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Objective: Optimizing cord blood donor selection based mainly on cell dose and human leukocyte antigen (HLA) disparities may further improve results of unrelated cord blood transplants (UCBT)., Materials and Results: We analyzed 550 UCBTs for hematologic malignancies reported to the Eurocord Registry. Main outcomes and prognostic factors were analyzed in univariable and multivariable analyses incorporating center and period effects and using death and relapse as competitive risks for nonfatal endpoints. Nucleated cell (NC) dose before freezing and number of HLA disparities had a significant influence on outcome. Cumulative incidence (CI) of neutrophil and platelet recovery was associated with the number of HLA mismatches, number of NC before freezing, and use of granulocyte colony-stimulating factor. Coexistence of HLA class I and II disparities and high CD34 cell dose in the graft were associated with graft-vs-host disease grades III-IV. CI of disease relapse was higher in matched transplants showing a graft-vs-leukemia effect increased in HLA-mismatched transplants. Overall 3-year survival was 34.4%. Prognostic factors for survival were recipient age, gender, and disease status., Conclusion: Our results provide indications for a better choice of cord blood units according to cord blood cell content and HLA.

Objective: We investigated the effect of interleukin-15 (IL-15) with Flt3 ligand (Flt3L) on the expansion and activation of NK cells derived from umbilical cord blood mononuclear cells (UCB-MNCs)., Materials and Methods: UCB-MNCs were cultured at 1 to 100 ng/mL of IL-15 + Flt3L (10 ng/mL) compared with 1 to 500 ng/mL of IL-2 + Flt3L (10 ng/mL). Cultured cells were assessed for surface marker and we calculated absolute number of NK cells and T cells. The cytotoxic activity was analyzed with purified NK cells., Results: After 2 weeks culture with 5 ng/mL of IL-15 + Flt3L, the fold inductions of absolute number of NK cells significantly increased to 20.9-fold +/- 9.3-fold of the number of NK cells on day 0 (p < 0.05), with 24.4-fold +/- 16.1-fold of T cells. But with 50 ng/mL of IL-15 + Flt3L, fold induction of NK cells decreased to 5.1-fold +/- 3.9-fold, while T cells showed 34.8-fold +/- 18.7-fold (n = 8). The proportion of NK vs T cells showed to be significantly higher (1.61 +/- 0.91) with 5 ng/mL of IL-15 than with 50 ng/mL of IL-15 (0.12 +/- 0.03). Such proportional change of NK/T cells could not be observed with IL-2. Immunophenotypes of CD56, CD16, LFA1, CD94, CD8, and perforin of cultured NK cells with 10 ng/mL of IL-15 + Flt3L showed the same pattern of those with 50 ng/mL of IL-2 + Flt3L. Cytotoxic activity against K562 of cultured NK cells resulted in the same level as adult peripheral blood (PB)-derived NK cells., Conclusions: Higher induction of NK cells derived from UCB-MNCs was achieved by low dose (5 to 10 ng/mL) rather than high dose (> 50 ng/mL) of IL-15.

Objective: The cause of delayed hematopoietic reconstitution after umbilical cord blood transplantation (UCBT) remains controversial. We hypothesized that hematopoietic stem/progenitor cells (HS/PCs) from UCB have some defects of the homing-related molecules responsible for their slow engraftment., Materials and Methods: A homing-related molecule repertoire expressed on HS/PCs from fresh and cryopreserved UCB, mobilized peripheral blood (mPB), and bone marrow (BM) were compared using sensitive, four-color fluorescence-activated cell sorting analysis. Purified CD34+ cells were subjected to ex vivo transmigration through double-coated transwell filter inserts, and an in vivo homing assay was performed in xenotransplanted NOD/SCID mice., Results: UCB-derived CD34(bright) cells expressed significantly lower levels of CD49e, CD49f, and CXCR-4 than their mPB and BM counterparts. CD34+ cells from UCB (and BM) exhibited significantly lower ex vivo transmigration than those from mPB, which were largely blocked by neutralizing antibodies to CD49e or CD49f. Recombinant human tumor necrosis factor-alpha treatment enhanced ex vivo transmigration of CD34+ cells from UCB and BM by inducing expression of the matrix metalloproteinases MMP-2/MMP-9. Short-term treatment of UCB-derived CD34+ cells with rHu-stem cell factor (rHuSCF) up-regulated levels of the homing-related molecules with their increased ex vivo transmigratory and in vivo homing potential., Conclusion: Our results indicate that disadvantageous transmigratory behavior of HS/PCs from UCB, which might partly explain the delayed reconstitution after UCBT, can be reversed by ex vivo manipulation with rHuSCF.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that are required for the initiation of the immune response. DCs have been shown to be generated from hematopoietic stem cells, but relatively little is known about the regulation underlying differentiation and activation of DCs. Here, we report that recombinant human (rh)IL-13 induces functional maturation of rhGM-CSF plus rhIL-4 generated monocyte-derived immature DCs. Incubation of these immature DCs with rhIL-13 or rhTNF-alpha for 2 days resulted in increased surface expression of CD1a, CD11c, CD86 and HLA-DR. The DCs treated with rhIL-13 or rhTNF-alpha, but not rhIL-4, for 2 days were more efficient than unstimulated DCs in the primary autologous/allogeneic T-cell response whereas the antigen (Ag)-specific T-cell response was suppressed. The treatment of DCs with rhIL-13 as well as rhTNF-alpha for 4 days down-modulated endocytic capacity for FITC-dextran (FITC-DX) and lucifer yellow (LY), and induced surface expression of CD83. Morphological, phenotypical, and functional analyses revealed that the monocytes cultured with rhGM-CSF plus rhIL-13 gave rise to a DC type more mature than rhGM-CSF plus rhIL-4-induced DCs. These findings revealed a new role for rhIL-13 in regulating both the maturation and activation of DCs.

We determined the appropriate incubation period to expand human peripheral blood (PB) CD34+ cells for clinical application and the role of recombinant human (rh) interleukin-3 (rhIL-3) in the expansion and differentiation of these cells. The cells were purified up to 40 +/- 16% and transitional changes in immunophenotypic subpopulations in suspension culture were examined following stimulation with three different combinations of rh colony-stimulating factors (rhCSFs): 1) rhIL-3 alone, 2) rhIL-6, rhSCF, rhG-CSF, plus rhGM-CSF, and 3) the four CSFs plus rhIL-3. With all three CSF combinations, the total cells increased continuously after day 5 until day 14, and a combination of the five CSFs always gave rise to the highest number of total cells. Immunophenotypic analysis of the expanded cells showed that with all three CSF combinations CD34+ cells reached a maximal rate on day 5 and then decreased in an inverse correlation between the logarithm of CD34 positive rate and the duration of suspension culture. The maximum expansion of CD34+ cells and PB progenitor cells (PBPC) with rhIL-3 alone, the four CSFs, or the five CSFs was observed on day 5, 10, or 7, respectively. The combination of the five CSFs was identified as the most potent stimulus for expansion of PBPC and CD34+ cells, as it increased non-erythroid PBPC 89 +/- 69-fold, with a range of 24 to 204-fold on day 7. However, differences in the expansion rate of these cells on days 5, 7, and 10 were not statistically significant. The majority of purified CD34+ cells coexpressed CD38 (91 +/- 3%) but were negative for CD33 (85 +/- 4%), and one-half coexpressed CD13. With all three combinations of CSFs, the mature CD34+ cells that coexpressed CD38, CD33, or CD13 expanded in parallel with the total CD34+ cells, while an increase in relatively immature CD34+ cells, which do not express CD38, CD33, or CD13, was only statistically significant with the five CSFs. Thus, rhIL-3 played a critical role when combined with the four CSFs by increasing both mature and immature CD34+ cells.

In a search for the optimal method to harvest peripheral blood progenitor cells (PBPC) without myeloablative chemotherapy, we administered recombinant human granulocyte colony-stimulating factor (G-CSF) to adult, healthy volunteers and investigated the mobilization rate of the PBPC. The consecutive subcutaneous administration of G-CSF in a dose of 2 micrograms/kg/d for 5 days significantly increased colony-forming units-granulocyte/macrophage (CFU-GM) up to 2340 +/- 980 per mL whole blood with a 30 +/- 21-fold mobilization of PBPC, range 11- to 76-fold. Burst-forming units-erythroid (BFU-E) and mixed erythroid progenitors (CFU-Mix) were also increased, and the mobilization rate was 8.4 +/- 4.5-fold and 7.6 +/- 4.7-fold, respectively. A time course study of PBPC after final G-CSF injection was done for 0 to 30 hours on days 1, 3, and 5. After a single administration of G-CSF (day 1), there was no increment of PBPC during the subsequent 30 hours, but the white blood cell count (WBC) markedly increased. Three days after the injection of G-CSF, no increase of CFU-GM was seen during the first 2 hours, then a significant, time-dependent increase occurred for up to 30 hours. Five days after the injection of G-CSF, there was no increase of CFU-GM during the first 2 hours, and the significant increase at 24 hours was maintained for up to 30 hours. BFU-E and CFU-Mix showed the same pattern of expansion as seen with CFU-GM. Thus, subcutaneous administration of 2 micrograms/kg/d G-CSF for 5 days will lead to a successful PBPC harvest for transplantation, and the most suitable period for this is 24 to 30 hours after the final G-CSF administration.