nervous system. Demyelinating disorders are debilitating and are often associated with failure of resident oligodendrocyte precursor cells (OPCs) to differentiate into mature, myelinating oligodendrocytes. Derivation of OPCs, from a safe source that evades ethical issues offers a solution to remyelination therapy. We therefore hypothesized that bone marrow stromal cells (BMSCs) harbour neural progenitor cells at a pre-commitment stage and that in vitro conditions can be exploited to direct differentiation of these cells along the oligodendroglial lineage. For the current study, adult rat BMSCs used were >90% immunopositive for CD90, CD73, STRO-1 (stromal cell markers), 10% for nestin (neural progenitor marker) but negligible for CD45 (haematopoietic cell marker) as measured by flow cytometry. Transfer of the culture from a highly adhesive substratum to a moderately adhesive substratum resulted in increase in proportion of p75-positive cells but CD49b-positive cells remained at 97% and Sox 10-positive cells remained negligible. Transfer of the culture to a non-adherent substratum fostered the generation of neurospheres comprising cells that were positive for the neural stem/progenitor cell (NP) marker, nestin, and for the neural crest markers CD49b, p75 and Sox10. Prior to this stage, the BMSCs were not yet committed to the neural lineage even though transient upregulation of occasional marker may suggest a bias towards the neural crest cell lineage. The BM-NPs were then maintained in adherent culture supplemented with beta-Heregulin (β-Her), basic fibroblast growth factor (bFGF) and platelet-derived growth factor-AA (PDGF-AA) to direct differentiation along the oligodendroglial lineage. Within two weeks of glial induction, cells expressing the OPC markers - NG2, Olig2, PDGFRa and Sox10, were detectable and these could be expanded in culture for up to 3 months with no observable decline in marker expression. These BM-OPCs matured into myelinating oligodendrocytes after 2 weeks in co-culture with either dorsal root ganglion neurons or cortical neurons. In vivo myelination by BM-OPCs was demonstrated by exploitation of the non-myelinated axons of retinal ganglion cells of adult rats. By 8 weeks post-injection of BM-OPCs into the retina, myelin basic protein-positive processes were also observable along the retinal axons. The results not only suppport our hypothesis, but also provide pointers to the adult bone marrow as a safe and accessible source for the derivation of OPCs towards transplantation therapy in acute demyelinating disorders.
published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy

Bibliography: leaves 152-223. Experiments were designed to identify and purify human bone marrow stromal precursor cells by positive immunoselection, based on the cell surface expression of the VCAM-1 and STRO-1 antigens. The data presented demonstrates a hierarchy of bone cell development in vitro.

This work is concerned with the characterization of mesenchymal stem cells (MSC) specifically from bone marrow samples derived from patients with osteoarthritis (OA). The multilineage potential of mesenchymal stem cells as well as their ease of exvivo expansion makes these cells an attractive therapeutic tool for applications such as autologous transplantation and tissue engineering. Bone marrow is considered a source of MSC. However, there is a general assumption that the occurrence of MSCs and their activity in bone marrow diminishes with age and disease. This prompted us to isolate and identify multipotential and self-renewing cells from patients with the degenerative disease osteoarthritis, with the view of using these cells for autologous cell therapies. It is therefore of great potential benefit to investigate the isolation and characterization of stem cell/progenitors from bone marrow samples of patients with osteoarthritis in greater detail. We employed a single cell clone culture method in order to develop clonal cell populations from three bone marrow samples and characterized them based on their proliferation and differentiation capabilities. The clonal populations were grouped into fast-growing and slow-growing clones based on their proliferation rates. The fastgrowing clones displayed 20-30% greater proliferation rate than the slow-growing clones. The study also revealed that the proliferation rates were directly proportional to their differentiation capacities. Most of the fast-growing clones were found to be tripotential for osteogenic, chondrogenic and adipogenic lineages, whereas the slow growing clones were either uni or bipotential. Flow cytometry analysis for the phenotype determination using putative MSC surface markers did not reveal any difference between the two clonal populations indicating a need for further molecular studies. Two approaches were employed to further investigate the molecular processes involved in the existence of such varying populations. In the first method gene expression studies were performed between the fast-growing (n=3) and slow-growing (n=3) clonal populations to identify potential genetic markers associated with cell 'sternness' using the Stem Cell RT2 ProfilerTM PCR Array comprising a series of 84 genes related to stem cell pathways. Ten genes were identified to be commonly and significantly over represented in the fast-growing stem cell clones when compared to slow-growing clones. This included expression of transcripts beyond MSC lineage specification such as SOX2, NOTCH1 and FOXA2 which signified that stem cell maintenance requires a coordinated regulation by multiple signalling pathways. The second study involved an extensive protein expression profiling of the fast growing (n=2) and slow growing (n=2) clonal populations using off-line Two Dimensional Liquid Chromatography (2D-LC)/Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). A total of 67 proteins were identified, of which 11 were expressed at significantly different levels between the subpopulations. Protein ontology revealed these proteins to be associated with cellular organization, cytokinesis, signal transduction, energy pathways and cell stress response. Of particular interest was the differential presentation of the proteins calmodulin, tropomyosin and caldesmon between fast- and slow-growing clones. Based on their reported roles in the regulation of cell proliferation and maintenance of cell integrity, we draw an association between their expression and the altered status in which the subpopulations exist. Based on our observations, these proteins may be prospective molecular markers to distinguish between the fast-growing and slow-growing subpopulations. In summary, this study demonstrated the existence of potential stem cells of therapeutic importance in spite of a supposedly smaller stem cell compartment in patients with osteoarthritis. Furthermore, the differentially expressed genes between the sub-populations highlight the 'sternness' of the potential clones, an observation supported by the expression of proteins which act as effective modulators in the maintenance of cell integrity and cell cycle regulation. This study provides a basis for more detailed investigations in search of selective cell surface markers

This work is concerned with the characterization of mesenchymal stem cells (MSC) specifically from bone marrow samples derived from patients with osteoarthritis (OA). The multilineage potential of mesenchymal stem cells as well as their ease of exvivo expansion makes these cells an attractive therapeutic tool for applications such as autologous transplantation and tissue engineering. Bone marrow is considered a source of MSC. However, there is a general assumption that the occurrence of MSCs and their activity in bone marrow diminishes with age and disease. This prompted us to isolate and identify multipotential and self-renewing cells from patients with the degenerative disease osteoarthritis, with the view of using these cells for autologous cell therapies. It is therefore of great potential benefit to investigate the isolation and characterization of stem cell/progenitors from bone marrow samples of patients with osteoarthritis in greater detail. We employed a single cell clone culture method in order to develop clonal cell populations from three bone marrow samples and characterized them based on their proliferation and differentiation capabilities. The clonal populations were grouped into fast-growing and slow-growing clones based on their proliferation rates. The fastgrowing clones displayed 20-30% greater proliferation rate than the slow-growing clones. The study also revealed that the proliferation rates were directly proportional to their differentiation capacities. Most of the fast-growing clones were found to be tripotential for osteogenic, chondrogenic and adipogenic lineages, whereas the slow growing clones were either uni or bipotential. Flow cytometry analysis for the phenotype determination using putative MSC surface markers did not reveal any difference between the two clonal populations indicating a need for further molecular studies. Two approaches were employed to further investigate the molecular processes involved in the existence of such varying populations. In the first method gene expression studies were performed between the fast-growing (n=3) and slow-growing (n=3) clonal populations to identify potential genetic markers associated with cell 'sternness' using the Stem Cell RT2 ProfilerTM PCR Array comprising a series of 84 genes related to stem cell pathways. Ten genes were identified to be commonly and significantly over represented in the fast-growing stem cell clones when compared to slow-growing clones. This included expression of transcripts beyond MSC lineage specification such as SOX2, NOTCH1 and FOXA2 which signified that stem cell maintenance requires a coordinated regulation by multiple signalling pathways. The second study involved an extensive protein expression profiling of the fast growing (n=2) and slow growing (n=2) clonal populations using off-line Two Dimensional Liquid Chromatography (2D-LC)/Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). A total of 67 proteins were identified, of which 11 were expressed at significantly different levels between the subpopulations. Protein ontology revealed these proteins to be associated with cellular organization, cytokinesis, signal transduction, energy pathways and cell stress response. Of particular interest was the differential presentation of the proteins calmodulin, tropomyosin and caldesmon between fast- and slow-growing clones. Based on their reported roles in the regulation of cell proliferation and maintenance of cell integrity, we draw an association between their expression and the altered status in which the subpopulations exist. Based on our observations, these proteins may be prospective molecular markers to distinguish between the fast-growing and slow-growing subpopulations. In summary, this study demonstrated the existence of potential stem cells of therapeutic importance in spite of a supposedly smaller stem cell compartment in patients with osteoarthritis. Furthermore, the differentially expressed genes between the sub-populations highlight the 'sternness' of the potential clones, an observation supported by the expression of proteins which act as effective modulators in the maintenance of cell integrity and cell cycle regulation. This study provides a basis for more detailed investigations in search of selective cell surface markers

La Leucemia Linfoblastica Acuta (LLA) è il tumore più frequente in età pediatrica. Nonostante l’alto successo terapeutico raggiunto, una percentuale di pazienti non risponde alle terapie convenzionali. Numerosi studi hanno mostrato come il microambiente midollare giochi un importante ruolo nella progressione tumorale. Il nostro scopo è stato quello di identificare i pahways cruciali coinvolti nel cross-talk tra le cellule leucemiche e il microambiente stromale e che possano essere dei potenziali target terapeutici. Abbiamo studiato AttivinaA, molecola della superfamiglia del TGF-β, all’interno della nicchia midollare leucemica. Questa molecola è nota nel contesto dei tumori solidi per la sua capacità di promuovere la progressione tumorale attraverso la regolazione della motilità e invasività cellulare. Abbiamo qui definito per la prima volta AttivinaA come un fattore associato alla leucemia: all’esordio di leucemia, questa molecola è altamente espressa nel midollo leucemico e risulta essere prodotta a più alti livelli dalle cellule stromali mesenchimali (MSC) in seguito a co-coltura con le cellule leucemiche. Anche nel contesto della B-LLA, abbiamo dimostrato che AttivinaA promuove la motilità ed invasività cellulare in presenza o meno del fattore chemiotattico CXCL12, fondamentale nella localizzazione delle cellule staminali ematopoietiche (HSC) e delle cellule leucemiche all’interno della nicchia midollare. In particolare, il meccanismo d’azione di AttivinaA dipende dall’incremento dei livelli di calcio intracellulare e della polimerizzazione dell’actina che sono importanti regolatori della riorganizzazione del citoscheletro e del movimento cellulare. Interessante, AttivinaA modula in modo specifico le proprietà biologiche delle cellule leucemiche in quanto svolge un effetto contrario sulle HSC, favorendo quindi le cellule leucemiche nella competizione per la nicchia. Come già riportato in letteratura, abbiamo trovato livelli ridotti di CXCL12 nel midollo leucemico e abbiamo osservato che AttivinaA è responsabile almeno in parte di questa riduzione a causa di un suo effetto diretto sulla secrezione della chemochina da parte delle MSC. Tuttavia, essendo AttivinaA capace di aumentare la migrazione cellulare anche verso concentrazioni subottimali di CXCL12, questi dati suggeriscono un possibile meccanismo tramite il quale le cellule leucemiche persistono all’interno della nicchia midollare distruggendo l’ematopoiesi sana. I nostri dati in vitro circa il ruolo pro-migratorio e pro-invasivo di AttivinaA sono stati confermati anche in vivo. I processi di regolazione dei flussi di calcio e della riorganizzazione del citoscheletro da parte di AttivinaA sono importanti per stimolare anche la vescicolazione da parte delle cellule. Abbiamo dimostrato che AttivinaA è in grado di aumentare il rilascio di vescicole extracellulari (VE) da parte delle cellule di B-LLA. Tali vescicole trasportano al loro interno l’oncogene t(1;19), tipico delle cellule dalle quali esse originano. Sia AttivinaA sia le VE da essa indotte conferiscono chemioresistenza alle cellule leucemiche, diminuendo in maniera significativa la loro sensibilità al farmaco Asparaginasi, che viene poi ripristinata bloccando la via di segnalazione di AttivinaA. Il meccanismo alla base della chemioprotezione esercitata dalle VE può essere spiegato dalla presenza al loro interno di microRNA differenzialmente espressi in seguito al trattamento con AttivinaA, tra cui il miRNA-491-5p precedentemente associato a chemioresistenza all’Asparaginasi nella leucemia pediatrica. Complessivamente, i nostri dati suggeriscono che AttivinaA è una molecola chiave della nicchia leucemica, che conferisce un vantaggio migratorio alle cellule leucemiche e le protegge dai farmaci convenzionali attraverso la produzione di VE. Il nostro lavoro potrebbe portare dunque allo sviluppo di nuovi farmaci in grado di agire sul cross-talk stroma-leucemia.
Acute Lymphoblastic Leukemia (ALL) is the most common type of cancer in children. About 80% of the cases arises from precursor B cells (BCP-ALL), which abnormally accumulate as a consequence of genetic alterations associated to differentiation inhibition and abnormal expansion. Despite the 85% survival rate, a total of 10-15% of patients retains leukemic stem cells and their progenitors in the bone marrow (BM), thereby relapsing following treatment cessation. The importance of BM microenvironment for cancer progression has been widely recognized in recent years. In this study, we aimed to identify the crucial pathways involved in the bi-directional leukemia-stroma cross-talk that could be an attractive target for future antileukemic therapy. We focused our attention on the characterization of ActivinA, a TGF-β family member, within the BM leukemic niche. Here, we identified ActivinA as a new crucial factor exploited by leukemic cells to create a self-reinforcing niche: indeed, this molecule was highly expressed in the BM plasma of leukemic patients. Furthermore, we reported that BCP-ALL cells, along with the highly pro-inflammatory environment of leukemic BM, induced a strong increase in the molecule secretion by Mesenchymal Stromal Cells (MSCs). In accordance with its protumoral role in solid tumors, ActivinA strongly induced both random and CXCL12-driven migration of cells also in the context of BCP-ALL. We observed that ActivinA selectively stimulated these leukemic cell biological properties with a calcium- and actin polymerization-mediated mechanism as this molecule showed an opposite effect on Hematopoietic stem cells (HSCs). According to the literature, we found reduced CXCL12 levels in the leukemic BM, but ActivinA enhanced cell migration also towards suboptimal CXCL12 concentrations, suggesting a possible mechanism by which leukemic cells could persist in the BM niche, displacing healthy hematopoiesis. Our in vitro data about the pro-migratory and pro-invasive role of ActivinA were confirmed also in vivo. By using a xenograft mouse model of human BCP-ALL, we demonstrated the ability of ActivinA to enhance both BM engraftment and metastatic potential intro extra-medullary sites of leukemic cells. Notably, the regulation of calcium influx and cytoskeleton organization by ActivinA is an important process to stimulate also cell vesiculation. Recent studies have shown that cancer extracellular vesicles (EVs) can mediate cell-cell communication and potentially contribute to tumor progression. Therefore, we investigated whether ActivinA was able to influence vesiculation by leukemic cells. We demonstrated that ActivinA increased the production of both exosomes and MVs by BCP-ALL cells. We found that EVs transport the t(1;19) fusion transcript, typical of cells from which they originate. We then studied the biological effects by which ActivinA-induced leukemia EVs can actively promote BCP-ALL disease, focusing our attention on resistance to therapy. Firstly, we demonstrated that ActivinA significantly decreased the sensitivity of leukemic cells to the anti-leukemic drug Asparaginase (ASNase) which was re-stored by blocking ActivinA signaling. Interestingly, also ActivinA-induced leukemia EVs conferred resistance to leukemic cells. To understand the mechanism underlying EV chemoprotection, we explored their miRNA cargo and identified differentially expressed miRNAs induced by ActivinA treatment. Of these, miR-491-5p has been previously reported to be associated with ASNase chemoresistance in childhood leukemia. The discovery of ActivinA signaling between BCP-ALL cells and MSCs adds significant insights into the mechanisms of communication in the leukemic niche. Moreover, ActivinA-induced leukemia EVs seem to play a crucial role in sustaining leukemic cells, by conferring them drug resistance. Our data provide a new concept to develop alternative therapeutic strategies that include targeting of the leukemic niche in BCP-ALL.

Mice homozygous for the scid (severe combined immunodeficiency) mutation are generally unable to produce B lymphocytes, a condition attributed to defective rearrangements of immunoglobulin genes in precursor B cells. Some early B lineage cells are present in the bone marrow (BM), however. In scid mice, we defined three subsets of early progenitor B cells lacking $ mu$ heavy chains (pro-B cells) based on the expression of terminal deoxynucleotidyl transferase (TdT) and B220 glycoprotein: (a) early (TdT+B220$-$), (b) intermediate (TdT+B220+), and (c) late (TdT$-$B220+). Double immunofluorescence labelling of BM cell suspensions has shown normal numbers of early and intermediate pro-B cells, substantially reduced numbers of late pro-B cells, and an absence of pre-B cells and B cells. Nearly all defective cells, thus, abort at the late pro-B cell stage. Early and intermediate pro-B cells accumulated in metaphase in near-normal numbers after intraperitoneal vincristine administration, apparently not regulated by feedback signals from the B cell pool.
The scid mouse provided a system for studies on regulation of pro-B cells and the effectiveness of the deletion mechanism. It was determined that the defective pro-B cells of scid mice were susceptible to the stimulatory effects of an exogenous agent, sheep red blood cells, and a stromal cell-derived growth factor for precursor B cells, interleukin-7, as previously observed in normal mice in vivo. In addition, pro-B cells of scid mice were driven to proliferate by the dysregulated constitutive expression of the c-myc oncogene. In each case, however, all cells aborted soon after reaching the late pro-B stage. Mechanisms by which aberrant cells are deleted from the B lineage, safeguarding against entry of potentially dysregulated cells into the peripheral B cell pool, thus, could not be overwhelmed or evaded by increased proliferative activity of pro-B cells.

The control of B lymphocyte production in bone marrow is of central importance in maintaining a supply of new B cells for primary humoral immune responses throughout life, while its perturbation may underlie states of immunodeficiency, neoplasia and autoimmunity. Molecules expressed by developing B cells and stromal cells within bone marrow, as well as systemic factors, may all play key roles in regulating proliferation and survival of precursor B cells in bone marrow. Candidate regulatory molecules investigated in the present work include surrogate light chains (SL) of immunoglobulin (Ig) expressed intracellularly by precursor B cells before and during the synthesis of CI heavy (H) chains of IgM, and interleukin (IL)-1, a systemic pleiotropic macrophage-derived factor. In vivo administration of radiolabeled mAbs specific for SL and IL-1 receptors type I and type II revealed that both SL and IL-1 receptors are normally expressed in bone marrow, displayed on the surface of lymphoid precursors and stromal cells, respectively. Ex vivo analysis of the DNA content of SL/$ mu$H$ sp+$ pre-B cells revealed a low incidence of apoptosis. Systemic administration of rIL-1 followed by analysis of precursor B cell dynamics using double immunofluorescence labeling and stathmokinetic techniques revealed that systemic IL-1 can either stimulate or depress B lymphopoiesis in a dose-dependent manner. Stimulation of cytoplasmic (c) $ mu sp+$ pre-B cell proliferation following activation of systemic macrophages by injecting sheep red blood cells (SRBC) is partially abrogated by rIL-1ra. Bone marrow IL-1 receptors, assayed by binding of radiolabeled anti-IL-1R monoclonal antibodies to plasma membrane fractions, undergo changes in levels of expression following SRBC administration. The work supports the hypotheses that surface SL is involved in the positive selection of precursor B cells with productively-rearranged $ mu$ chain genes, and suggests that the elevated proliferative level o

The possible roles of the anti-apoptotic protein, Bcl-2, and pro-apoptotic Fas receptor-ligand interactions in cell death decisions during the development of B lymphocytes in mouse bone marrow and their maturation in the spleen have been evaluated in gene-modified and mutant mice.
In Emu-bcl-2 transgenic mice, the population dynamics and tissue organisation of phenotypically defined precursor B cells, have been evaluated by immunofluorescence labeling, mitotic arrest, BrdU uptake, flow cytometry and in vivo radioimmunolabeling combined with light and electron microscope radioautography. In bone marrow of Emu-bcl-2 mice, the number and production rate of proliferating precursor B cells were increased. Immature B lymphocytes also increased in number, accumulating extravascularly around the central venous sinus, and the total rate of production of these rapidly-renewed IgM + B cells was increased. Phenotypically mature B lymphocytes and B lymphocytes having a slow turnover rate greatly increased in number. Many mature B cells were located within the lumen of venous sinusoids and in perisinusoidal locations. In the spleen, the usual population of rapidly-renewed IgM + B cells was undetectable. In contrast, both slowly renewing B cells and a further stable population of very long-lived B cells were greatly increased in numbers but had unchanged longevity. The rates of apoptosis among B cell subsets in short term bone marrow cultures from bcl-2 transgenic mice were reduced, while bcl-2/scid mice accumulated many B220+mu- pro-B cells in bone marrow. The results indicate that overexpression of bcl-2 inhibits apoptosis during B cell development in bone marrow and promotes survival of newly-formed B cells in the spleen and their entry into a long-lived recirculating B cell pool.
In Ipr and gld mutant mice lacking functional Fas and Fas ligand, respectively, pre-B cells were increased in number and production rate in bone marrow, while in spleen, in addition to an increase in number of mature B cells, a population of B220+mu - cells was expanded. Thus, Fas ligation may contribute to B cell death in bone marrow.
The findings suggest that Bcl-2 and Fas can help to regulate the developmental stage-specific apoptosis of B cells designed to prevent the persistence of nonfunctional, preneoplastic or autoreactive cells.

Aggregatibacter actinomycetemcomitans está associado à periodontite agressiva, caracterizada pela intensa reabsorção do osso alveolar. Esta espécie produz a toxina distensora citoletal (AaCDT) que possui atividade de DNAse, e promove o bloqueio das células alvo na fase G2 ou G1/ G2. Por outro lado, CDT ativa a cascata apoptótica pela atividade de PIP3, regulando a proliferação e sobrevivência de linfócitos, pelo bloqueio de Akt. Em monócitos, AaCDT induz aumento da produção de citocinas pró-inflamatórias e inibe a produção de óxido nítrico e fagocitose. Células precursoras de osteoclastos têm origem hematopoiética e sofrem diferenciação em osteoclastos, mediada pelo RANKL, mas outros fatores co-estimulatórios estão envolvidos. A AaCDT induz a produção de RANKL por fibroblastos. Assim, formulamos a hipótese se CDT influenciaria a homeostase óssea por afetar a diferenciação de células precursoras de osteoclastos. O estudo visou determinar o efeito de AaCDT sobre a sobrevivência, diferenciação e atividade em RAW264.7 e BMC. Os dados sugerem que a CDT interfere na homeostase óssea, favorecendo a indução da diferenciação de células precursoras de osteoclastos e alterando o perfil de citocinas produzidas.
Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis, characterized by severe alveolar bone resorption. This species produces a distending toxin cytolethal (AaCDT) which has DNase activity, and promotes the blocking of target cells in G2 or G1 / G2 phase. On the other hand, CDT activates the apoptotic cascade by PIP3 activity, regulating lymphocyte proliferation and survival by blocking Akt. In monocytes, AaCDT enhances the production of proinflammatory cytokines and inhibits nitric oxide production and phagocytosis. Osteoclast precursor cells are of hematopoietic origin and must undergo differentiation into osteoclasts mediated by RANKL although other co-stimulatory factors are involved. AaCDT induces the production of RANKL by fibroblasts. Thus, CDT is hypothesized to influence bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. This study aimed to determine the effect of AaCDT on survival, differentiation and activity of osteoclasts precursor cells. The data suggested that CDT interfere in bone homeostasis, favoring the differentiation of osteoclasts precursors cells and by altering their cytokines profile.

Normal wound healing process is a regulated biological response of multiple events whose common aim is to restore the integrity of injured tissues. Fibroblasts exert an important role in this process as target cells that allow either tissue remodeling through the predominant production of proteases (i.e., matrix metalloproteinases) or tissue fibrosis through the over-expression of extracellular matrix (ECM) components such as collagens. However, it remains unclear which factors activate such diversity of fibroblast responses and how this decision making process is made. Currently, there is no a well established model that integrally explains the diversity of responses from minimal to hypertropic scarring and keloids. Previous reports have demonstrated that some recruited cells can be locally transformed into fibrocytes, a pro-fibrogenic cells that stimulate resident fibroblasts to produce collagen accumulation and tissue fibrosis. However, recruited cells with anti-fibrogenic profile that can compete with and eventually reverse the local effects of fibrocytes have not been identified. How the skin maintains the cell transdifferentiation balance in normal wound healing and how this balance is modified in fibro-proliferative cutaneous disorders cannot be entirely explained by pro-fibrogenic fibrocytes without anti-fibrogenic counterparts. This doctoral thesis hypothesizes a novel mechanism in which bone marrow-derived cells recruited to the injured area modulate the expression of ECM components produced by resident fibroblasts. As a result of the tissue injury, a repertoire of systemic and local cytokines and growth factors induce epigenetic changes and cell transdifferentiation in circulating recruited cells. Thus, these locally transformed cells can acquire either pro- or anti-fibrogenic profile, and more importantly, they can induce the production of either collagens or MMPs by dermal fibroblasts. This study demonstrates that circulating stem cells and monocytes have the capacity to transdifferentiate into keratincoyte-like cells (KLCs), anti-fibrogenic cells that increase the expression MMPs by themselves and by the stimulation of dermal fibroblasts. The transdifferentiation of pro-fibrogenic fibrocytes into KLCs was also studied. In all cases the TGF-β deprivation was identified as crucial factor in the anti-fibrotic commitment of recruited cells. Finally, adipofascial groin flaps in rats were utilized as in vivo model to study the role of the tissue repair microenvironment in the cell transdifferentiation of recruited bone marrow-derived cells. The findings presented in this thesis are consistent with the existence of a "seesaw mechanism" in the regulation of MMPs/collagen production by dermal fibroblasts. Thus, during the wound-healing response, the local environment may induce epigenetic changes in recruited bone marrow-derived cells to follow either pro- or anti-fibrogenic pathways. Subsequently, these committed cells may trigger a fibro-proliferative switch on resident fibroblasts to predominantly develop either collagen accumulation with tissue fibrosis or collagen breakdown with tissues remodeling. Findings of this doctoral thesis provide new insights into the role of cell transdifferentiation and local environment not only in wound healing, but may also in other fibro-proliferative processes such as lung fibrosis, asthma, liver cirrhosis, chronic pancreatitis, and atherosclerosis, among others.