Impact of mesenchymal stem cells and rapamycin in xenogeneic graft-versus-host disease

Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSC) could exert potent immunosuppressive effects.In first time, the aim of our study was to develop xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rgamma;null (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMC). Secondly, we assessed human bone marrow derived MSC to prevent xenogeneic GVHD in these mouse models.We observed that injection of 200 × 106 human PBMC intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM1 antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 106 MSC IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMC. Similarly, injection of 30 × 106 human PBMC IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 106 MSC IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMC.The administration of RAPA (or sirolimus) is one of the other therapeutic strategies considered in the context of the prevention of GVHDa. Rapamycin (Sirolimus; RAPA) is an inhibitor of the mammalian target of rapamycin (mTor) which can induce tolerance by inhibiting activated T cells without diminishing CD4+CD25+Foxp3+ regulatory T cells Treg) proliferation and suppressive function. In second time, the aim of the current study was to assess the impact of RAPA administration on xenogeneic graft-versus-host disease (xGVHD) in NSG mice.NSG mice were given 2.5 Gy total body irradiation on day-1 and an intravenously (i.v.) injection of 2 × 106 human peripheral blood mononuclear cells (PBMC) on day 0. RAPA was administered intraperitoneally (i.p.) five days/week at a dose of 1.5 mg/kg from day -1 to day 14, and at 1 mg/kg from day 15 to day 21.We observed that RAPA administration was associated with a lower proportion of conventional T cell in cell cycle on day 20 after transplantation and a reduction in xGVHD lethality. The beneficial impact of RAPA on survival was significantly decreased when CD25-depleted PBMC were transplanted instead of unmanipulated PBMC, suggesting that the beneficial effect of RAPA was mediated at least in part by Treg. However, RAPA did not induce long-term tolerance since RAPA treated mice that survived beyond day 60 developed signs of chronic GVHD

Production d'une Protéine de Morphogenèse Osseuse 2 pure et fonctionnelle

Bone Morphognetic Protein-2 (BMP-2), which belongs to the transforming growth factor β (TGF-β) superfamily, plays an important role during bone regeneration and repair, and also during various stages of embryonic development. Noggin is a BMP-specific antagonist implicated in the regulation of BMP signaling pathways. Both BMP-2 and Noggin are dimeric molecules with each subunit containing several intramolecular disulfide bonds. This complex disulfide bonds network combined with the presence of large hydrophobic patches on the surface of these proteins cause their low solubility in aqueous solutions. As a result, BMP-2 and Noggin are commonly produced as inclusion bodies in Escherichia coli. The recovery of soluble and active molecules through in vitro refolding is difficult and the overall yield is low. Here we propose a new and simple method to produce biologically active BMP-2 and Noggin. Refolding of BMP-2 and Noggin was achieved in a one-step dilution and hydrophobic or ion exchange chromatography were used to purify the refolded proteins. The best conditions yielded significant quantities of dimeric BMP-2 and Noggin, with purity above 95%. Both BMP-2 and Noggin were characterized by optical spectroscopies and its biological activity was tested by measuring alkaline phosphatase activity in ATDC5 cells. These experiments indicated that our BMP-2 and Noggin display structural and functional properties similar to their commercial counterparts.AUTOBMP

Co-transplantation of mesenchymal stem cells might mitigate acute GvHD without abrogating graftversus- tumour alloreactivity after allogeneic transplantation with non-myeloablative conditioning

peer reviewedBackground: Results of nonmyeloablative HCT in pts with HLA-mismatched donors have been disappointing due to high incidence of graft rejection and severe acute GVHD. Recent studies have suggested that infusion of mesenchymal stem cells (MSC) the day of HCT might promote engraftment and prevent acute GVHD after myeloablative allogeneic HCT. However, some studies suggested that MSC co-infusion might abrogate graft-versus-host alloreactivity and graft-versus-tumor effects. This prompted us to investigate whether MSC infusion a few hours before HCT could allow nonmyeloablative HCT from HLA-mismatched donors to be performed safely (i.e. with a 100-day incidence of nonrelapse mortality < 35%). Methods: 20 patients with hematological malignancies were given MSC (1-2 x 10E6 cells/kg) from third party donors a few hours before PBSC from HLA-mismatched unrelated donors, after conditioning with 2 Gy TBI and fl udarabine 90 mg/m. Postgrafting immunosuppression included tacrolimus (day -3 to +180; tapered by day +365) and mycophenolate mofetil (tid days 0 to +42). HLA-compatibility was assessed at the HLA-A, -B, -C, -DRBI and DQBI loci: 13 pairs were mismatched for at least one HLA class I antigen (including 4 pairs who were also mismatched for 1 HLA-class II antigens (n=3) or 1 HLA-class I allele (n=1)), 1 pair was mismatched for 2 HLA class II alleles, while 6 pairs were mismatched for a single HLA class I (n=3) or HLA class II (n=3) alleles. Results: Median follow-up for surviving patients was 288 (range, 76-571) days. One patient with secondary AML had primary graft rejection, while the remaining 19 patients had sustained engraftment. Median donor T-cell chimerism levels on days 28, 100, 180 and 365 after HCT were 90%, 98%, 96%, and 98%, respectively. Grade II, III and IV acute GVHD were seen in 5, 2 and 1 patients, respectively, while 7 experienced NIH moderate/severe chronic GVHD. Three of 7 patients with measurable disease at transplantation achieved complete remission on days 41, 104 and 353 after HCT. Two patients died of nonrelapse causes on days 74 and 114 after HCT, while 3 died of disease progression. Projected 1-yr overall and progressionfree survivals were 77% and 61%, respectively. Conclusions: HLA-mismatched nonmyeloablative HCT with MSC co-infusion appeared to be safe, with MSC co-infusion possibly mitigating graft-versus-host alloreactivity without abrogating graft-versus-tumor effects. Survival is encouraging

Colony formation in presence of tyrosine kinase inhibitors were decreased due to the inhibition of cell cycle entry.

<p>Human cord blood CD34⁺ were cultured for 48 hours in presence/absence of either imatinib or nilotinib and then stained with propidium iodide using the CycleTEST™ Plus DNA Reagent Kit. Results are expressed as mean percentages relative to control experiments without TKIs ± SD. n = 3, *p<0.05, **p<0.01 versus CTL, Student’s paired t tests. CTL: control condition without TKIs.</p

Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

Background aims. Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects. Methods. The ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rg(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed. Results. Injection of 200 106 human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM1 antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 106 MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 106 human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 106 MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Conclusions. Injection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models