Optimizing dose and scheduling of filgrastim (granulocyte colony- stimulating factor) for mobilization and collection of peripheral blood progenitor cells in normal volunteers [see comments]

To define an optimal regimen for mobilizing and collecting peripheral blood progenitor cells (PBPC) for use in allogeneic transplantation, we evaluated the kinetics of mobilization by filgrastim (recombinant met- human granulocyte colony-stimulating factor [r-metHuG-CSF]) in normal volunteers. Filgrastim was injected subcutaneously for up to 10 days at a dose of 3 (n = 10), 5 (n = 5), or 10 micrograms/kg/d (n = 15). A subset of volunteers from each dose cohort underwent a 7L leukapheresis on study day 6 (after 5 days of filgrastim). Granulocyte-macrophage colony-forming cell (GM-CFC) numbers in the blood were maximal after 5 days of filgrastim; a broader peak was evident for CD34+ cells between days 4 and 6. The 95% confidence intervals (CI) for mean number of PBPC per milliliter of blood in the three dose cohorts overlapped on each study day. However, on the peak day, CD34+ cells were significantly higher in the 10 micrograms/kg/d cohort than in a pool of the 3 and 5 micrograms/kg/d cohorts. Mobilization was not significantly influenced by volunteer age or sex. Leukapheresis products obtained at the 10 micrograms/kg/d dose level contained a median GM-CFC number of 93 x 10(4)/kg (range, 50 x 10(4)/kg to 172 x 10(4)/kg). Collections from volunteers receiving lower doses of filgrastim contained a median GM- CFC number of 36 x 10(4)/kg (range, 5 x 10(4)/kg to 204 x 10(4)/kg). The measurement of CD34+ cells per milliliter of blood on the day of leukapheresis predicted the total yield of PBPC in the leukapheresis product (r = .87, P < .0001). Assuming a minimum GM-CFC requirement of 50 x 10(4)/kg (based on our experience with autologous PBPC transplantation), all seven leukapheresis products obtained at the 10 micrograms/kg/d dose level were potentially sufficient for allogeneic transplantation purposes. We conclude that in normal donors, filgrastim 10 micrograms/kg/d for 5 days with a single leukapheresis on the following day is a highly effective regimen for PBPC mobilization and collection. Further studies are required to determine whether PBPC collected with this regimen reliably produce rapid and sustained engraftment in allogeneic recipients.     

ADAMTS13 Endopeptidase Protects against Vascular Endothelial Growth Factor Inhibitor–Induced Thrombotic Microangiopathy

Thrombotic microangiopathy (TMA) is a life-threatening condition that affects some, but not all, recipients of vascular endothelial growth factor (VEGF) inhibitors given as part of chemotherapy. TMA is also a complication of preeclampsia, a disease characterized by excess production of the VEGF-scavenging soluble VEGF receptor 1 (soluble fms-like tyrosine kinase 1; sFlt-1). Risk factors for VEGF inhibitor–related TMA remain unknown. We hypothesized that deficiency of the VWF-cleaving ADAMTS13 endopeptidase contributes to the development of VEGF inhibitor–related TMA. ADAMTS13^−/− mice overexpressing sFlt-1 presented all hallmarks of TMA, including thrombocytopenia, schistocytosis, anemia, and VWF-positive microthrombi in multiple organs. Similar to VEGF inhibitor–related TMA in humans, these mice exhibited severely impaired kidney function and hypertension. In contrast, wild-type mice overexpressing sFlt-1 developed modest hypertension but no other features of TMA. Recombinant ADAMTS13 therapy ameliorated all symptoms of TMA in ADAMTS13^−/− mice overexpressing sFlt-1 and normalized BP in wild-type mice. ADAMTS13 activity may thus be a critical determinant for the development of TMA secondary to VEGF inhibition. Administration of recombinant ADAMTS13 may serve as a therapeutic approach to treat or prevent thrombotic complications of VEGF inhibition.     

Expression of bcr-abl abrogates factor-dependent growth of human hematopoietic M07E cells by an autocrine mechanism

The introduction of a retrovirus vector expressing p210bcr-abl (P210) into the human factor-dependent cell line M07E resulted in the rapid outgrowth of factor-independent cells. Early after infection, four factor-independent clones were isolated and analyzed in greater detail along with mass populations obtained from separate infections. High levels of P210 tyrosine kinase activity were measured in the factor- independent cells. The mass populations and three of the four clones remained responsive to exogenous growth factors. Concentrated conditioned media isolated from the factor-independent populations and from all clones contained biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF); interleukin-3 (IL-3) was detected at low levels in the mass population and in two of the clones. Neutralizing antibodies to IL-3, GM-CSF, and mast cell growth factor inhibited proliferation of the factor responsive clones by 60% to 90%. These results indicate that the growth autonomy of the P210-expressing M07E cells was acquired via an autocrine mechanism. In addition to factor-independent growth, P210-expressing M07E cells readily acquired a more mature megakaryocytic phenotype compared with control M07E cells. These data provide experimental evidence that expression of P210 tyrosine kinase in human hematopoietic cells induced growth factor secretion resulting in a pleiotropic effect on growth factor dependence and differentiation.