Plasma levels of SDF-1 and expression of SDF-1 receptor on CD34+ cells in mobilized peripheral blood of non-Hodgkin's lymphoma patients

CXCR4 is the receptor for the chemokine stromal derived factor-1 (SDF-1), is expressed on CD34+ cells, and has been implicated in the process of CD34+ cell migration and homing. We studied the mobilization of CD34/CXCR4 cells and the plasma levels of SDF-1 and flt3-ligand (flt3-L) in 36 non-Hodgkin's lymphoma patients receiving cyclophosphamide (Cy) plus G-CSF (arm A), Cy plus GM-CSF (arm B), or Cy plus GM-CSF followed by G-CSF (arm C) for peripheral blood stem cell (PBSC) mobilization and autotransplantation. We observed lower plasma levels of SDF-1 in PBSCs compared to premobilization bone marrow samples. The mean plasma SDF-1 levels were similar in PBSC collections in the three arms of the study. In contrast, SDF-1 levels in the apheresis collections of the "good mobilizers" (patients who collected a minimum of 2 x 10(6) CD34+ cells/kg in one to four PBSC collections) were significantly lower than the apheresis collections of the "poor mobilizers" (> or = 0.4 x 10(6) CD34+ cells/kg in the first two PBSC collections; 288 +/- 82 pg/ml versus 583 +/- 217 pg/ml; p = 0.0009). The mean percentage of CD34+ cells expressing CXCR4 in the apheresis collections was decreased in the PBSC collections compared with premobilization values from 28% to 19.4%. Furthermore, the percentage of CD34+ cells expressing CXCR4 in the good mobilizers was significantly lower compared with the poor mobilizers (14.7 +/- 2.1% versus 33.6 +/- 2.1%; p = 0.002). The good mobilizers had also significantly lower levels of flt3-L compared with the poor mobilizers (34 +/- 4 pg/ml versus 106 +/- 11 pg/ml; p = 0.006), Finally, the levels of flt3-L strongly correlated with SDF-1 levels (r = 0.8; p < 0.0001). We conclude: A) low plasma levels of SDF-1 and low expression of CXCR4 characterize patients with good mobilization outcome, and B) the levels of SDF-1 correlate with flt3-L, suggesting an association of these cytokines in mobilization of CD34+ cells.     

Immunologic profiles of effector cells and peripheral blood stem cells mobilized with different hematopoietic growth factors

BACKGROUND: Peripheral blood stem cells (PBSC) have become the preferred source of stem cells for autologous transplantation because of the technical advantage and the shorter time to engraftment. Mobilization of CD34(+) cells into the peripheral blood can be achieved by the administration of G-CSF or GM-CSF, or both, alone or in combination with chemotherapy. G-CSF and GM-CSF differ somewhat in the number and composition of CD34(+) cells and effector cells mobilized to the peripheral blood. However, the molecular mechanism underlying the release and engraftment of CD34(+) cells is poorly understood. PURPOSE: The purpose of this review is to give a recent update on the type and immunological properties of effector cells and CD34(+) cells mobilized by the different growth factors with emphasis on A) mobilization of T cells, natural killer cells, and dendritic cells; B) coexpression of adhesion molecules such as VLA-4 and L-selectin in mobilized PBSC collection, and C) coexpression of CXCR4-the receptor for the stromal-derived differentiation factor 1-with latest information shedding light on the molecular mechanism underlying the release and subsequent engraftment of CD34(+) cells. CONCLUSIONS: A) The reported suppression of T cell and NK cell functions in PBSC apheresis collections in patients primed with G-CSF or GM-CSF is controversial and may merely reflect low effector cell activity before mobilization. B) A decrease in the expression of adhesion molecules such as VLA-4 and L-selectin is a necessary requirement for the release of CD34(+) cells to the peripheral blood. C) A decrease in the expression of CXCR4 is a necessary requirement for the release of CD34(+) cells to the peripheral blood and correlates with mobilization success.     

Cell cycle-dependent change of the adhesive character of CD34+ progenitor cells and their VLA-4 expression]

We identified the cell cycle status of CD34+ cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of BM and apheresis PB samples collected from donors who had been administered granulocyte colony-stimulating factor (G-CSF). Regardless of whether G-CSF treatment was undergone, more than 10% of CD34+ cells in the BM was in the S + G2/M phase. In contrast, less than 2% of CD34+ cells in the PB was cycling. After co-culturing BM CD34+ cells with a monolayer of the stromal cell line MS-5 for 1 hour, some cells adhered to the stroma. The percentage of cells in the S + G2/M phase among these adherent cells was higher than that among the non-adherent cells. Flow cytometric analysis revealed that CD34+ cells in mobilized PB expressed less VLA-4 than those in BM and that in in vitro-cultured non-adherent cells exhibited a lower level of VLA-4 expression than adherent cells. In addition, CD34+ cells in the G0/G1 phase expressed lower levels of VLA-4 than those in the S + G2/M phase. These findings suggested that the reduced expression of adhesion molecules such as VLA-4 by the progenitor cells in the G0/G1 phase of the cell cycle result in the release of progenitor cells from the hematopoietic microenvironment to peripheral blood.     

Quantitative expression of the adhesion receptors VLA-4, VLA-5, L-selectin, MAC-1, and ICAM-1 on the surface of CD34+ cells]

The aim of this work was to quantify by flow cytometry the main adhesion receptors on CD34+ cells. These cells were isolated from bone marrow (BM) or mobilized peripheral blood (PB). The proportions of CD34+/CD49d+ and CD34+/CD49e+ are weaker on PB cells, without quantitative expression variation. This phenotypic variation may induce CD34+ cells exist from BM into circulation, promoting the mobilization. The homing to the BM implicate the CD62L receptor, which expression was found more frequently and stronger on PB cells than on BM. The CD11b, CD18 and CD54 receptors are implicated in CD34+ cells adhesion to BM micro-environment. No significant variation in CD34+/CD11b+ and CD34+/CD18+ cells frequency was noted. Moreover, CD54 receptor was more frequently expressed on PB cells. Quantitative analysis revealed that CD18 was more strongly expressed on BM than on PB cells. This quantitative variation could promote progenitor adhesion by interacting with stromal cells. Finally, quantitative expression of the main receptors on CD34+ cells provides an original option for studying CD34+ cells during the mobilization, the homing or the adhesion to BM micro-environment.     

Endothelial-like vascular progenitor cells (VPCs) from allogeneic and autologous donors: mobilization features distinct from hematopoietic progenitors.

Endothelial-like progenitor cells circulate in the peripheral blood (PB) and can be enumerated using cell culture-based progenitor assays. These circulating vascular progenitor cells (VPCs) are implicated in new vessel formation and regenerative potential in several animal and human models of tissue injury. Given the emerging role of VPCs in regenerative processes and the limited information on the availability of such progenitor cells, we sought to determine baseline circulating VPC levels in healthy allogeneic donors and autologous hematopoietic transplant patients. VPC numbers were also measured in peripheral blood stem cell (PBSC) grafts from both graft types. Immunohistochemistry revealed that VPC clusters obtained under our culture conditions were CD45(+) and acquired endothelial features (CD31 and vascular endothelial-cadherin) in vitro upon angiogenic stimulation and gradually lost monocytic surface markers (CD14). Before PBSC mobilization, VPCs levels varied substantially in healthy donors and were markedly lower in patients with hematologic malignancies compared with healthy allogeneic donors with 27 +/- 15 versus 99 +/- 21 VPCs/mL (mean +/- SEM), respectively (P = .001). In patients undergoing stem cell mobilization, VPCs in the PB increased from 7 +/- 2 on day 0 to 51 +/- 9 by day 7 of mobilization (P = .05), representing a median fold increase of 8.9 (range, 3.0-29.8). Although autologous transplant patients underwent more intensive mobilization, VPCs were higher in allogeneic (7.2 +/- 1.4 x 10(3)/kg) than in autologous (2.6 +/- 1.5 x 10(3)/kg) mobilized PB grafts (P = .045). To identify predictors of VPC content, graft VPCs were compared with levels of CD34(+) cells, total colony forming unit (CFU), or granulocyte-macrophage colony forming unit (GM-CFU). None of these hematopoietic progenitors correlated with VPC numbers in PBSC grafts (P = NS). However, PB monocyte levels were highly correlated with circulating VPC levels (r = 0.71, P < .0001). Thus, our analysis identified significant variability in VPCs at baseline and in PBSC grafts from healthy donors. Nevertheless, these donors remain a better source of VPCs than do autologous transplant patients. Importantly, VPC mobilization occurs independently of hematopoietic mobilization. In view of the potential role of VPCs in recovery from transplant-related tissue injury, angiogenic mobilization strategies that complement hematopoietic mobilization will need to be specifically designed.     

Kinetics of peripheral blood stem cell mobilization following G-CSF-supported chemotherapy

It would be a clinical and economical advantage if the optimal time point of peripheral blood stem cell (PBSC) mobilization following G-CSF-supported chemotherapy (CT) was known in advance. Therefore, we retrospectively analyzed mobilization parameters in 113 adult tumor patients treated in our institution within 1 year. The start of apheresis was guided by CD34(+) cell measurements in the PB and occurred on or after day 11 after start of mobilization CT in 97% of patients. The median peak (p)CD34(+) cell count in PB uniformly occurred on day 14-15 (range: 6-32 days) after the start of CT, irrespective of the diagnosis (multiple myeloma n = 76, other histology n = 37), the type, but not the amount, of premobilization CT or radiotherapy (RT), the mobilization regimen, or the G-CSF dosage administered. Among more heavily pretreated patients (>six cycles of prior CT or RT), a higher proportion mobilized late (pCD34(+) cell count later than day 20 in 12% and 13%, respectively, versus 2%-5% in the other groups). Therefore, we propose to start measuring CD34(+) cells in the PB on day 11 after the start of mobilization therapy. The wide range of optimal mobilization time points argues for an individualized rather than a preset start of apheresis.     

Comparison of CD34+ subsets and clonogenicity in human bone marrow, granulocyte colony-stimulating factor-mobilized peripheral blood, and cord blood

To compare the clonogenicity and distribution of CD34+ subsets in bone marrow (BM), granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood (PB) and cord blood (CB), we analyzed in vitro colony formation and CD34+ cells co-expressing differentiation molecules (CD38, HLA-DR), myeloid associated molecules (CD13, CD33), a T-cell associated molecule (CD3), and a B-cell associated molecule (CD19) from mononuclear cells (MNCs) in the three compartments. The proportions of CD34+CD38- cells (BM: 4.4+/-2.8%, PB: 5.3+/-2.1%, CB: 5.9+/-3.9%) and CD34+HLA-DR cells (BM: 4.7+/-3.4%, PB: 5.5+/-2.3%, CB: 6.1+/-3.7%) did not differ significantly among the compartments. In contrast, a significantly higher proportion of CD34 cells of PB and CB co-expressed CD13 (75.0+/-11.4%, 77.7+/-17.3%) and CD33 (67.1 +/-5.7%, 56.8+/-10.3%) compared with those of BM (43.0+/-6.3%, 27.6+/-5.1%) and a significantly higher number of granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) were detected in MNCs derived from PB and CB compared with those from BM (p<0.01). The proportion of CD34+CD19+ cells was higher in BM (34.9+/-11.9%) than those in PB (5.6+/-3.0%) and CB (4.7=2.1%) (p<0.05). The proportion of CD34+CD3+ was comparable in all three compartments. In conclusion, our findings show that MNCs of mobilized PB and CB display similar phenotypic profiles of CD34+ subsets and clonogenicity, different from those of BM.     

Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity

The replacement of bone marrow (BM) as a conventional source of stem cell (SC) by umbilical cord blood (UCB) and granulocyte-colony stimulating factor-mobilized peripheral blood SC (PBSC) has brought about clinical advantages. However, several studies have demonstrated that UCB CD34(+) cells and PBSC significantly differ from BM CD34(+) cells qualitatively and quantitatively. Here, we quantified the number of SC in purified BM, UCB CD34(+) cells, and CD34(+) PBSC using in vitro and in vivo assays for human hematopoietic SC (HSC) activity. A cobblestone area-forming cell (CAFC) assay showed that UCB CD34(+) cells contained the highest frequency of CAFC(wk6) (3.6- to tenfold higher than BM CD34(+) cells and PBSC, respectively), and the engraftment capacity in vivo by nonobese diabetic/severe combined immunodeficiency repopulation assay was also significantly greater than BM CD34(+), with a higher proportion of CD45(+) cells detected in the recipients at a lower cell dose. To understand the molecular characteristics underlying these functional differences, we performed several DNA microarray experiments using Affymetrix gene chips, containing 12,600 genes. Comparative analysis of gene-expression profiles showed differential expression of 51 genes between BM and UCB CD34(+) SC and 64 genes between BM CD34(+) cells and PBSC. These genes are involved in proliferation, differentiation, apoptosis, and engraftment capacity of SC. Thus, the molecular expression profiles reported here confirmed functional differences observed among the SC sources. Moreover, this report provides new insights to describe the molecular phenotype of CD34(+) HSC and leads to a better understanding of the discrepancy among the SC sources.     

Mobilization kinetics of CD34(+) cells in association with modulation of CD44 and CD31 expression during continuous intravenous administration of G-CSF in normal donors

The aim of the present study is to evaluate the kinetics of CD34(+) cells and investigate the potential modulation of CD44 and CD31 expression on CD34(+) cells during continuous i.v. administration of G-CSF, thus to elucidate the possible mechanism of peripheral blood progenitor cell (PBPC) mobilization. Fifteen healthy donors were enrolled in this study. G-CSF (10 microg/kg/day) was administered for four consecutive days through continuous 24-h i.v. infusion. For measurement of complete blood counts, CD34(+) cell levels and their expression of CD44 and CD31, PB sampling was performed immediately before the administration of G-CSF (steady-state) and after 4, 8, 24, 48, 72, 96, and 120 h of G-CSF administration. The percentage and absolute number of CD34(+) cells significantly increased at day 3 (0. 55 +/- 0.09%, 51.12 +/- 24.83 x 10(3)/ml) and day 4 (0.47 +/- 0.09%, 46.66 +/- 24.93 x 10(3)/ml), compared to the steady-state level (0. 06 +/- 0.09%, 2.03 +/- 5.69 x 10(3)/ml). At day 3 to day 5 following the onset of G-CSF administration, a strong decrease of CD44 and CD31 expression was observed on mobilized CD34(+) cells compared to controls: the relative fluorescence intensity of CD44 and CD31 was, respectively, 50%-70% and 40%-90% lower than that of controls. We conclude that continuous i.v. administration of G-CSF apparently results in more rapid mobilization of CD34(+) cells, and downregulation of CD44 and CD31 on CD34(+) cells is likely to be involved in the mobilization of PBPC after treatment with G-CSF.     

Cytokine expansion culture of cord blood CD34+ cells induces marked and sustained changes in adhesion receptor and CXCR4 expressions

Recent studies have demonstrated defective bone marrow homing of hematopoietic stem cells after cytokine expansion culture. Adhesion receptors (ARs) are essential to the homing process, and it is possible that cytokine culture modulates AR expression. We studied changes in expression of very late antigen-4 (VLA-4), VLA-5, L-selectin, leukocyte function-associated antigen-1 (LFA-1), CD44, and the stromal cell-derived factor-1 (SDF-1) receptor, CXCR4, during cytokine culture of cord blood (CB) CD34(+) cells. Expression of ARs was studied by flow cytometry on CB CD34(+) cells in whole blood, after purification and during culture for up to 10 days. Cells were cultured with stem cell factor (SCF), thrombopoietin (TPO), Flt3-ligand (Flt3), and G-CSF. Results showed that 80% or more of uncultured CD34(+) cells were positive for VLA-4, L-selectin, LFA-1, CD44, and CXCR4 while 50% were positive for VLA-5. Purification of CD34(+) cells did not affect AR expression, but cytokines increased expression three- to nine-fold throughout the 10-day culture period. In contrast, expression of CXCR4 decreased. Expression changes of ARs and CXCR4 on CD34(+)/CD38(-) cells mirrored those of the total CD34(+) population. The results indicate that cytokine culture significantly increases AR expression on CB CD34(+) cells, which may be related to the decrease in homing of cytokine-cultured hematopoietic stem cells.     

Lymphocytes expressing alpha1beta1 integrin (very late antigen-1) in peripheral blood of patients with arthritis are a subset of CD45RO(+) T-cells primed for rapid adhesion to collagen IV

We report that very late antigen-1 (VLA-1(+)) CD3(+)CD45RO(+) T-cells are selectively segregated from VLA-1(-) peripheral blood (PB) mononuclear cells (MC), in which CD3(+) T-cells are evenly CD45RO(+) and CD45RO(-), when PBMC are stained with a monoclonal antibody (mAb) to VLA-1 and passaged on immunomagnetic columns. In contrast, both VLA-1(+) and VLA-1(-) MC isolated from synovial fluid (SF) are mainly CD45RO(+)CD3(+) T-cells. VLA-1(+) MC formed 13 +/- 5.3% of MC eluting from columns loaded with PBMC of patients with seropositive rheumatoid arthritis (n = 6) and 2.3 +/- 1.6% of patients (n = 4) with other arthritides (P < 0.022). Importantly, only the VLA-1(+) MC from PB and SF adhered to collagen IV upon triggering with phorbol 12-myristate 13-acetate. Moreover, adhesion and migration on collagen IV were preferentially maintained in lines cultured from VLA-1(+) T-cells, and both were inhibited by mAb to the VLA-1 alpha1 I domain. These results suggest that VLA-1(+) CD45RO(+) T-cells in patients with arthritis could play a role in both systemic and local inflammation by rapidly adhering to collagen IV.     

Plerixafor Plus Granulocyte Colony-Stimulating Factor versus Placebo Plus Granulocyte Colony-Stimulating Factor for Mobilization of CD34(+) Hematopoietic Stem Cells in Patients with Multiple Myeloma and Low Peripheral Blood CD34(+) Cell Count: Results of a Subset Analysis of a Randomized Trial

Preapheresis peripheral blood (PB) CD34(+) cell count is a strong predictor of hematopoietic stem cell (HSC) mobilization and is routinely used to optimize the timing, cost, and success of HSC collection in patients with multiple myeloma. However, a uniform PB CD34(+) cell count that predicts mobilization failure has not been defined, resulting in the development of institute-specific algorithms for mobilization, particularly regarding the decision of when to use the novel stem cell mobilization agent plerixafor. In this post hoc analysis, we evaluated the mobilization efficacy of plerixafor plus granulocyte colony-stimulating factor (G-CSF) versus placebo plus G-CSF in patients with multiple myeloma, stratified by preapheresis PB CD34(+) cell count: <10, <15, <20, and ≥20 cells/μL. Regardless of the PB CD34(+) cell count, the total yield of CD34(+) cells from apheresis was significantly higher in the plerixafor group than in the placebo group, and significantly more patients in the plerixafor group collected the minimum (≥2?×?10(6) cells/kg) and optimum (≥6?×?10(6) cells/kg) stem cell yields on each day of apheresis. As a corollary, the greater stem cell collection in plerixafor-treated patients resulted in the need for significantly fewer days of apheresis to reach minimum and optimum cell doses across all cell count groups. For all CD34(+) cell count groups, the proportion of patients proceeding to transplantation and the median time to platelet and neutrophil engraftment were similar in the plerixafor and placebo groups. Our findings demonstrate that in patients with multiple myeloma who might be predicted to fail mobilization based on low PB CD34(+) cell count, the addition of plerixafor to G-CSF allows for collection of the minimal and optimal cell doses in a greater proportion of patients compared with G-CSF alone. In addition, plerixafor plus G-CSF significantly improves the likelihood of optimal HSC collection in patients with higher preapheresis PB CD34(+) cell counts (≥20 cells/μL) compared with placebo plus G-CSF. Collectively, this analysis of predicted poor mobilizers validates the superiority of plerixafor plus G-CSF compared with G-CSF alone, which had been demonstrated previously in the overall patient population.     

Peripheral blood CD34+ cell enumeration as a predictor of apheresis yield: an analysis of more than 1,000 collections

The role of the peripheral blood (PB) CD34(+) cell count in predicting the CD34(+) cell yield in hematopoietic progenitor cell apheresis collections is well established. However, sometimes unexpectedly poor CD34(+) cell yields are obtained. To determine the effect, if any, of a range of factors on the ability of the PB CD34(+) count to predict collection CD34(+) cell count, we performed a retrospective analysis on consecutive hematopoietic progenitor cell apheresis collections between 2004 and 2008. Factors investigated included mobilization regimen, PB white blood cell count, body weight, and disease. After exclusion of collections involving apheresis complications, a total of 1,225 PB CD34(+) cell results with corresponding collection CD34(+) cell results from 458 patients were analyzed. Although differences in the median PB CD34(+) cell counts and collection CD34(+) cell counts were seen between mobilized collections with chemotherapy plus granulocyte colony-stimulating factor and those with granulocyte colony-stimulating factor alone, the predictive capability of the PB CD34(+) cell count for the collection CD34(+) cell yield remained similar. Although poorer collection efficiencies were observed in the myelodysplastic syndrome/myeloproliferative disorder diagnostic subgroup, our findings confirm that PB CD34(+) cell analysis remains a powerful and irreplaceable tool for predicting hematopoietic progenitor cell apheresis CD34(+) cell yield.     

Hematopoietic repopulating ability of cord blood CD34(+) cells in NOD/Shi-scid mice

Although umbilical cord blood (CB) is increasingly being used as an alternative to bone marrow (BM) as a source of transplantable hematopoietic stem cells (HSC), information on the hematopoietic repopulating ability of CB HSC is still limited. We recently established a xenotransplantation system in NOD/Shi-scid mice to evaluate human stem cell activity. In the present study, we transplanted 5 to 10 x 10(4) CB CD34(+) cells into six NOD/Shi-scid mice treated with anti-asialo GM1 antiserum to investigate the hematopoietic repopulating ability of CB. The BM of all recipients contained human CD45(+) cells 10 to 12 weeks after the transplantation (43.8 +/- 17.7%). Clonal culture of the recipient BM cells revealed the formation of various types of human hematopoietic colonies, including myelocytic, erythroid, megakaryocytic, and multilineage colonies, indicating that CB HSC can differentiate into hematopoietic progenitors of various lineages. However, the extent of the differentiation and maturation differed with each lineage. CD13(+)/CD14(+)/CD33(+) myelocytic cells were mainly repopulated in BM and peripheral blood (PB). While CD41(+) megakaryocytic cells and platelets were present, few glycophorin A(+)CD71(+) or hemoglobin alpha-containing erythroid cells were detected. CD19(+) B cells were the most abundantly repopulated in NOD/Shi-scid mice, but their maturational stage differed among the hematopoietic organs. Most of the BM CD19(+) cells were immature B cells expressing CD10 but not surface immunoglobulin (Ig) M, whereas more mature CD19(+)CD10(-) surface IgM(+) B cells were predominantly present in spleen and PB. CD3(+) T cells were not detected even in the recipient thymus. The transplantation to the NOD/Shi-scid mouse may provide a useful tool for evaluating the repopulating ability of transplantable human HSC.     

Correlation between plasma Flt3-ligand concentration and hematopoiesis during G-CSF-induced CD34+ cell mobilization

This study aimed to correlate blood Flt3-ligand (FL) concentration with CD34(+) cell number in blood and bone marrow (BM) during granulocyte colony-stimulating factor (G-CSF) mobilization. Nonhuman primates were injected with 10 microg/kg of G-CSF (Lenograstim) daily over a period of 5 days. Daily blood sampling and repeated BM sampling showed that FL concentration before mobilization was negatively correlated to the absolute number of BM CD34(+) cells, but also to the number of G-CSF-mobilized CD34(+) cells on days 3-5 of treatment. This showed that FL concentration in the blood reflected BM status before mobilization, and suggested that this parameter could be used as a predictive indicator of G-CSF-induced CD34(+) cell mobilization.     

Hematopoietic stem cells from fancc(-/-) mice have lower growth and differentiation potential in response to growth factors

Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow (BM) failure. We have previously shown that stem cells from the FA group C mouse model have lower long-term primary and secondary reconstitution ability, and that bone marrow of Fancc(-/-) mice contained fewer lineage-negative (Lin(-))Thy1.2(low)Sca-1(+)c-kit(+) CD34(+) cells but normal levels of Lin(-)Thy1.2(low)Sca-1(+)c-kit(+)CD34(-) primitive cells. These data suggest that CD34(+) primitive cells have either a lower growth or differentiation potential, or that these cells have greater apoptosis levels. To investigate the role Fancc might have on the growth and differentiation potentials of primitive hematopoietic stem cells, we used a single-cell culture system and monitored cell viability, doubling potential, and apoptosis levels of Fancc(-/-) primitive Lin(-)Thy1.2(-)Sca-1(+) (LTS)-CD34(+) and LTS-CD34(-) stem cells. Results showed that Fancc(-/-) LTS-CD34(-) and LTS-CD34(+) cells had altered growth and apoptosis responses to combinations of stimulatory cytokines, most dramatically in response to a combination of factors that included interleukin-3 (IL-3) and IL-6. In addition, Fancc(-/-) LTS-CD34(-) and LTS-CD34(+) cells showed a lower differentiation potential than Fancc(+/+) cells. These results support a role for Fancc in the growth and differentiation of primitive hematopoietic cells and suggest that an altered response to stimulatory cytokines may contribute to BM aplasia in FA patients.     

In Situ Activation of Helper T Cells in the Lung

To better understand the lung and systemic responses of helper T cells mediating memory immunity to Mycobacterium tuberculosis, we used three- and four-color flow cytometry to study the surface phenotype of CD4(+) lymphocytes. Bronchoalveolar lavage (BAL) fluid and peripheral blood (PB) samples were obtained from a total of 25 subjects, including 10 tuberculosis (TB)-infected subjects, 8 purified-protein-derivative-negative subjects, and 7 purified-protein-derivative-positive subjects. In marked contrast to CD4(+) lymphocytes from PB (9% +/- 5% expressing CD45RA and CD29), the majority (55% +/- 16%) of CD4(+) lymphocytes in BAL (ALs) simultaneously expressed CD45RA, a na?ve T-cell marker, and CD29, members of the very late activation family. Further evaluation revealed that CD4(+) ALs expressed both CD45RA and CD45RO, a memory T-cell marker. In addition, the proportion of CD4(+) lymphocytes expressing CD69, an early activation marker, was drastically increased in BAL fluid (83% +/- 9%) compared to PB (1% +/- 1%), whereas no significant difference was seen in the expression of CD25, the low-affinity interleukin 2 receptor (34% +/- 15% versus 40% +/- 16%). More importantly, we identified a minor population of CD69(bright) CD25(bright) CD4(+) lymphocytes in BAL (10% +/- 6%) that were consistently absent from PB (1% +/- 1%). Thus, CD4(+) lymphocytes in the lung paradoxically coexpress surface molecules characteristic of na?ve and memory helper T cells as well as surface molecules commonly associated with early and late stages of activation. No difference was observed for ALs obtained from TB-infected and uninfected lung segments in this regard. It remains to be determined if these surface molecules are induced by the alveolar environment or if CD4(+) lymphocytes coexpressing this unusual combination of surface molecules are selectively recruited from the circulation. Our data suggest that ex vivo experiments on helper T-cell subsets that display distinctive phenotypes may be pivotal to studies on the human immune response to potential TB vaccines.     

CD34(+) hematopoietic progenitor cell selection of bone marrow grafts for autologous transplantation in pediatric patients.

CD34(+)-selection of hematopoietic grafts for patients undergoing autologous hematopoietic stem cell transplantation (HSCT) is frequently used to obtain a tumor-free graft. The majority of published experience is with peripheral blood stem cell (PBSC) products; only scant information has been published on bone marrow (BM) grafts. We reviewed our experience using CD34(+) selection of BM grafts in children undergoing autologous BM transplantation. After obtaining institutional approval, we performed a retrospective review of the medical records of patients who underwent autologous stem cell collection at St. Jude. From January 1, 1999, to December 31, 2003, 373 patients underwent autologous HSCT; 131 received marrow grafts, 237 received PBSC grafts, and 5 received a combination. Seventeen patients underwent BM harvests for CD34(+) selection of their stem cell grafts. Sixteen patients received 19 CD34 purified grafts processed on the Isolex 300i Magnetic Cell Selection System device. Four patients were not included in the engraftment analysis as 1 did not receive the collected product, 1 received a tandem product, and 2 received products that were composed of 2 or 3 combined purified products. Following selection, marrow grafts contained a median of 1.4 x 10(6) CD34(+) cells/kg (range: 0.09-8.3 x 10(6)/kg) and a median of 0.014 x10(8) total nucleated cell cells/kg (range: 0.001-0.09 x 10(8)/kg). The median CD34% recovery was 30.9% (range: 9.3%-57.1%), with the median CD34 purity being 95.5% (range: 62.2%-98.8%). All patients engrafted. The median time to absolute neutrophil count > or = 500/mm(3) was 19 days (range: 12-35 days), and to platelet recovery was 28 days (range 18-37 days). No patient died from transplant-related complications. Our study demonstrates that CD34(+)-selection of marrow grafts is feasible, and these grafts are able to successfully reconstitute hematopoiesis in patients undergoing autologous BMT.     

Cost-Effectiveness Analysis of a Risk-Adapted Algorithm of Plerixafor Use for Autologous Peripheral Blood Stem Cell Mobilization

Historically, up to 30% of patients were unable to collect adequate numbers of peripheral blood stem cells (PBSCs) for autologous stem cell transplantation (ASCT). Plerixafor in combination with granulocyte colony-stimulating factor (G-CSF) has shown superior results in mobilizing peripheral blood (PB) CD34+ cells in comparison to G-CSF alone, but its high cost limits general use. We developed and evaluated risk-adapted algorithms for optimal utilization of plerixafor. In plerixafor-1, PBSC mobilization was commenced with G-CSF alone, and if PB CD34 on day 4 or day 5 was <10/μL, plerixafor was administered in the evening, and apheresis commenced the next day. In addition, if on any day, the daily yield was <0.5 × 10⁶ CD34/kg, plerixafor was added. Subsequently, the algorithm was revised (plerixafor-2) with lower thresholds. If day-4 PB CD34 <10/μL for single or <20/μL for multiple transplantations, or day-1 yield was <1.5 × 10⁶ CD34/kg, or any subsequent daily yield was <0.5 × 10⁶ CD34/kg, plerixafor was added. Three time periods were analyzed for results and associated costs: January to December 2008 (baseline cohort; 319 mobilization attempts in 278 patients); February to November 2009 (plerixafor-1; 221 mobilization attempts in 216 patients); and December 2009 to June 2010 (plerixafor-2; 100 mobilization attempts in 98 patients). Plerixafor-2 shows a significant improvement in PB CD34 collection, increased number of patients reaching minimum and optimal goals, fewer days of apheresis, and fewer days of mobilization/collection, albeit at increased costs. In conclusion, although the earlier identification of ineffective PBSC mobilization and initiation of plerixafor (plerixafor-2) increases the per-patient costs of PBSC mobilization, failure rates, days of apheresis, and total days of mobilization/collection are lower.