Rationalizing infection control in the dental office

We performed a prospective study in 17 consecutive patients following Autologous bone marrow (BM) or rhG-CSF primed peripheral blood item cell (PBSC) transplantation, with the objective of comparing immune recovery between both procedures and to evaluate results in rhG-CSF mobilized peripheral blood stem cell transplantation (PBSCT). Kinetics of immune reconstitution showed differences, with a faster recovery of CD3+ and CD8+ T cells, and a more rapid and sustained recovery of CD8+/-/CD56+ natural killer (NK) cells in the PBCSCT group. Autologous bone marrow transplantation (ABMT) was associated with a improved reconstitution of the CD19+/CD5+/-subpopulation. Moreover, rhG-CSF mobilized PBSCT generated a greater recovery of CD8+/-/CD56+ cells than previous data concerning transplantation with peripheral blood (PB) progenitors collected after myelosuppressive chemotherapy or myelosuppressive therapy plus rhG-CSF. Our results show differences in the rate and pattern of B and T lymphocytes reconstitution after ABMT and PBSCT. Additionally, we state an enhancement of CD56+ cells in patients undergoing PBSCT mobilized solely using rhG-CSF.     

Generation and functional characterization of human dendritic cells derived from CD34 cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM-CSF and TNF-alpha with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+ CD14- cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediatelate-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.     

Efficacy and onset of action of fluticasone propionate aqueous nasal spray on nasal symptoms, eosinophil count, and mediator release after nasal allergen challenge in patients with seasonal allergic rhinitis

In order to determine if peripheral blood stem cells (PBSC) collected after priming with G-CSF in AML in first complete remission (CR) can be used for autologous transplantation and to evaluate the efficacy of early intensification therapy as in vivo purging, we studied 35 consecutive patients with AML in first CR. After standard induction and consolidation chemotherapy, 24 of them were treated with one (10 patients) or two (14 patients) cycles of high-dose cytarabine plus etoposide prior to PBSC collection. G-CSF was used as the priming agent. Of the 35 patients scheduled for peripheral blood stem cell transplantation (PBSCT), three relapsed before transplantation, and the 32 remaining underwent PBSCT. High-dose therapy consisted of either total body irradiation plus cyclophosphamide or busulphan plus cyclophosphamide. The median number of CD34+ cells infused was 3.24 x 10(6)/kg (range 0.15-14). The median times to reach a PMN count of 0.5 x 10(9)/l and a platelet count of 50 x 10(9)/l were 12 (8-28) and 30 (11-345) days, respectively. There was no transplant-related mortality. Twelve patients relapsed between 2 and 21 months post-PBSCT. With a median follow-up of 28 months, actuarial disease-free survival (DFS) is 52.41 +/- 9% in the intent-to-treat group and 57.4 +/- 9.8% in patients who underwent PBSCT. The probability of DFS is significantly higher for patients who receive early intensification therapy prior to both PBSC collection and PBSCT as compared with patients that do not: 68.8 +/- 10.27% vs 35.5 +/- 12.6%, P = 0.0418. These results indicate the feasibility of PBSCT in AML using G-CSF-mobilized PBSC. The use of intensification treatment as 'purging in vivo' prior both to collection of PBSC and PBSCT significantly reduces the risk of relapse in this group of patients.     

O-glucuronidation, a newly identified metabolic pathway for topotecan and N-desmethyl topotecan

BACKGROUND AND OBJECTIVE: CD34+ hematopoietic progenitor cells (HPCs) constitute a heterogeneous population both in size and in immunological features. Lack of CD38, HLA-DR and lineage committed antigens as well as the co-expression of Thy-1 (CDw90) and c-kit receptor (CD117), are able to identify the so-called stem cells. A flow cytometric study was carried out to investigate the co-expression of Thy-1 and c-kit receptors, both members of Ig superfamily adhesion molecules, involved in cell to cell and cell to stroma interactions, on bone marrow (BM), mobilized peripheral blood (PB) and human umbilical cord blood (HUCB) CD34+ HPCs. DESIGN AND METHODS: Lysed whole blood from 15 BM, 25 mobilized PB and 25 HUCB samples were used to perform a five-dimensional flow cytometric evaluation of both CDw90 and CD117 on CD34+ cells. RESULTS: Few CD34+ cells co-expressed Thy-1 antigen in all three compartments (BM: 11.2 +/- 7.2%; PB: 6.2 +/- 3.6%; HUCB: 6 +/- 2.9%; BM vs PB < 0.04; BM vs HUCB < 0.008; PB vs HUCB ns). c-kit receptor was detected on the majority of CD34+ HPCs, particularly in HUCB (HUCB: 80.7 +/- 8.2%; BM: 72.3 +/- 13.1%; PB: 64.2 +/- 17%; HUCB vs BM < 0.03; HUCB vs PB < 0.0001; BM vs PB ns). CD34+Thy-1+ and CD34+c-kit+ HPCs generally displayed HLA-DR antigen, as expression of early cell commitment. However, the most immature CD34+Thy-1+HLA-DR- (HUCB: 1 +/- 0.6%; BM: 0.4 +/- 03%; PB: 0.7 +/- 0.5%; HUCB vs BM < 0.0001; BM vs PB < 0.04; HUCB vs PB ns) and CD34+c-kit+HLA-DR- HPCs (HUCB: 6.5 +/- 4.4%; BM: 6.3 +/- 4.8%; PB: 2.2 +/- 1.8%; HUCB vs BM ns; BM vs PB < 0.0001; HUCB vs PB < 0.0001) were mainly detected in HUCB. Finally, the greatest percentage of CD34+Thy-1+c-kit+ cells was found in BM (6.9 +/- 4.1%) followed by leukapheretic samples (4.4% +/- 2.7) and then by HUCB (3.7 +/- 1.2%; BM vs PB ns; BM vs HUCB < 0.001; HUCB vs PB ns). INTERPRETATION AND CONCLUSIONS: Since the blood release or HPCs is probably due to a perturbation of the adhesive interactions between these cells and the marrow stroma, the different pattern of Thy-1 and c-kit receptor expression on CD34+ HPCs found in the three hemopoietic compartments evaluated can lead to new knowledge about the mobilization kinetics in which the Ig superfamily adhesion molecules are involved.     

Population dynamics of the monogenean Anoplodiscus cirrusspiralis on the snapper, Pagrus auratus

Mobilized peripheral blood progenitor cells (PBPC) have been shown to differ qualitatively from bone marrow (BM) progenitors. The released progenitor cells are predominantly in G0/G1 and show a relatively high percentage of rhodamine dull cells. Within the BM these last two features are characteristic of the more primitive progenitors. Although the mobilized PB cells can give rise to long-term repopulation and thus contain stem cells, the frequency of stem cells is not much higher if long-term initiating cell (LTC-IC) assays are used. To determine whether quiescent stem cells are selectively released or the low-cycle status of PB progenitors is related to the release from the BM microenvironment, the cell cycle status and rhodamine content in the PB and BM during mobilization were studied and compared with steady-state BM. More differentiated and more primitive progenitors were separated based on differentiation markers and cloned in single cell assay. In mobilized PB 54% of the CD34+ cells (n=5) were rhodamine dull compared to 22% in steady-state BM (P=0.014) [n=6]. The percentage of CD34+ cells in the S/G2M phases of the cell cycle was 2.1% in the mobilized PB (n=11), and 18% in steady-state BM (n=11) [P=0.002]. During mobilization the fraction of cells in the S/G2M phase of the cell cycle was 16% in BM (n=7), similar to steady-state BM (P=0.34). The released progenitors represented a selection of BM progenitors, with significantly more primitive progenitors (CD34+/13+/33dim) and less lymphoid precursors (CD34+/19+). Within the more differentiated CD34+113+/33bright, myelomonocytic precursors, both in PB as well as in BM, the percentage S/G2M was relatively higher than in the CD34+/13+/33dim subfraction: in normal BM: median 18% vs 8% (P=0.006) [n=8]; in mobilized PB 3% vs 2% (P=0.03) [n=10]; and in BM during mobilization 24% vs 7% (P=0.01) [n=6]. The cycle status of mobilized PB progenitors was low both in the primitive and more differentiated subfractions. During the mobilization period the BM progenitors are cycling as in steady-state BM. The low-cycle status of the mobilized PB progenitors may be related to the loss of contact with the micro-environment.     

Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): evidence for a phenotypic series involving three chondrodysplasias

Flow cytometric DNA analysis was performed in combination with three-colour immunological staining of cell surface antigens on density-separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell-cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5/micrograms per kg body weight of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-simulating factor (GM-CSF). Despite considerably increasing numbers of CD34+ PB MNC, the latter were not found to be in S/G2M phase, whereas, among the CD34- MNC, the proportion of cells in S/G2M phase increased from < 0.1% to 0.75 +/- 0.4% (GM-CSF) and to 1.34 +/- 0.75% (G-CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34+ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G2M phase. The non-cycling status of PB and progenitor cells was confirmed by the analysis of CD34+ cells enriched from the two cells sources. However, in vitro stimulation of these progenitor cells using IL3, GM-CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34+ cells were in S/G2m phase. The fact that cycling CD34+ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized 'stem cells' which may require the BM micro-environment for adequate proliferation in vivo.     

The role of granulocyte colony-stimulating factor in mobilization and transplantation of peripheral blood progenitor and stem cells

The article provides a review of the role of granulocyte colony-stimulating factor (G-CSF) for mobilization and transplantation of peripheral blood progenitor and stem cells. Recombinant gene technology has permitted the production of highly purified material for therapeutic use in humans. Progenitor cells can be assessed using semisolid and liquid culture assays or direct immunofluorescence analysis of cells expressing CD34. This antigen is found on lineage-determined hematopoietic progenitor cells as well as on more primitive stem cells with extensive self-renewal capacity. Administration of G-CSF during steady-state hematopoiesis or following cytotoxic chemotherapy leads to an increase of hematopoietic progenitor cells in the peripheral blood. The level of circulating CD34+ cells post-chemotherapy is greater compared with G-CSF administration during steady state. On the other hand, CD34+ cells harvested post-chemotherapy contain a smaller proportion of more primitive progenitor cells (CD34+/HLA-DR- or CD34+/CD38-) compared with G-CSF treatment alone. Independent of the mobilization modality, the amount of previous cytotoxic chemo- and radiotherapy adversely affects the yield of hematopoietic progenitor cells. While continuous subcutaneous administration of G-CSF between 5 and 16 micrograms/kg bodyweight is preferred, additional dose-finding studies may be helpful to optimize current dose schedules. Adhesion molecules like L-selectin, VLA (very late antigen)-4 and LFA (leukocyte function antigen)-1 are likely to play a role in mobilization, since these antigens are expressed on CD34+ cells from bone marrow in different densities compared with blood-derived CD34+ cells collected following G-CSF-supported cytotoxic chemotherapy. It is also relevant for transplantation that during G-CSF-enhanced recovery post-chemotherapy, peripheral blood is enriched with a greater proportion of CD34+ cells expressing Thy-1 in comparison with CD34+ cells from bone marrow samples obtained on the same day or before the mobilization therapy was started. The early nature of the CD34+/Thy-1+ cells is very likely since this phenotype has been found on stem cells from human fetal liver and bone marrow and on cord blood cells. As a result, G-CSF-mobilized blood stem cells provide rapid and sustained engraftment following high-dose therapy, including myeloablative regimens. Positive selection of CD34+ cells as well as ex vivo expansion using different cytokines are currently being investigated for purging and improvement of short-term recovery post-transplantation. Future developments include the use of blood-derived hematopoietic stem cells for somatic gene therapy. The availability of growth factors has been an important prerequisite for the development of these new avenues for cell therapy.     

Mifepristone modulation of ACTH and CRH regulation of bovine adrenocorticosteroidogenesis in vitro

In this article, we review neoplastic contamination in the peripheral blood (PB) of patients with multiple myeloma (MM) upon stem cell mobilization. We first evaluated PB samples from pretreated MM patients following administration of high-dose cyclophosphamide (Cy, 7 g/m2 or 4 g/m2) and granulocyte colony-stimulating factor (G-CSF) for the presence of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic immunoglobulin (cIg) were counted by immunofluorescence microscopy after incubation with appropriate antisera against light and heavy chain Ig. Flow cytometry studies were performed to determine the presence of malignant B lineage elements, using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Prior to PBSC mobilization, circulating plasma cells were detected in all MM patients at 0.1%-1.8% of the mononuclear cell (MNC) fraction (mean value 0.7 +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after administration of chemotherapy and G-CSF. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors, with the peak coinciding with the optimal period for the collection of PBSC. The absolute number of plasma cells showed a 10-50-fold increase over the baseline value. Apheresis products contained 0.7 +/- 0.2% SD myeloma cells (range 0.2%-2.7%), which demonstrated the capacity of plasma cells to proliferate, differentiate, and mature in response to c-kit ligand (SCF), IL-3, IL-6, and a combination of IL-3 and IL-6. Subsequently, in an attempt to reduce tumor cell contamination prior to autologous transplantation, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute bone marrow (BM) function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51%-94%), with a 75-fold enrichment compared with the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33%-95%) and 45% (range 7%-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log depletion of plasma cells and CD 19+ B lineage cells as determined by immunofluorescence studies, although DNA analysis of the CDR III region of the IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high-dose melphalan (140 mg/m2) or melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis. The median time to 0.5 x 10(9) neutrophils, 20 x 10(9) and 50 x 10(9) platelets/L of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. Our data demonstrate the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring normal hematopoiesis following a TBI-containing conditioning regimen.     

The remission status before and the PCR status after high-dose therapy with peripheral blood stem cell support are prognostic factors for relapse-free survival in patients with follicular non-Hodgkin's lymphoma

It was the aim of our study to examine the clinical significance of t(14;18)-positive cells in samples from 47 patients with follicular non-Hodgkin's lymphoma (NHL) who underwent high-dose therapy with autologous peripheral blood stem cell (PBSC) transplantation. At the time of PBSC mobilization, 25 patients were in first remission, while 22 patients had a history of previous treatment failure. At the same time, 43 patients had polymerase chain reaction (PCR)-positive cells in samples from bone marrow (BM) and/or peripheral blood (PB). Independent of the remission status, high-dose cytarabine and mitoxantrone with granulocyte colony-stimulating factor (G-CSF) support were administered for PBSC mobilization. Following high-dose conditioning therapy which consisted of cyclophosphamide (200 mg/kg) and hyperfractionated total body irradiation (TBI, 14.4 Gy) or BEAM (carmustine, etoposide, cytarabine, melphalan), 34 patients received PCR-positive and 13 patients received PCR-negative autografts. After a median follow-up time of 20 months (range, 6-50) post-transplantation, 33 patients were in remission, while 14 patients had relapsed after a median time of 14.5 months (range, 10-42). Using the Andersen-Gill proportional hazards regression model for the analysis of relapse-free survival, we found that PCR-positive findings in samples from BM and/or PB at any given time-point after transplantation were associated with an increased estimated hazard ratio of 4.5 in comparison with a PCR-negative finding (P=0.013). On the other hand, patients included while they were in first remission had a smaller estimated hazard ratio of 0.3 when compared with patients with a history of previous treatment failure (P=0.048). For the latter group of patients, this translates into a significantly smaller probability of relapse-free survival in comparison to patients who were in first remission at the time of PBSC-mobilization (P=0.012). In conclusion, the remission status of the patients before autografting and the PCR status as assessed on the occasion of follow-up examinations are significant prognostic parameters for relapse-free survival in patients with follicular lymphoma undergoing high-dose therapy with PBSC autografting.     

What''s new in syndactyly?

The collection of peripheral blood stem cells (PBSC) is a crucial step for successful PBSC transplantation. Routine hematological parameters utilized for predicting the optimal timing of collection include white blood cell (WBC) counts, high fluorescence ratio (HFR) of reticulocytes, and platelet counts. We compared these parameters with the CD34-positive rates in the peripheral blood. In regimen with high-dose chemotherapy where the WBC count at nadir was lower than 1,000/microliter, we found that the maximum mobilization of PBSC was observed on the day when the WBC count reached 10,000/microliter. This coincidence was within about one day (mean 0.44, standard deviation 0.53). However, the reliability of the WBC count as a marker of PBSC mobilization varied among different harvest regimens. In the regimen with regular-dose chemotherapy where the WBC count at nadir was above 1,000/microliter, we could not find such a tight coincidence between the WBC count and PBSC mobilization. These results suggested that, in some situations, the measurement of the CD34-positive rate is mandatory for an efficient PBSC collection. We also found that the number of CD34-positive cells in the peripheral blood correlated (x) well to the amount of the CD34-positive cells actually harvested (y) (y = 0.524x + 0.249, r = 0.787). Thus, rapid fluorescence activated cell sorter (FACS) analysis of peripheral CD34-positive rates seemed to be extremely useful to predict the yield of PBSC collection.     

Triploidy: antenatal sonographic features with post-mortem correlation

The CD34 antigen is expressed by human hematopoietic progenitor and stem cells. These cells are capable of reconstituting marrow function after marrow-ablative chemo-radiotherapy. Several different technologies have been developed for the separation of CD34+ cells from bone marrow or peripheral blood stem cell (PBSC) components. We used an immunomagnetic separation technique to enrich CD34+ cells from PBSC components in anticipation of autologous transplantation for patients with B lymphoid malignancies. Twenty-nine patients enrolled on this study and received mobilization chemotherapy followed by G-CSF. Of these, 21 achieved a peripheral blood CD34+ cell level of at least 2.0 x 10(4)/l required by protocol for separation of the stem cell components. A median of three components per patient was collected for processing. The average CD34+ cell concentration in the components after apheresis was 1.0 +/- 1.2%. After the CD34+ cell selection, the enriched components contained 0.6 +/- 0.6% of the starting nucleated cells. The recovery of CD34+ cells, however, averaged 58.4 +/- 19.2% of the starting cell number, with a purity of 90.8 +/- 6.5%. Overall depletion of CD34- cells was 99.96 +/- 0.06%. Nineteen patients were treated with marrow-ablative conditioning regimens and received an average of 6.2 +/- 2.0 x 10(6) CD34+ cells/kg body weight. These patients recovered to an ANC >0.5 x 10(9)/l at a median of 11 days (range 8-14), and platelet transfusion independence at a median of 9 days (range 5-13). Four patients died of transplant-related complications or relapse before 100 days after transplantation. No patient required infusion of unseparated cells because of failure of sustained bone marrow function. These data demonstrate that peripheral blood-derived CD34+ cells enriched by use of an immunomagnetic separation technique are capable of rapid engraftment after autologous transplantation.     

Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse as a model system to study the engraftment and mobilization of human peripheral blood stem cells

Mobilized CD34(+) cells from human peripheral blood (PB) are increasingly used for hematopoietic stem-cell transplantation. However, the mechanisms involved in the mobilization of human hematopoietic stem and progenitor cells are largely unknown. To study the mobilization of human progenitor cells in an experimental animal model in response to different treatment regimens, we injected intravenously a total of 92 immunodeficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with various numbers of granulocyte colony-stimulating factor (G-CSF) -mobilized CD34(+) PB cells (ranging from 2 to 50 x 10(6) cells per animal). Engraftment of human cells was detectable for up to 6.5 months after transplantation and, depending on the number of cells injected, reached as high as 96% in the bone marrow (BM), displaying an organ-specific maturation pattern of T- and B-lymphoid and myeloid cells. Among the different mobilization regimens tested, human clonogenic cells could be mobilized from the BM into the PB (P = .019) with a high or low dose of human G-CSF, alone or in combination with human stem-cell factor (SCF), with an average increase of 4.6-fold over control. Therefore, xenotransplantation of human cells in NOD/SCID mice will provide a basis to further study the mechanisms of mobilization and the biology of the mobilized primitive human hematopoietic cell.     

Antimicrobial substances produced by Staphylococcus aureus strains isolated from cattle in Brazil

Many studies have documented faster engraftment after transplantation with peripheral blood stem cells (PBSC) compared to bone marrow (BM) stem cells. Most comparisons, however, have been between unprimed BM and primed PBSC. We have collected engraftment data on 39 patients from 4 Danish centres and compared G-CSF primed BM with G-CSF primed PBSC in malignant lymphoma and solid tumours. In the lymphoma group 6 BM transplants were compared with 8 PBSC transplants, whereas in the testicular cancer group 16 BM transplants were compared with 9 PBSC transplants. In the lymphoma group, the time to platelet engraftment (platelets >20x10(9)/l unsupported) was median 15 d in PBSC transplants and median 34 d in BM transplants (p=0.003). In the solid tumour patients the difference in time to platelet engraftment was 11 and 18 d in PBSC and BM transplants, respectively (p<0.0001). In an attempt to explain this difference we performed CD34+ subset analysis of BM and PBSC. This analysis revealed a higher content of lineage restricted cells (CD34+CD61+ and CD34+GlyA+) in PBSC compared to BM. In conclusion, G-CSF mobilized PBSC seems to result in faster engraftment than G-CSF primed BM, which could be explained by an increased number of lineage specific progenitors in PBSC compared to BM.     

High-dose granulocyte-colony stimulating factor (G-CSF) in vitro induces the growth of high proliferative potential colony forming cells (HPP-CFC) in patients undergoing blood stem cell mobilization

We evaluated the role of high-dose granulocyte colony stimulating factor (G-CSF) in vitro, in inducing the generation of high-proliferative potential colony forming cells (HPP-CFC), from either mononuclear cells or purified CD34+ cells. Both normal controls and patients undergoing peripheral blood stem cell (PBSC) mobilization and transplantation were studied. In serum-driven agar cultures, G-CSF stimulated the proliferation of HPP-CFC in a dose dependent manner (r = 0.92). The number of HPP-CFC was four-fold greater in mobilized patients than in normal controls. Purified CD34+ cells yielded 11-fold more colonies than mononuclear cells. HPP-CFC from mobilized patients showed replating capacity, giving rise to secondary colonies of more mature appearance. In serum-free cultures, the effect of G-CSF appeared to be mediated by synergistic interaction with stem cell factor. Our results suggest that G-CSF stimulates primitive hematopoietic cells that are detectable in increased amounts in patients receiving mobilization therapy. Therefore, determination of G-CSF induced HPP-CFC could be a useful tool in the evaluation of mobilization strategies.     

Immune reconstitution after autologous progenitor hemopoietic cell transplantation. A study comparing autologous bone marrow and autologous peripheral blood transplantation

BACKGROUND: In order to find out the effect of peripheral blood (PB) hematopoietic progenitor cells on immune reconstitution the present study compares, through a randomized trial, some lymphoid subsets after peripheral blood (PBT) or bone marrow (BMT) autologous transplantation. MATERIAL AND METHODS: Twelve patients suffering from malignant hematological disorders were included (6 BMT and 6 PBT). From these patients 14 lymphoid and natural killer (NK) subsets were sequentially analyzed using appropriate dual staining. NK activity was analyzed by measuring Cr51 release from the K562 cell line. Studies were done in days and -6, +10, +17, +24, +31, +38, +52, +66, +90, +120, +180 and +360 after transplantation. RESULTS: The CD8+ cell regeneration was produced mainly by activated cells (CD38+), and no differences were observed between BMT and PBT, but CD8+ HLADR+ cells were higher in the PBT group. During the first year after transplantation CD4+ lymphoid cells were never within normal range, and its recovery was due to the memory subset (CD4+/CD45RO+). The CD19+ lymphocytes began their regeneration after the first month and it was produced mainly by by the CD19+/CD5+ subset. NK cells recovered faster in patients who underwent PBT, but NK activity was similar in both subgroups of patients and it was within normal range from day +17 until the end of the study. CONCLUSION: T, B and NK lymphoid reconstitution do not differ significantly between patients that receive BM or PB as hemathopoietic rescue, but PB seems influence a faster reconstitution of cytotoxic subsets (CD8+/HLADR+ and NK lymphoid cells).     

Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function

Despite the wide use of mobilized peripheral blood (PB) progenitor cells (PBPC) for clinical transplantation the mechanism(s) underlying their mobilization and subsequent engraftment are still unknown. We compared the adhesive phenotype of CD34(+) colony-forming cells (CFC) in bone marrow (BM) and PB of normal donors before and after administration of granulocyte colony-stimulating factor (G-CSF) for 5 d. G-CSF-mobilized PB CFC cells adhered significantly less to BM stroma, fibronectin, and to the alpha4 beta1 binding fibronectin peptide, CS1, because of decreased expression of the alpha4 integrin. Since incubation of BM CD34(+) cells for 4 d with G-CSF at concentrations found in serum of G-CSF- treated individuals did not affect alpha4-dependent adhesion, G-CSF may not be directly responsible for the decreased alpha4-mediated adhesion of PB CFC. Culture of G-CSF-mobilized PB CD34(+) cells with cytokines at concentrations found in BM stromal cultures upregulated alpha4 expression and restored adhesion of mobilized PB CFC to stroma, fibronectin, and CS1. Adhesion of cultured, mobilized PB CFC to stroma and CS1 could not be further upregulated by the beta1 activating antibody, 8A2. This indicates acquisition of a maximally activated alpha4 beta1 integrin once PB CFC have been removed from the in vivo mobilizing milieu. Thus, decreased alpha4 expression on CD34(+) CFC in PB may be responsible for the aberrant circulation of mobilized PB CD34(+) cells. Reexpression of a maximally activated alpha4 beta1 integrin on mobilized PB CFC removed from the mobilizing in vivo milieu may contribute to the early engraftment of mobilized PBPC.     

Effect of a vitamin D3 analog, EB1089, on hematopoietic stem cells from normal and myeloid leukemic blasts

The seco-steroid 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induces differentiation and inhibits clonal proliferation of HL-60 cells. We analyzed the effect of a novel vitamin D3 analog, EB1089, on normal myeloid and leukemic cells as well as CD34+ cells. EB1089 showed an extraordinary inhibition of clonal growth of HL-60 cells (ED50 = 5 x 10(-11) M) and AML blast cells (ED50 = 9 x 10(-10) M) compared to 1,25(OH)2D3 without suppression of growth of normal human bone marrow CFU-GM. The CD34+ cells from acute myeloid leukemia (AML) blasts were inhibited in a dose-dependent fashion by 1,25(OH)2D3 with an ED50 of 1.2 x 10(-9) M; and even more strikingly, 10(-10) M of EB1089 inhibited all clonal growth of human CD34+ leukemic colony-forming cells. In contrast, both EB1089 and 1,25(OH)2D3 (10(-8) M) showed little or only mild inhibition of CD34+ clongenic hematopoietic cells from normal human peripheral blood (PB); and in liquid culture, EB1089 stimulated the proliferation of normal human CD34+ cells about 2.5 times as compared to control cultures. In order to evaluate the potential use of EB1089 for purging leukemic cells from normal CD34+ progenitor cells for PB stem cell transplantation (PBSCT), normal human PB mononuclear cells (PBMNC) were contaminated with HL-60 cells, and then CD34+ cells purified and treated with EB1089. We found that CD34+ purification and EB1089 purging was able to eliminate approximately 100% of HL-60 leukemic cells with no toxicity to normal CD34+ hematopoietic progenitor cells. These data suggested that purification of CD34+ cells and ex vivo treatment with EB1089 might provide an effective therapeutic approach for PBSCT.     

Mobilization of peripheral blood stem cells in children using G-CSF after carboplatin containing myelosuppressive chemotherapy

PURPOSE: Peripheral blood stem cell (PBSC) apheresis provides an alternative to autologous marrow harvest as a source of hematologic stem cells for transplantation in children with solid tumors. PATIENTS AND METHODS: Eight children with metastatic or recurrent solid tumors underwent 27 apheresis procedures. Recovery from myelosuppressive chemotherapy occurred without continuous daily growth factor support prior to mobilization. Granulocyte colony stimulating factor (G-CSF) at 16 microgs/kg/day was used to increase stem cells in the peripheral circulation. CD 34 positive cells, mononuclear cells (MNC), and CFU-GM were measured in the apheresis products. Prior chemotherapy was examined as a clinical factor that affected PBSC yield. RESULTS: A significant correlation was found between CD 34+/kg and CFU-GM/kg of the products (r = 0.758, P < 0.001). Patients receiving cumulative doses of carboplatin over 1,600 mg/m2 produced adequate MNC (1 x 10(8)/kg) but yielded significantly less CD 34+ cells or CFU-GM than those patients receiving less carboplatin. Prior doses of etoposide and ifosfamide did not effect PBSC yield. CONCLUSIONS: The mobilization technique was well tolerated, and the products obtained produced trilineage engraftment in the patients that underwent peripheral blood stem cell transplantation. Peripheral blood stem cell apheresis in children can be optimized by selection of appropriate candidates and mobilization with G-CSF after an absence of hematopoietic growth factor support.     

Changes of immunological markers after autologous peripheral blood stem cell transplantation

PURPOSE: Recently high-dose chemotherapy with peripheral blood stem cell transplantation (PBSCT) has become an important treatment for hematological and solid tumors. METHODS: Immunological parameters were examined before and after PBSCT in 9 patients with lung cancer and 13 patients with malignant lymphoma. Findings were compared with those for bone marrow transplantation (BMT). Peripheral blood cells were analyzed for phenotype and the levels of cytokines and soluble factors were measured. RESULTS: After PBSCT, activated T cells (CD3+HLA-DR+ cells, CD8+HLA-DR+ cells) and suppressor/cytotoxic T cells (CD8+CD11b- cells) were significantly higher in the patients with lung cancer than in those with malignant lymphoma. Serum levels of interleukin-4 and soluble interleukin-2 receptor were also significantly higher in the patients with lung cancer than in those with lymphoma. On the other hand, the serum levels of interferon gamma, tumor necrosis factor alpha, interleukin-6, soluble human leukocyte antigen class 1, and soluble thrombomodulin were significantly increased after bone marrow transplantation. The transfused peripheral stem cells of lung cancer and lymphoma patients had a similar number of granulocyte/macrophage-colony-forming units, but lung cancer patients had significantly more CD34-positive cells. CONCLUSION: By reinfusing large numbers of autologous immune cells, PBSCT may accelerate immune reconstitution, with T cells being likely to have a marked therapeutic potential. The changes after PBSCT were greater in patients with lung cancer than in lymphoma patients. These blood cells are potent mediators of anticancer activity and could play an important role in the elimination of autologous malignant cells.     

Dendritic cell-based vaccines in the setting of peripheral blood stem cell transplantation: CD34+ cell-depleted mobilized peripheral blood can serve as a source of potent dendritic cells

We are investigating the use of tumor-pulsed dendritic cell (DC)-based vaccines in the treatment of patients with advanced cancer. In the current study, we evaluated the feasibility of obtaining both CD34+ hematopoietic stem/ progenitor cells (HSCs) and functional DCs from the same leukapheresis collection in adequate numbers for both peripheral blood stem cell transplantation (PBSCT) and immunization purposes, respectively. Leukapheresis collections of mobilized peripheral blood mononuclear cells (PBMCs) were obtained from normal donors receiving granulocyte colony-stimulating factor (G-CSF) (for allogeneic PBSCT) and from intermediate grade non-Hodgkin's lymphoma or multiple myeloma patients receiving cyclophosphamide plus G-CSF (for autologous PBSCT). High enrichment of CD34+ HSCs was obtained using an immunomagnetic bead cell separation device. After separation, the negative fraction of mobilized PBMCs from normal donors and cancer patients contained undetectable levels of CD34+ HSCs by flow cytometry. This fraction of cells was then subjected to plastic adherence, and the adherent cells were cultured for 7 days in GM-CSF (100 ng/ml) and interleukin 4 (50 ng/ml) followed by an additional 7 days in GM-CSF, interleukin 4, and tumor necrosis factor alpha (10 ng/ml) to generate DCs. Harvested DCs represented yields of 4.1+/-1.4 and 5.8+/-5.4% of the initial cells plated from the CD34+ cell-depleted mobilized PBMCs of normal donors and cancer patients, respectively, and displayed a high level expression of CD80, CD86, HLA-DR, and CD11c but not CD14. This phenotypic profile was similar to that of DCs derived from non-CD34+ cell-depleted mobilized PBMCs. DCs generated from CD34+ cell-depleted mobilized PBMCs elicited potent antitetanus as well as primary allogeneic T-cell proliferative responses in vitro, which were equivalent to DCs derived from non-CD34+ cell-depleted mobilized PBMCs. Collectively, these results demonstrate the feasibility of obtaining both DCs and CD34+ HSCs from the same leukapheresis collection from G-CSF-primed normal donors and cancer patients in sufficient numbers for the purpose of combined PBSCT and immunization strategies.