ALBERT

Description: Gain-of-function (GOF) mutations of protein tyrosine phosphatase nonreceptor type 11 Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling pathways, are associated with childhood leukemias and solid tumors. The underlying mechanisms are not fully understood. Here, we report that Ptpn11 GOF mutations disturb mitosis and cytokinesis, causing chromosomal...

Description: Experience with unrelated allogeneic UCB stem cell transplantation in the non-myeloablative setting is limited because of concerns of graft failure due to limited cell content. Dual UCB graft infusion has been explored to overcome cell dose limitations, but influence on engraftment is unclear. This single institution phase I trial examined RIC followed by single UCB transplantation for patients unable to tolerate fully myeloablative regimens and lacking a matched adult donor. Conditioning consisted of TBI 200 cGy, fludarabine 175 mg/m², cyclophosphamide 2 gm/m², ATG 60 mg/kg. Trial design specified selection of UCB grafts to be matched at 4/6 HLA loci or better with one unit infused if cryopreserved nucleated cell dose exceeded 2.5 x 107/kg recipient weight. If no adequate single UCB unit to meet these criteria was available, study patients received 2 UCB units containing a combined cell dose exceeding 1.5 x 107/kg. Grafts were analyzed via flow cytometry post thaw for stem cell and lymphocyte expression of surface antigens, among them CD4, CD8, CD7, CD34, CD38, CD45RA, CD45RO. 23 patients have been enrolled, the majority diagnosed with AML (n=17). Median age was 47 (range 25–68) years. Single UCB units meeting stated criteria were identified for all but one study patient, who was excluded from graft analysis. The time to engraftment was measured from the date of transplantation to the date of 3 successive days ANC≥500/μL attained. In addition, we assessed time to peripheral blood donor lymphocyte chimerism ≥ 60%. Patients were censored at the date of last follow-up or the date of death. 94% (95%CI: 77-.99%) of patients achieved ANC≥500/μL by T+36 with median of 26.5 ( range 11–55) days. 68% (95%CI: 48–85%) of patients (n=15) achieved 〉60% donor derived chimerism by median of 22 (range 14–35) days. Median cryopreserved and infused total nucleated cell dose was 2.85 x 107 and 2.5 x 107 cells/kg, respectively. Median infused CD34 cell dose was 1.71 (range 0.21–5.39) x 105 cells/kg. By univariate analysis the dose of infused UCB graft T-cells including CD4 and CD8 T-cells co-expressing CD45RO, and CD34 stem cells co-expressing CD7 and CD38 correlated with neutrophil recovery, p=0.046, 0.008, and 0.033, respectively. Median overall survival at this early interim analysis has not been reached; with 86% and 65% survival at 3 and 24 months, respectively. Event free survival is 78% and 44.5% at 3 and 24 months, respectively. In summary, in this heavily pretreated group of advanced age patients, RIC and single unit unrelated allogeneic UCB stem cell transplantation is safe. Identification of a single UCB graft of HLA match 4/6 meeting cell dose requirement 2.5x107/kg is identified in the vast majority of full size adult patients. Engraftment and survival rates are higher than reported in single UCB transplants following administration of myeloablative regimens and similar to recent reports for RIC with double UCB graft infusion. The potential benefit of infusion of 1 vs. 2 UCB units after RIC in adult patients remains to be fully evaluated.

Description: Correlative laboratory studies were developed in a phase I trial to evaluate the safety of intracoronary injection of escalating doses of bone marrow (BM) CD133+ cells in patients with chronic coronary ischemia. Concurrent with patient cellular therapy, CD133+ cells were phenotyped and tested functionally with endothelial cell colony formation and in vitro and in vivo transmigration. BM (194 ± 11 ml) was isolated from patients meeting study inclusion criteria. CD133+ cells (20 ± 13 x 106, 84 ± 7% purity and 76 ± 7% viability (7AAD)) were isolated using the CliniMACS device (Miltenyi). Contaminating cells following the CliniMACS selection were: 〈 5% of CD3, CD3neg/CD56, CD19 (immature/mature), CD14, and CD71 cells with 5% CD61, 8% CD13+ SSChigh. BM, PB (peripheral blood), cord blood (CB)-derived endothelial progenitor cells (EPC) were assessed by a culture assay (StemCell Technologies) scoring early outgrowth CFU-EC. SEACOAST patients yielded significantly less colonies compared to controls of matched PB and BM (donors 28–48 yrs) and CB: normal donor (ND) PB, 65; ND BM, 40; CB, 43; SEACOAST patient PB, 2, SEACOAST patient BM, 1. Transmigration assays were used to evaluate the functionality of selected CD133+ cells to chemotactic agents stromal derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF). Selected CD133+ cells were recovered, resuspended in DMEM/1% HSA media and after a 37°C incubation for 16–20 hrs, 5 x 104 CD133+ cells were added to transwells (5 mm) for 3 hours. Transmigrated cells were quantitated by flow cytometry using anti-CD45, anti-CD133 antibodies, and Fluorosphere beads. Surface expression on ND BM CD133+ cells of CXCR4 and VEGF-R2 was 0–16.4% and 1.2–4.3%, respectively. Transmigration was effected by 200 ng/ml (range of 16–62%) but not to 10 ng/ml VEGF. For CD133+ cells devoid of the expression of CXCR4, SDF-1-induced transmigration was absent. Expression of CXCR4 and VEGF-R2 on clinical trial patient-selected CD133+ cells was 0–5% and 0–2%, respectively, and transmigration was 5–19% to 200 ng/ml SDF-1 but not to 10 ng/ml VEGF. Patient selected CD133+ cells or PB mononuclear cells (PBMC), ND CD133+ cells, or a vehicle control were injected via a left intraventricular route into NOD/SCID mice with a femoral artery ligation immediately after injury. Doppler flow measurements were obtained weekly for 6 weeks comparing the perfusion ratio of ischemic/healthy limbs. At 28 days, perfusion ratios were statistically higher in study groups receiving ND CD133+ cells (0.51 ± 0.06) compared to controls (0.37 ± 0.03, p=0.025). Mice receiving patient CD133+ cells (0.46 ± 0.04) or PBMC (0.37 ± 0.08) did not show statistically significant improvement over control animals (p= 0.07, p= 0.94, respectively). BM was harvested to assess human engraftment by cytometric analysis. Mice injected with 0.5 x 106 patient BM CD133+ cells showed 70% purity and 〉70% viability) to chronic ischemic patients via an intracoronary route, important correlative in vitro and in vivo assays has demonstrated the diminished potency of BM-derived CD133+ cells as compared to CB and ND PB and BM-derived cells.

Description: Early clinical studies have demonstrated benefit in patients receiving cellular therapy utilizing bone marrow derived (BM)-CD133+ hematopoietic stem cells (HSC) for treatment of cardiovascular disease. Umbilical cord blood (UCB) is a potential source of CD133+ cells for use as an allogeneic cell source for therapeutic angiogenesis, however, the benefits must be weighed against potential adverse immunologic responses. We tested the hypothesis that allogeneic CD133+ cells derived from UCB may be defective as professional antigen-presenting-cells (APC) and thus may mediate TH2 T-cell immune response. CD133+ cells were isolated from UCB mononuclear cells (MNC) by magnetic autoMACs bead selection (Miltenyi Biotech, Auburn CA) and analyzed for purity and surface expression of MHC and co-stimulatory antigens by flow cytometry. We have demonstrated induced immune reactivity by mixed lymphocyte reactions (MLR) using healthy adult peripheral blood MNC as responders stimulated by irradiated CD133+ from UCB and BM (ratio 3:1) with both 3H-thymidine and CFSE staining. Surface expression of both MHC class I (57.6±17.2%) and MHC class II (66.5±12.5%) are present on the majority of UCB CD133+ cells. To test the ability of CD133+ cells to function as APC, a modified MLR was performed. Briefly, isolated UCB CD133+ cells were cultured for 96h in the presence of adult peripheral blood (AB) derived-MNC or isolated CD3 AB T-cells as responders at a 3:1 ratio (responder:effector) in RPMI 1640 supplemented with 10% FBS and 1% L-glutamine. CD133+ cells induced proliferation in both non-selected AB MNC (21.7±6.4e3cpm) and selected AB T-cell (85.6±15.1e3 cpm) cultures as measured by 3H-thymidine incorporation. UCB CD133+ cells were noted to lack surface expression of co-stimulatory antigens including: CD40 (0.93±1.0%), CD80 (0.85±0.15%), and CD86 (0.88±0.64%). Because primary T-cell receptor stimulation in the absence of a co-stimulatory response is known to induce TH2 responses we compared UCB MNC and CD133+ derived from the same unit as stimulators for responding HLA mismatched allogeneic AB MNC. Preliminary results demonstrate elevated levels of IL-4 and IL-10 produced by responder AB MNC stimulated by CD133+ cells as compared to UCB MNC, with measured increases of 964±538pg/mL (IL-10) and 141±45.7pg/mL (IL-4). In conclusion, though CD133+ cells derived from UCB express both MHC class I and II they lack surface expression of co-stimulatory receptors, and are defective as professional APC. Additionally, UCB CD133+ cells induce elevated levels of IL-4 and IL-10 by responding allogeneic adult MNC in modified mixed lymphocyte in vitro cultures. MNC stimulation by selected UCB CD133+ cells elicit elevated levels of tolerance-associated cytokines IL-4 and IL-10 as compared to UCB MNC used as stimulator cells. Taken together, UCB CD133 lack co-stimulatory receptor expression and elicit TH2 T-lymphocyte responses, and may allow for immune tolerance responses when used as a source of allogeneic stem cells for therapeutic use in vasculogenesis.

Description: Manual on launching of epe-d spacecraft