Cynthia E. Dunbar

Serial Cardiac Magnetic Resonance Imaging of Injected Mesenchymal Stem Cells

Background—Delivery and tracking of endomyocardial stem cells are limited by the inability to image transplanted cells noninvasively in the beating heart. We hypothesized that mesenchymal stem cells (MSCs) could be labeled with a iron fluorophore particle (IFP) to provide MRI contrast in vivo to assess immediate and long-term localization. Methods and Results—MSCs were isolated from swine. Short-term incubation of MSCs with IFP resulted in dose-dependent and efficient labeling. Labeled cells remained viable for multiple passages and retained in vitro proliferation and differentiation capacity. Labeled MSCs (104 to 106 cells/150 &mgr;L) were injected percutaneously into normal and freshly infarcted myocardium in swine. One, 3, and 1 animals underwent serial cardiac MRI (1.5T) for 4, 8, and 21 days, respectively. MRI contrast properties were measured both in vivo and in vitro for cells embedded in agar. Injection sites containing as few as 105 MSCs could be detected and contained intact IFP-bearing MSCs on histology. Conclusions—IFP labeling of MSCs imparts useful MRI contrast, enabling ready detection in the beating heart on a conventional cardiac MR scanner after transplantation into normal and infarcted myocardium. The dual-labeled MSCs can be identified at locations corresponding to injection sites, both ex vivo using fluorescence microscopy and in vivo using susceptibility contrast on MRI. This technology may permit effective in vivo study of stem cell retention, engraftment, and migration.

Dysregulated interleukin 6 expression produces a syndrome resembling Castleman's disease in mice.

Interleukin 6 (IL-6) is an important regulator of the acute phase response, T cell function, and terminal B cell differentiation. Excessive or inappropriate production of this cytokine may be involved in a variety of autoimmune and neoplastic disorders. To investigate the consequences of dysregulated synthesis of IL-6 in vivo, a high-titer recombinant retroviral vector produced in psi-2 packaging cells was used to introduce the coding sequences of murine IL-6 into mouse hematopoietic cells. Congenitally anemic W/Wv mice reconstituted with bone marrow cells transduced with the retroviral vector developed a syndrome characterized by anemia, transient granulocytosis, hypoalbuminemia, and polyclonal hypergammaglobulinemia, with marked splenomegaly and peripheral lymphadenopathy. Extensive plasma cell infiltration of lymph nodes, spleen, liver, and lung was noted. The similarity of these findings to those of multicentric Castlemans disease, taken together with the observation that lymph nodes from these patients elaborate large amounts of this cytokine, suggest that the inappropriate synthesis of IL-6 has a primary role in the pathogenesis of this systemic lymphoproliferative disorder.

Eltrombopag and Improved Hematopoiesis in Refractory Aplastic Anemia

BACKGROUND Severe aplastic anemia, which is characterized by immune-mediated bone marrow hypoplasia and pancytopenia, can be treated effectively with immunosuppressive therapy or allogeneic transplantation. One third of patients have disease that is refractory to immunosuppression, with persistent, severe cytopenia and a profound deficit in hematopoietic stem cells and progenitor cells. Thrombopoietin may increase the number of hematopoietic stem cells and progenitor cells. METHODS We conducted a phase 2 study involving patients with aplastic anemia that was refractory to immunosuppression to determine whether the oral thrombopoietin mimetic eltrombopag (Promacta) can improve blood counts. Twenty-five patients received eltrombopag at a dose of 50 mg, which could be increased, as needed, to a maximum dose of 150 mg daily, for a total of 12 weeks. Primary end points were clinically significant changes in blood counts or transfusion independence. Patients with a response continued to receive eltrombopag. RESULTS Eleven of 25 patients (44%) had a hematologic response in at least one lineage at 12 weeks, with minimal toxic effects. Nine patients no longer needed platelet transfusions (median increase in platelet count, 44,000 per cubic millimeter). Six patients had improved hemoglobin levels (median increase, 4.4 g per deciliter); 3 of them were previously dependent on red-cell transfusions and no longer needed transfusions. Nine patients had increased neutrophil counts (median increase, 1350 per cubic millimeter). Serial bone marrow biopsies showed normalization of trilineage hematopoiesis in patients who had a response, without increased fibrosis. Monitoring of immune function revealed no consistent changes. CONCLUSIONS Treatment with eltrombopag was associated with multilineage clinical responses in some patients with refractory severe aplastic anemia. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT00922883.).

T cell-depleted bone marrow transplantation and delayed T cell add- back to control acute GVHD and conserve a graft-versus-leukemia effect

Thirty-eight patients with hematological malignancies, received T cell-depleted marrow transplants (BMT) and cyclosporine to prevent acute graft-versus-host disease (aGVHD), followed by delayed add-back of donor lymphocytes to prevent leukemia relapse. In 26 patients scheduled for donor T cell add-back of 2 × 106 cells/kg on day 30 and 5 × 107 cells/kg on day 45 (schedule 1), the overall probability of grade ⩾II aGVHD developing was 31.5%, with a 15.5% probability of aGVHD occurring after T cell add-back. In 12 patients receiving 107 donor T cells/kg on day 30 (schedule 2), the probability of grade ⩾II aGVHD was 100%. The incidence of grade III–IV aGVHD was higher in schedule 2 than in schedule 1 (P = 0.02). Of 24 evaluable patients, 10 (46%) developed chronic GVHD which was limited in eight and extensive in two. Current disease-free survival for 18 patients at standard risk for relapse (chronic myeloid leukemia (CML) in chronic or accelerated phase, acute myeloid leukemia in remission) vs 20 patients with more advanced leukemia or multiple myeloma were respectively 72% vs 12% (P < 0.01) with a 29% vs 69% probability of relapse (P = 0.08). In 12 CML patients surviving more than 3 months, PCR analysis of the BCR/ABL transcript showed that minimal residual disease after T cell add-back was transient except in two patients who developed hematological relapse. Results indicate that the risk of acute GVHD is low following substantial T cell doses, transfused 45 days after transplant, using cyclosporine prophylaxis. Furthermore a graft-versus-leukemia effect was conserved.

American Society of Gene Therapy (ASGT) Ad Hoc Subcommittee on Retroviral-Mediated Gene Transfer to Hematopoietic Stem Cells

Gene transfer using retroviral vectors has been under clinical study for more than 12 years1. Many studies have targeted hematopoietic stem cells (HSCs) as a potentially enduring and renewable source of gene-modified blood cells for the treatment of specific genetic diseases, cancer, leukemia, and HIV-1 infection2. Although initial studies were hampered by very low levels of gene transfer to HSCs, incremental progress has been realized in the efficiency of gene transfer to HSCs. These advances have culminated in the report of clinically significant restoration of immunity in patients with the X-linked form of severe combined immune deficiency (XSCID) by Alain Fischer, Marina Cavazzana-Calvo, and colleagues at the Hopital Necker Enfants Malades in Paris3. Their study and those conducted by Adrian Thrasher and colleagues at the Great Ormond Street Childrens Hospital in London for XSCID and by Claudio Bordignon and colleagues at the Hospital San Raffaele in Milan for children with SCID due to deficiency of adenosine deaminase (ADA) provide incontrovertible proof that gene therapy can ameliorate genetic diseases4,5.

Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells

Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine stromal cell derived factor-1 (SDF-1) to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4+ human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, whereas β2-adrenergic antagonist inhibited both steady state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady-state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.

Eltrombopag restores trilineage hematopoiesis in refractory severe aplastic anemia that can be sustained on discontinuation of drug

About a quarter of patients with severe aplastic anemia remain pancytopenic despite immunosuppressive therapy. We have previously demonstrated that eltrombopag has efficacy in this setting with 44% (11/25) of patients having clinically significant hematologic responses. We now report safety and efficacy data on a further 18 patients and long-term follow-up on the entire cohort of 43 patients. The overall response rate was 17 of 43 patients (40%) at 3 to 4 months, including tri- and bilineage responses. The majority of patients who remained on eltrombopag in an extension study (14/17) continued to show improvement, and 7 eventually had significant increases in neutrophil, red cell, and platelet lineages. Five patients with robust near-normalization of blood counts had drug discontinued at a median of 28.5 months after entry (range, 9-37 months), and all maintained stable counts a median of 13 months (range, 1-15 months) off eltrombopag. Eight patients, including 6 nonresponders and 2 responders, developed new cytogenetic abnormalities on eltrombopag, including 5 with chromosome 7 loss or partial deletion. None evolved to acute myeloid leukemia to date. Eltrombopag is efficacious in a subset of patients with aplastic anemia refractory to immunosuppressive therapy, with frequent multilineage responses and maintenance of normalized hematopoiesis off treatment. This study is registered at www.clinicaltrials.gov as #NCT00922883.

High-dose cyclophosphamide in severe aplastic anaemia: A randomised trial

BACKGROUND High-dose cyclophosphamide has been proposed as an alternative immunosuppressive agent for treatment of severe aplastic anaemia, with a response rate similar to that with regimens containing antithymocyte globulin (ATG) but neither relapse nor clonal haematological complications. We undertook a phase III, prospective, randomised trial to compare response rates to immunosuppression with either high-dose cyclophosphamide plus cyclosporin or conventional immunosuppression with ATG plus cyclosporin in previously untreated patients. METHODS Between June, 1997, and March, 2000, 31 patients were enrolled. 15 were assigned cyclophosphamide (1 h intravenous infusion of 50 mg/kg daily for 4 days) and 16 were assigned ATG (40 mg/kg daily for 4 days); both groups received cyclosporin, initially at 12 mg/kg daily with adjustment to maintain concentrations at 200-400 microg/L, for 6 months. The primary endpoint was haematological response (no longer meeting criteria for severe aplastic anaemia). The trial was terminated prematurely after three early deaths in the cyclophosphamide group. Analyses were by intention to treat. FINDINGS Median follow-up was 21.9 months (range 1-33). There was excess morbidity in the cyclophosphamide group (invasive fungal infections, four cyclophosphamide vs no ATG patients; p=0.043) as well as excess early mortality (three deaths within the first 3 months cyclophosphamide vs no ATG patients; p=0.101). There was no significant difference at 6 months after treatment in the overall response rates among evaluable patients (six of 13 [46%] cyclophosphamide vs nine of 12 [75%] ATG). INTERPRETATION A longer period of observation will be necessary to assess the secondary endpoints of relapse and late clonal complications as well as disease-free and overall survival. However, cyclophosphamide seems a dangerous choice for treatment of this disorder, given the good results achievable with standard therapy.

Collection of hematopoietic stem cells from patients with autoimmune diseases

We reviewed data from 24 transplant centers in Asia, Australia, Europe, and North America to determine the outcomes of stem cell collection including methods used, cell yields, effects on disease activity, and complications in patients with autoimmune diseases. Twenty-one unprimed bone marrow harvests and 174 peripheral blood stem cell mobilizations were performed on 187 patients. Disease indications were multiple sclerosis (76 patients), rheumatoid arthritis (37 patients), scleroderma (26 patients), systemic lupus erythematosus (19 patients), juvenile chronic arthritis (13 patients), idiopathic autoimmune thrombocytopenia (8 patients), Behcets disease (3 patients), undifferentiated vasculitis (3 patients), polychondritis (1 patient) and polymyositis (1 patient). Bone marrow harvests were used in the Peoples Republic of China and preferred worldwide for children. PBSC mobilization was the preferred technique for adult stem cell collection in America, Australia, and Europe. Methods of PBSC mobilization included G-CSF (5, 10, or 16 μg/kg/day) or cyclophosphamide (2 or 4 g/m2) with either G-CSF (5 or 10 μg/kg/day) or GM-CSF (5 μg/kg/day). Bone marrow harvests were without complications and did not affect disease activity. A combination of cyclophosphamide and G-CSF was more likely to ameliorate disease activity than G-CSF alone (P < 0.001). G-CSF alone was more likely to cause disease exacerbation than the combination of cyclophosphamide and G-CSF (P = 0.003). Three patients died as a result of cyclophosphamide-based stem cell collection (2.6% of patients mobilized with cyclophosphamide). When corrected for patient weight and apheresis volume, progenitor cell yields tended to vary by underlying disease, prior medication history and mobilization regimen. Trends in the approaches to, and results of, progenitor cell mobilization are suggested by this survey. While cytokine-based mobilization appears less toxic, it is more likely to result in disease reactivation. Optimization with regard to cell yields and safety are likely to be disease-specific and prospective disease-specific studies of mobilization procedures appear warranted. Bone Marrow Transplantation (2001) 28, 1–12.

Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates

Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34(+) cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.