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Objective To optimize transduction efficiency of mobilized CD34 + cells with serotype-5 adenoviruses (Ad5s), we investigated the activity of the chemical cocktail BoosterExpress Reagent in enhancing Ad5-mediated gene transfer into CD34 + cells. Methods Enriched CD34 + cells were transduced with three different Ad5s at increasing multiplicity of infections (MOIs) in the presence and absence of BoosterExpress Reagent. Percentages of transduced cells and levels of transgene expression were quantified by flow cytometry. Propidium iodide staining and colony growth were used to assess the toxicity of the transduction protocol. Expression of adenovirus receptors was investigated by flow cytometry. Results Irrespective of the Ad5 used, transduction with BoosterExpress Reagent using an MOI of 500 resulted in an average six- to seven-fold increase of transduction efficiencies and 1.5- to 2-fold increase of the levels of transgene expression, which could be detected up to 7 days post-transduction. Although BoosterExpress Reagent alone did not affect the plating efficiency of CD34 + cells, adenovector transduction plus BoosterExpress Reagent significantly reduced the plating efficiency due to the marked increase of transduced cells. However, adenoviral transduction in the presence of BoosterExpress Reagent failed to significantly reduce the recovery of CD34 + cells as compared with transduction in the absence of the compound. Coxsackievirus and adenovirus receptor as well as α v β 3 , α v β 5 , α 5 , and β 1 integrins were upregulated by BoosterExpress Reagent. Conclusions BoosterExpress Reagent allows high-levels of durable transgene expression, thus making CD34 + cells a suitable target for Ad5-mediated gene transfer.

Objective To explore new treatments specifically targeting malignant plasma cells (PCs), we examined CD52 antigen expression on primary PCs as well as multiple myeloma (MM) cell lines, and investigated in vivo the antimyeloma activity of alemtuzumab. Materials and Methods PCs were enriched from the marrow of MM patients (n = 39) according to CD138 expression and then analyzed by 3-color flow cytometry and quantitative PCR. The in vivo activity of alemtuzumab was evaluated in a xenotransplant model of MM in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Results CD52 expression revealed a substantial heterogeneity in terms of both percentage of positive cells and fluorescence intensity, with 25/39 (64%) MM patients showing ≥30% CD138+ PCs expressing the CD52 antigen (mean = 79%; range, 33–100%). Similarly to primary cells, cell lines showed heterogeneous CD52 expression. Expression of CD52 mRNA by quantitative PCR analysis strongly correlated with CD52 antigen detection by flow cytometry. In vivo, alemtuzumab treatment significantly increased the median survival of animals with an early- (64 vs 77 days, p ≤ 0.0005) or advanced-stage (66 vs 75 days, p ≤ 0.02) disease. Conclusion We conclude that: 1) CD52 is expressed on PCs of a significant proportion of MM patients; 2) alemtuzumab used as a single agent exerts a good antitumor activity in NOD/SCID mice bearing an early-stage disease; and 3) alemtuzumab might have therapeutic potential in a subset of MM patients.

Objective The aim of this study was to evaluate the capacity of defibrotide in enhancing cytokine-induced hematopoietic mobilization in rhesus monkeys. Materials and methods Animals received recombinant human granulocyte colony-stimulating factor (rhG-CSF, 100 μg/kg/day SC for 5 days) and, after a 4- to 6-week washout period, were remobilized with defibrotide (15 mg/kg/hour continuous intravenous for 5 days) plus rhG-CSF. Hematopoietic mobilization was evaluated by complete blood counts, differential counts, as well as frequency and absolute numbers of colony-forming cells (CFCs), high-proliferative potential CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). Results Compared to baseline values, rhG-CSF increased circulating CFCs, HPP-CFCs, and LTC-ICs by 158-, 125-, and 67-fold, respectively; the same figures for defibrotide/rhG-CSF were 299-, 1452-, and 295-fold, respectively. Defibrotide/rhG-CSF treatment compared to rhG-CSF alone increased CFCs, HPP-CFCs, and LTC-ICs by 1.4- (35,089 vs 25,825, p ≤0.02), 6- (4358 vs 748, p ≤0.02), and 5-fold (884 vs 168, p ≤0.04), respectively. We then evaluated the effects of a 2-day defibrotide treatment associated with a 5-day rhG-CSF treatment. Compared to rhG-CSF, defibrotide/rhG-CSF increased the mobilization of CFCs, HPP-CFCs, and LTC-ICs by 2- (31,128 vs 15,527, p ≤0.05), 8- (5361 vs 660, p ≤0.01), and 8-fold (954 vs 119, p ≤0.01), respectively. Conclusions Our data demonstrate that in nonhuman primates: 1) defibrotide enhances rhG-CSF–elicited mobilization of primitive and committed progenitors; and 2) a 2-day defibrotide injection is as effective as a 5-day injection.

Objective The aim of this story was to evaluate the activity of recombinant human (rh) growth hormone (GH) in restoring bone marrow progenitor cell growth as well as cytokine-elicited stem cell mobilization in aged BALB/c mice with impaired marrow hematopoietic function and reduced stem cell mobilizing capacity. Materials and Methods BALB/c mice included in this study were either naturally aged (group I) or aged after having been used for radioprotective assays (group II). Mice were treated for 5 weeks with either rhGH [2.5 mg/kg/day intraperitoneally (IP)] or phosphate-buffered saline (PBS). Subsequently, colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) were evaluated. In addition, progenitor cell mobilization elicited by granulocyte colony-stimulating factor (rhG-CSF) was analyzed. Results Compared with young controls, the growth of marrow CFCs and LTC-ICs was significantly reduced ( P ≤0.05) in group I and II mice. Treatment with rhGH significantly enhanced marrow hematopoiesis in mice of both groups, as demonstrated by a complete restoration of marrow cellularity, and CFC and LTC-IC growth. To further evaluate the hematopoietic potential of rhGH, aged mice treated with rhGH or PBS were mobilized with rhG-CSF (10 μg/day IP for 5 days). Compared with PBS-injected mice, rhGH-treated mice showed a significant improvement of rhG/CSF–elicited stem cell mobilization, with significant increases of white blood cell counts (5633 vs 8133, P ≤0.05), frequency of circulating CFCs per 10 5 mononuclear cells (36 vs 67, P ≤0.009), as well as absolute numbers per mL of blood of circulating CFCs (783 vs 2288, P ≤0.001) and LTC-IC (21 vs 64, P ≤0.001). Conclusion Our data demonstrate in mice that a 5-week treatment with rhGH restores age- and irradiation-associated loss of marrow primitive and committed progenitors.