ISSN:1061-6128    

DOI:10.1089/scd.1.1997.6.1

年代:1997

数据来源: MAL

摘要:

ABSTRACTThe Amgen Cell Selection Device (ACSD) is a fully automated system based on the research scale magnetic-activated cell separation (MACS) system (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) for the selection of CD34+ cells. Leukapheresis products (LP) (n= 30) from normal donors mobilized with recombinant human granulocyte colony-stimulating factor (rhG-CSF) were selected with the ACSD to evaluate the performance of this system. The starting LP contained a median of 0.51% CD34+ cells (range 0.21%–1.54%) and a median WBC count of 3.0 × 1010(range 1–4.7 × 1010 cells). After selection on the ACSD a mean purity of 91.5% ± 0.6% CD34+ cells was obtained, with a median purity of 95.5% CD34+ cells. A median of 98 × 106total CD34+ cells were recovered postselection, with a range of 31–323 × 106cells collected from the LP. This represented a mean recovery of 81.7% ± 6% of CD34+ cells and a median of 78% compared with starting CD34+ cell numbers in the LP. FACS analysis of the selected products demonstrated a 4–5 log depletion of T cell subsets, including CD3, CD4, CD8, and CD56 subsets. These data demonstrate the high performance obtained with the ACSD resulting in a final product of greater than 90% purity of CD34+ cells. CD34+ cells selected with the ACSD represent an ideal product for clinical applications, such as tumor cell purging, T cell depletion for allogeneic transplant, ex vivo expansion, and

ISSN:1061-6128    

DOI:10.1089/scd.1.1997.6.5

年代:1997

数据来源: MAL

摘要:

ABSTRACTThe value of daily monitoring of the blood CD34+ cell concentration as a guide to the optimal timing of stem cell harvests was studied in 60 patients who underwent 66 stem cell mobilizations and 189 leukaphereses. There was a highly significant correlation between the blood CD34+ count and the CD34+ cell content in the apheresis product of the same day (r = 0.904,p<0.01). Thus, the target yield of 4 × 106CD34+ cells/kg can be harvested in one or two leukaphereses when the blood CD34+ cell count exceeds 50 × 106/L. However, an insufficient harvest is to be expected when the blood CD34+ cell count is below 20 × 106/L. The data from 35 autologous blood cell transplantations with a minimum CD34+ cell yield of 1.5 × 106/kg showed that the recovery of blood neutrophil counts to 1.0 × 109/L occurred in all patients within 9–14 days, but the time to recovery of the platelet counts to 20 × 109/L may exceed 14 days, especially if the CD34+ cell content is below 4 × 106/kg. Daily monitoring of blood CD34+ cell counts is a rapid and reliable means to guide the timing of stem cell collections. The count predicts well the CD34+ cell content of the harvests, the number of leukaphereses needed, and the speed of hematopoietic

ISSN:1061-6128    

DOI:10.1089/scd.1.1997.6.13

年代:1997

数据来源: MAL

摘要:

ABSTRACTThe evaluation of contaminating breast cancer cells in hematopoietic grafts is of considerable importance for monitoring the efficiency of purging procedures. We report a comparison of three systems for the in vitro detection and enumeration of metastatic breast cancer cells. Breast cancer cells from established cell lines were mixed with Daudi cells at dilutions ranging from 1:10 to 1:1,000,000, and a predetermined number were fixed in defined areas on microscope slides coated with one of the following attachment factors: (i) Cell-Tak® Cell and Tissue Adhesive, (ii) 0.1% solution of Poly-L-Lysine, or (iii) Cel-Line HTC Super Cured® slides. We employed a specificity-proven pancytokeratin antibody (A45-B/B3) and the alkaline phosphatase-antialkaline phosphatase (APAAP) staining technique. In multiple experiments, one breast cancer cell in 1,000,000 Daudi cells could reliably be detected in the Cell-Tak and Cel-Line systems and 1 in 100,000 with the Poly-L-Lysine system. The observed number of seeded cells showed a highly significant correlation with the number of cells seeded (p<0.0001 in all cases). Finally, we used the Cell-Tak method to evaluate clinical material from various sources: from patients with primary carcinomas of the breast, prechemotherapy, and during various chemotherapeutic regimens, as well as from patients with metastatic disease. The system consistently detected tumor cells in bone marrow samples from these patients. All peripheral blood samples from patients with metastatic disease tested positive at incidences ranging from 5 to 19/106peripheral blood mononuclear cells. This is a simple and reliable technique that allows rapid screening of large cell numbers with high resolution of positive cell

ISSN:1061-6128    

DOI:10.1089/scd.1.1997.6.21

年代:1997

数据来源: MAL

摘要:

ABSTRACTMerocyanine 540 (MC540)-mediated photodynamic therapy (PDT) inactivates experimental leukemia, lymphoma, and neuroblastoma cells by a singlet oxygen-mediated mechanism but is relatively well tolerated by normal pluripotent hematopoietic stem cells and granulocyte/macrophage progenitors (CFU-GM). MC540 is currently undergoing phase I clinical testing for the extracorporeal purging of autologous bone marrow and peripheral blood stem cells. We report here that performing MC540-mediated PDT at 4.7°C (hypothermia) instead of at ambient temperature enhanced the photoinactivation of L1210 cells and CFU-GM but left the photoinactivation of K562 cells unchanged. Hypothermia reduced dye binding in K562 but not in L1210 cells, whereas the photogeneration of lipid hydroperoxides (LOOH) was affected in neither cell line. Post-PDT incubation at 4°C delayed the decay of LOOH and enhanced the photoinactivation of CFU-GM as well as L1210 and K562 cells. Taken together, these results suggest that hypothermia interfered with the repair of potentially lethal photodynamic damage. They stress the importance of temperature control during and immediately after the photochemical purging of autologous bone marrow and peripheral blood stem cell

ISSN:1061-6128    

DOI:10.1089/scd.1.1997.6.31

年代:1997

数据来源: MAL

摘要:

ABSTRACTThe genetic modification of tumor cells to secrete immune regulatory molecules can elicit a potent antitumor immune response. Bacterial superantigens are among the most potent T cell mitogens. Activation of tumor-sensitized T cells by bacterial superantigens can lead to immune effector cells with potent and specific in vivo antitumor activity. Retrovirus vectors encoding for the bacterial superantigens SEA and SEC2 were constructed, and recombinant retrovirus stocks were generated. SEA and SEC2 could be detected in the culture supernatant of tumor cells after a single exposure to retrovirus. Molecular analysis of the genetically modified cells revealed intact proviral DNA and abundant vector-derived superantigen RNA. Biologic activity was apparent for both superantigens. Secretion of biologically active superantigen by mammalian cells has not been reported previously, and this will enable investigating the potential for superantigen gene therapy.

ISSN:1061-6128    

DOI:10.1089/scd.1.1997.6.41

年代:1997

数据来源: MAL

摘要:

ABSTRACTIsolation of CD34+ cells from bone marrow, umbilical cord blood, and mobilized peripheral blood stem cell (PBSC) collections has many potential clinical benefits. The aim of this study was to evaluate the use of the ISOLEX 300 system to select hematopoietic precursors and determine the effectiveness at depleting contaminating tumor cells from cryopreserved/thawed PBSC. Median recovery of CD34+ cells and CFU-GM colonies was 71% and 51.5%, respectively, using a protocol optimized for our laboratory. A mean 2.9 log10 decrease in contaminating breast carcinoma cells was seen after the selection process. Selected CD34+ cells underwent a second round of cryopreservation/thawing while retaining 85.6% viability and 72.3% recovery of CFU-GM colonies.

ISSN:1061-6128    

DOI:10.1089/scd.1.1997.6.53

年代:1997

数据来源: MAL

摘要:

ABSTRACTThe use of CFU-GM and CD34+ cell enumeration for assessing harvest quality and factors affecting peripheral blood progenitor cell (PBPC) harvest and engraftment were investigated in 45 women with high-risk and metastatic breast cancer scheduled for dose-intensive cyclophosphamide, thiotepa, and carboplatin (CTCb). PBPC were mobilized with standard breast cancer regimens or cyclophosphamide (1.5 g/m2) and 5 μg/kg/day G-CSF and used together with G-CSF for hematopoietic support post-CTCb. There was a significant correlation between peripheral blood CD34+ cells/μl and harvest CD34+/kg (r = 0.73,p<0.0001) and between harvest CFU-GM and CD34+ cells/kg (r = 0.5,p<0.0001). CFU-GM clonogenic assays were of no clinical use beyond that of CD34+ cell enumeration, with the latter allowing for real-time decisions regarding harvesting. Multiple stepwise regression identified the number of prior chemotherapy cycles as the only significant clinical predictor of CD34+ cell yield. For 34 patients proceeding to CTCb with PBPC support, multiple stepwise regression identified as the best predictors for engraftment CFU-GM and CD34+ cells/kg for neutrophils and CFU-GM, CD34+ cells/kg, and the number of prior cycles of chemotherapy for platelets, respectively. A threshold dose of 1 × 106CD34+ cells/kg, obtained in 87% of these heavily pretreated breast cancer patients, was adequate to ensure engraftment within 15 days. There was no significant difference in length of hospital stay or blood product use between patients receiving 1–2.5 × 106CD34+ cells/kg and greater than 2.5 × 106CD34+ cells/kg, although median time to engraftment of neutrophils (9 days versus 8 days,p= 0.007) and platelets (12 days versus 9 days,p= 0.006) was significantly longer. The established threshold of ≥1 × 106CD34+ cells/kg will allow for more confident consideration of using aliquots of this threshold dose for hematopoietic support in sequential high-dose regimens inclusiv

ISSN:1061-6128    

DOI:10.1089/scd.1.1997.6.61

年代:1997

数据来源: MAL

摘要:

ABSTRACTHuman CD34+ cells purified from frozen mobilized peripheral blood apheresis products (n= 7) were studied immediately (freshly isolated) or refrozen and studied after>30 days storage in liquid nitrogen (refrozen/thawed). The proliferation and differentiation of freshly isolated or refrozen/thawed CD34+ cells were examined after 10 days of serum-supplemented suspension culture with recombinant human hematopoietic growth factors. The proliferative capacity (fold increase) of the refrozen/thawed CD34+ cells (mean ± SD, 54.3 ± 34.3) was comparable to the freshly isolated CD34+ cell cultures (49.0 ± 42.4). Two-color flow cytometry of the CD34+ cultured cell populations, fresh and refrozen/thawed, displayed typical patterns of neutrophil differentiation into CD15/CDllb neutrophil precursors. The colony-forming ability of freshly isolated and refrozen/thawed CD34+ cells showed no significant differences (p>0.05) in the total number or type of colony-forming units (CFU-GM, CFU-M, BFU-E, CFU-GEMM) obtained. In addition, the cloning efficiencies of freshly isolated (19.5 ± 7.6%) and refrozen/thawed CD34+ cells (21.9 ± 12.7%) were comparable (p= 0.366). These data suggest that CD34+ cells enriched from frozen apheresis blood products can be either used immediately or stored in liquid nitrogen and thawed with minimal effect on their ability to proliferate and differentiate in liquid cul

ISSN:1061-6128    

DOI:10.1089/scd.1.1997.6.69

年代:1997

数据来源: MAL

ISSN:1061-6128    

DOI:10.1089/scd.1.1997.6.77

年代:1997

数据来源: MAL