Effectiveness of cytopenia prophylaxis for different filgrastim and pegfilgrastim schedules in a chemotherapy mouse model.

OBJECTIVES: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used to treat neutropenia during cytotoxic chemotherapy. The optimal scheduling of rhG-CSF is unknown and can hardly be tested in clinical studies due to numerous therapy parameters affecting outcome (chemotherapeutic regimen, rhG-CSF schedules, individual covariables). Motivated by biomathematical model simulations, we aim to investigate different rhG-CSF schedules in a preclinical chemotherapy mouse model. METHODS: The time course of hematotoxicity was studied in CD-1 mice after cyclophosphamide (CP) administration. Filgrastim was applied concomitantly in a 2 x 3-factorial design of two dosing options (2 x 20 mug and 4 x 10 mug) and three timing options (directly, one, and two days after CP). Alternatively, a single dose of 40 mug pegfilgrastim was applied at the three timing options. The resulting cytopenia was compared among the schedules. RESULTS: Dosing and timing had a significant influence on the effectiveness of filgrastim schedules whereas for pegfilgrastim the timing effect was irrelevant. The best filgrastim and pegfilgrastim schedules exhibited equivalent toxicity. Monocytes dynamics performed analogously to granulocytes. All schedules showed roughly the same lymphotoxicity. CONCLUSION: We conclude that effectiveness of filgrastim application depends heavily on its scheduling during chemotherapy. There is an optimum of timing. Dose splitting is better than concentrated applications. Effectiveness of pegfilgrastim is less dependent on timing.

Allogeneic non-adherent bone marrow cells facilitate hematopoietic recovery but do not lead to allogeneic engraftment.

BACKGROUND: Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4(+), murine CD4(-/-), HLA-DR3(+) mice. METHODOLOGY/PRINCIPAL FINDINGS: Bone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45(+) showed depletion of CD4(+), CD8(+), CD19(+), and CD117(+) cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4(+) donor chimerism of 5.8+/-2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death. CONCLUSIONS/SIGNIFICANCE: NA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies.

Duplex polymerase chain reaction quantification of human cells in a murine background.

Studies of the regenerative potential of human stem cells commonly involve their transplantation into immune-deficient mice or in vitro coculture with mouse cells. The optimal use of such models requires the detection and quantification of relatively low numbers of human cells in a murine background. We report here a duplex polymerase chain reaction (PCR) approach involving the coamplification of human-and mouse-specific repetitive sequences. The determination of product ratios compensates against variations in sample quality and enables quantitation from >50% down to 0.01% human-in-mouse from a single reaction. Product ratios are determined by standard electrophoresis of end-stage PCR reactions followed by image analysis techniques using freely available software, with no requirement for real-time PCR. The approach has been used to analyze tissue from mice transplanted with human cells and cocultures between differentiating mouse embryonal stem cells and human umbilical cord blood cells.