Il trapianto delle cellule staminali emopoietiche umane CD34+ in combinazione con le cellule T regolatorie CD4+/CD25+ FoxP3+ (Tregs) potrebbe prevenire l'alloreattivita verso le cellule staminali emopoietiche e ridurre il rischio di rigetto in trapianti allogenici HLA non correlati.
Per dimostrare questa ipotesi abbiamo messo in coltura le cellule CD34+ e le cellule CD4+/CD25+ isolate con metodica immunomagnetica (Miltnyi Biotec)da sangue periferico non manipolato,sangue periferico mobilizzato con G-CSF o da Cordone ombelicale.
Gli esperimenti svolti in vitro, hanno evidenziato che le cellule Tregs arricchite, ottenute dalla stessa fonte delle cellule CD34+( autologhe), mostravano un effetto inibitorio maggiore sulle celulle T alloreattive, rispetto alle cellule Tregs ottenute da un donatore terzo(allogenico).Inoltre l'attivita immunosoppressoria delle Tregs era mantenuta dopo stimolazione con cellule CD34+ allogeniche e i Tregs non modificavano l'attivita clonogenica delle cellule staminali CD34+. Avendo ottenuto questi dati in vitro abbiamo trapiantato in topi NOD/SCID le cellule Tregs e le cellule CD34+ in rapporto 1:1 o 1:2 ed e stato osservato un normale attecchimento delle cellule staminali.
Incubando queste cellule con dosi fisiologiche di timoglobulina derivata da coniglio (nota molecola immunosopressoria) non veniva modificato il numero dei Tregs dopo 6 giorni di coltura. Dopo l'esposizione alla Timoglobulina, inoltre, i Tregs mantenevano la loro attivita soppressoria, aumentava l'espressione del recettore chemochinico CCR7, e venivano rilasciate diverse citochine principalmente l'interleuchina 10(IL-10). Tali dati dimostrano come sia le cellule tregs autologhe che quelle allogeniche potrebbero essere trapiantate insieme alle cellule staminali CD34+ dopo un regime preparatorio di terapia che include la timoglobulina. A tale scopo sono state eseguite selezioni di Tregs da aferesi di donatori sani mobilizzati con G-CSF su scala clinica utilizzando biglie immunomagnetiche Clinical grade (Miltenyi Biotec) e sono state confrontate 2 modalita di selezione con due o tre passaggi con o senza la deplezione dei monociti CD14+.Si e dimostrato cosi che e possibile selezionare un numero uguale di CD34+ e Tregs ,che con la metodica che prevede la deplezione dei monociti si ottiene una popolazione di cellule Tregs piu pura ( > 80%) e infine le applicazioni possibili di questi risultati includo: trapianto di cellule CD34+ del donatore insieme a cellule Tregs in trapianti aploidentici ; infusione of cellule tregs isolate da PBSC mobilizzati con G-nei casi di pazienti con GVHD steroide-resistente; infusione di G-Tregs del donatore nei casi di rigetto di organo trapiantato da of donatore ancora vivente.
The aim of this study was to analyze the temporal and spatial localization of dentin matrix protein 1 (DMP1) and dentin sialoprotein (DSP) in human dental pulp cells differentiated in vitro towards odontoblast-like cells. Following to a notable injury to the tooth, the odontoblasts die and a new generation of odontoblast-like cells differentiate from pulp progenitor cells [1]. They are responsible for the secretion of the reparative dentin matrix. In this study, morphological changes and the analysis of DMP1 and DSP were investigated starting from human dental pulp cells differentiated in vitro. Alizarin red staining, transmission and scanning electron microscopy in combination with Real Time PCR and immunofluorescence techniques were performed to demonstrate the odontoblast-differentiated phenotype and the specific expression and localization of DMP1 and DSP. The results showed a gradual calcium deposition to demonstrate a mineralization matrix. Electron microscopy analysis showed fibroblast-like cells characterized by a large number of mitochondria and a developed rough endoplasmic reticulum and Golgi apparatus involved in intense protein synthesis. A large extracellular matrix was detectable in which collagen type I fibers were detected. The DMP1 and DSP showed low expression and nuclear localization at the beginning of the odontoblast-like cell differentiation. After 21 days, elevated cytoplasm expression was detectable for both odontogenic markers.
Not all cases of autoimmune hemolytic anemia (AIHA) are diagnosed by the direct antiglobulin test (DAT). We present and discuss a simple method of enhancing the sensitivity of the standard DAT.We report the case of a five-month-old child diagnosed with a severe IgG-mediated AIHA, characterized by quick DAT negativization despite clinical worsening. Warm AIHA with negative DAT, possibly due to a low affinity autoantibody, unresponsive to conventional therapy, was hypothesized.The DAT resulted strongly positive with anti-IgG serum using a 4C saline for erythrocyte washing, to reduce the dissociation of the supposed low affinity autoantibody. Very intensive cytoreductive treatment was administered twice until clinical remission was obtained.The clinical course of AIHA can be dissociated by the DAT. Since autoantibody-mediated hemolysis with negative DAT rarely occurs, once other causes of high reticulocyte count anemia have been ruled out, the DAT after ice-cold saline washing could be a useful and easy means of corroborating the diagnosis of AHIA, when traditional methods fail.
Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated the anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without as a control. Engraftment of human CD 45+ cells was analyzed by flow cytometry at 1, 2, 4, or 8 weeks. Marrow engraftment of human CD45+cells was significantly decreased in mice transplanted at any CD34:T cell ratio compared to control mice that did not receive T cells. Transplantation of T cells resulted in graft failure since >98% of human CD45+ cells in the marrow and spleen of NSG mice were CD3+. A normal CD4:CD8 T cell ratio was detected and CD4+ cells were mostly CD45RA+. Mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio were sacrificed at 1, 2, or 4 weeks to analyze the kinetics of human cell engraftment in the bone marrow and spleen. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. We found that treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13 + 7% engraftment of human CD45+ marrow cells (n=5). These included 53 + 9% myeloid CD33+ cells, 22 + 3% CD14+ monocytes, 7 + 2% CD1c myeloid dendritic cells, and 4 + 1% CD34+ cells while CD 19 B cells were only 3% and CD3+ T cells 0.5 + 1%. Persistent myeloid engraftment was found at d+56 after stopping CTLA4-Ig on d+28. Here we demonstrate that costimulatory blockade with CTLA4-Ig can prevent T cell mediated rejection of incompatible human CD34+ stem cells. We also show that the NSG model can be utilized to test in-vivo immunotherapy strategies aimed at engrafting human stem cells across HLA barriers. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia.
