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Query: UMLS:C0432222 (SEM)
 47,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enriched progenitor cell fractions from human bone marrow were induced to undergo myeloid maturation in culture using recombinant human interleukin-3 (rhIL-3) and granulocyte-macrophage colony stimulating factor (rhGM-CSF). A negative selection method using the murine monoclonal antibodies (MABs) PM81 (anti-CD15), AML-2-23 (anti-CD14), PC251 (anti-CD33), OKT11 (anti-CD2), and SCCL-1 (anti-CD71) and immunomagnetic beads coated with sheep anti-mouse IgG (Dynal A.S., Oslo, Norway) was used to remove the more mature cellular components of mononuclear cells from normal donor bone marrow samples. The resulting fraction of cells contained 35 to 40% CD34-positive cells, and less than 1% of cells expressed the receptors for the constant portion of immunoglobulin G Fc gamma RI or Fc gamma RII. A small population (3-5%) expressed Fc gamma RIII on day 0, and these cells were found by two-color flow cytometry to be primarily natural killer (NK) cells. The level of Fc gamma R expression was determined every 2 to 3 days on aliquots of the differentiating cells. Thirteen percent of the cultured bone marrow cells expressed Fc gamma RII after 48 hours in liquid culture with rhIL-3 and rhGM-CSF. The percent of cells expressing Fc gamma RII increased to a peak of 78% of the gated population on day 10. The mean fluorescence intensity (MFI) remained low for the first 8 to 10 days of culture, but at that time the MFI more than doubled. Fc gamma RI and Fc gamma RIII expression remained low throughout the culture period. The ability of the differentiating cells to perform antibody-dependent cellular cytotoxicity (ADCC) was determined at a single-cell level in a modified plaque assay using monolayers of ox erythrocyte (oxE) target cells. The purified progenitor cells, when placed in oxE monolayers sensitized with polyclonal rabbit anti-oxE antibody (AB), showed no plaque formation over control oxE layers. No increase in ability to generate cytolytic plaques in antibody-sensitized oxE layers was seen compared with unsensitized oxE layers until after 10 days of incubation in liquid culture. At that time, the percent of cells forming plaques in the AB-sensitized oxE layers was 34.4 +/- 10.7% (average +/- standard error of the mean [SEM]; n = 4) compared with 10.0 +/- 0.7% on the control oxE layers. The peak plaque formation appeared to coincide with the increase in MFI of a large population of the cultured cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The effect of recombinant human interleukin-3 and recombinant human granulocyte-macrophage colony-stimulating factor on Fc gamma receptor expression and antibody-dependent cellular cytotoxicity of hematopoietic progenitor cells during in vitro myeloid maturation. 750 91

It was the objective of the study to characterize CD34+ hematopoietic progenitor cells from peripheral blood (PB) and bone marrow (BM) in a group of 24 cancer patients. After cytotoxic chemotherapy, R-metHu granulocyte colony-stimulating factor (R-metHuG-CSF; filgrastim, 300 micrograms daily, subcutaneously) was given to shorten the time of neutropenia as well as to increase the rebound of peripheral blood progenitor cells (PBPC) for harvesting. The proportion of CD34+ cells in the leukapheresis products (LPs) was 1.4-fold greater than in BM samples that were obtained at the same day (LP: median, 1.4% v BM: median, 1.0%, P < .01). Two- and three-color immunofluorescence showed that blood-derived CD34+ cells comprised a greater proportion of a particular early progenitor cell than CD34+ cells of bone marrow. Blood-derived progenitor cells tended to have a higher mean fluorescence intensity of CD34 and expressed significantly lower levels of HLA-DR (mean fluorescence intensity of HLA-DR: 442.6 +/- 44.9 [LP] v 661.5 +/- 64.6 [BM], mean +/- SEM, P < .01). Furthermore, the blood-derived CD34+ cells comprised a 1.7-fold greater proportion of Thy-1+ cells (LP: median, 24.4% v BM: median, 14.4%, P < .001) and expressed significantly less c-kit (LP: median, 20.5% v BM: median, 31.0%, P < .01). Three-color analysis showed that high levels of Thy-1 expression were restricted to CD34+/HLA-DRdim or CD34+/HLA-DR- cells confirming the early developmental stage of this progenitor cell subset. The proportion of CD34+/CD45RA(bright) cells representing late colony-forming unit granulocyte-macrophage (CFU-GM) was smaller in LPs compared with BM (P < .05). For an examination of BM CD34+ cells before the mobilization chemotherapy, samples of 16 patients were available. The mean proportion of c-kit expressing CD34+ cells in the bone marrow during G-CSF-stimulated reconstitution decreased 1.8-fold compared with baseline values. There was no difference in the proportion of BM-derived CD34+/Thy-1+ cells and CD34+/CD45RA+ cells between steady-state hematopoiesis and G-CSF-supported recovery. Our data suggest that during G-CSF-enhanced recovery, CD34+ cells in the PB are enriched with more primitive progenitor cells to evenly replenish the BM after the chemotherapy-related cytotoxic damage.
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PMID:Blood-derived autografts collected during granulocyte colony-stimulating factor-enhanced recovery are enriched with early Thy-1+ hematopoietic progenitor cells. 753 95

In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell-free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis. 753 90

The purpose of this study was to evaluate the antigenic profile of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) in patients with non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and multiple myeloma (MM). The mobilization regimens consisted of high-dose cytarabine/mitoxantrone for patients with NHL, DexaBEAM for patients with HD, and high-dose cyclophosphamide (4 or 7 g per m2) for patients with MM. Cytotoxic therapy was supported by recombinant human G-CSF (Filgrastim, 300 micrograms/day sc) to shorten the period of neutropenia and to increase the number of circulating hematopoietic progenitor cells. The mean numbers of circulating CD34+ cells/microliters during leukocyte recovery were different between patient groups, 80.5 +/- 9.8 (mean +/- SEM) in low-grade NHL and 51.2 +/- 9.7 in high-grade NHL compared with 31.3 +/- 6.9 in HD and 24.4 +/- 4.1 in patients with MM. As a result, the greatest numbers of CD34+ cells/kg collected per leukapheresis were observed in patients with NHL, whereas the collection efficiency was substantially lower in patients with HD or MM. Patients with MM had also the smallest proportion of CD34+ cells in the mononuclear cell fraction (mean 0.79 +/- 0.10% versus 2.15 +/- 0.19% in low-grade NHL) but the greatest proportion of early CD34+ HLA-DR- progenitor cells (mean 2.38 +/- 0.51 versus 0.84 +/- 14% in low-grade NHL). Patients with MM had a mean proportion of CD34/c-kit+ cells that was twofold greater than that observed in patients with high- or low-grade NHL.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of peripheral blood progenitor cells mobilized by cytotoxic chemotherapy and recombinant human granulocyte colony-stimulating factor. 753 8

Stroma-dependent long-term bone marrow cultures (LTBMC) assay the ability of primitive haematopoietic stem cells (HSC) for long-term production of clonable progenitors. We have developed a limiting dilution type LTBMC assay allowing frequency analysis of transiently repopulating HSC and long-term culture initiating cells (LTC-IC) without the necessity to replate large numbers of wells. Normal or 5-FU-treated Ficoll bone marrow cells (BMC), or BMCs sorted on CD34 or HLA-DR expression, or Rh123 retention, (input range 40-70,000 CFU-GM/BFU-E/10(5) cells) were plated at limiting dilution on unirradiated adherent layers formed by a novel murine preadipose cell line (FBMD-1). The percentage of wells with at least one phase-dark haematopoietic clone (cobblestone area, CA) beneath the stromal layer was weekly determined for at least 8 weeks, and CA-forming cell (CAFC) frequencies were calculated using Poisson statistics. Parallel LTBMCs of the same samples were weekly assessed for supernate CFU-GM/BFU-E production. Weekly addition of rhIL-3 with rhG-CSF supported a high average clonogenic output per CA and dramatically increased CA size, but did not significantly alter the apparent CAFC frequency. The generation of CFU-GM per CA was constant over a period of 6 weeks with weekly means of eight normal BM samples, ranging between 5-16. At week 6 the mean CAFC frequency was 29 (1 SEM, 8.8)/10(5). Early appearing CAFC were highly sensitive to 5-FU, and were contained over the full Rh123 and HLA-DR fluorescence profile of CD34pos cells, whereas CAFC week 5-8 were predominantly contained in the CD34pos Rh123dull HLA-DRlow fraction in agreement with previously reported LTC-IC characteristics. In conclusion, the CAFC assay enumerates LTC-IC using a direct visual endpoint and allows study of LTC-IC heterogeneity with respect to progenitor cell generation per stem cell clone in various haematologic diseases.
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PMID:Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay. 803 1

We investigated the effects of interleukin-4 (IL-4) on the survival of leukemic and normal B-cell progenitors cultured on bone marrow stroma. IL-4 (at 100 U/mL) was cytotoxic in 16 of 21 cases of B-lineage acute lymphoblastic leukemia, causing reductions in CD19+ cell numbers that ranged from 50% to greater than 99% (median 83.5%) of those in parallel cultures not exposed to the cytokine. All nine cases with the t(9;22)(q34;q11) or the t(4;11)(q21;q23), chromosomal features that are often associated with multidrug resistance and a fatal outcome, were susceptible to IL-4 toxicity. IL-4 cytotoxicity resulted from induction of programmed cell death (apoptosis); there was no evidence of cell killing mediated by T, natural killer, or stromal cells. IL-4 cytotoxicity extended to a proportion of normal B-cell progenitors. After 7 days of culture with IL-4 at 100 U/mL, fewer CD19+, CD34+ normal lymphoblasts (the most immature subset) survived: in five experiments the mean (+/- SEM) reduction in cell recoveries caused by IL-4 was 60.0% +/- 6.0%. By contrast, reductions in recovery of more differentiated bone marrow B cells (CD19+, CD34-, surface Ig+) were low (6.6% +/- 2.2%; P < .001 by t-test). Our findings indicate that IL-4 is cytotoxic for human B-cell precursors and support clinical testing of IL-4 in cases of high-risk lymphoblastic leukemia resistant to conventional therapy.
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PMID:Interleukin-4 induces programmed cell death (apoptosis) in cases of high-risk acute lymphoblastic leukemia. 814 40

The mechanisms that underlie progestogen-induced endometrial breakthrough bleeding are poorly understood. The aim of the present study was to quantify endometrial microvascular density in 54 controls and 42 women with 3-12 months' exposure to Norplant (levonorgestrel subdermal contraceptive implant) and to correlate it with bleeding pattern, endometrial histology, and peripheral plasma oestradiol and progesterone concentrations. Endometrial biopsies were processed routinely and sections immunostained using anti-CD34 antibody to identify vascular endothelial cells. Menstrual record card data were analysed using World Health Organization definitions. The mean microvascular density (+/- SEM) for control samples was 186 +/- 8 vessels/mm2, and there were no significant differences across the cycle. Norplant user's endometrial microvascular density was significantly elevated above controls (294 +/- 18 vessels/mm2, P = 3.36 x 10(-8)). Endometrial microvascular density in Norplant users did not correlate with oestrogen concentrations prior to biopsy, bleeding patterns or endometrial histology. The results from this study show that women receiving Norplant have significantly increased endometrial microvascular density compared to controls. Another finding from this study was that bleeding in Norplant users often occurred from thin atrophic endometrium. These results provide new insights into the physiological mechanisms that may be involved in progestogen-induced endometrial bleeding.
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PMID:Endometrial microvascular density during the normal menstrual cycle and following exposure to long-term levonorgestrel. 825 25

Transfer of the multidrug resistance-1 (MDR1) gene into hematopoietic progenitor cells may reduce myelotoxicity of MDR1-related cytotoxic agents and therefore allow dose intensification. Mobilized peripheral blood progenitor cells (PBPC) can be obtained in ample quantity and are a suitable target cell population. CD34-selected PBPC samples (n = 6) were transduced with cell-free supernatant (SNT) of a cell line producing recombinant retrovirus containing the human MDR1 gene. Limiting-dilution long-term cultures were employed that allow continuous monitoring of stroma-adherent cobblestone areas (CA) and comparison of their frequency in a 5-log range over time. MDR1 provirus integration in CA-containing wells followed single-hit kinetics. According to Poisson statistics, proviral DNA was contained in 22% of unselected cobblestone area-forming cells (CAFC) at week 6, which represent primitive hematopoietic precursors. In comparison, 1.0 +/- 0.44% (mean +/- SEM) of week-6 CAFC were expressing P-glycoprotein at sufficient levels to convey vincristine resistance, suggesting low expression of the retroviral vector or splicing of the vector-drived mRNA in hematopoietic progenitor cells. Next we analyzed lineage-committed progenitors. The proviral DNA was detectable in 20-66% of colony-forming units granulocyte-macrophage (CFU-GM) while corresponding percentages (25-52%) of CD34+ PBPC were in the S/G2M phase of the cell cycle at the end of the transduction period. The proportion of vincristine-resistant CFU-GM was similar to the CAFC data and no significant differences were found between various MDR1-SNT transduction schedules whereas MDR1 co-cultivation, which served as a positive control, yielded significantly higher proportions of resistant colonies (5.3 +/- 1.4%, IL-3, 96 hr, p < or = 0.05). Assessment of rhodamine-123 (Rh-123) efflux in the myelo-monocytic progeny of MDR1-transduced cells mirrored the colony assay results in the SNT and co-cultivation groups. Less culture effort was required in the Rh-123 assay and functional characterization of the transferred P-glycoprotein was possible using cyclosporin A. Further development toward an effective MDR1 gene therapy should be facilitated by the CAFC assay, which allows estimation of the retroviral gene transfer frequency into primitive hematopoietic cells, and by the Rh-123 assay, which permits tractable side-by-side assessments of numerous MDR1 transduction protocols or different MDR1-SNT lots.
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PMID:Frequency analysis of multidrug resistance-1 gene transfer into human primitive hematopoietic progenitor cells using the cobblestone area-forming cell assay and detection of vector-mediated P-glycoprotein expression by rhodamine-123. 879 46

High levels of expression of the bcl-2 oncoprotein in acute myeloblastic leukaemia (AML) cells have been associated with low complete remission rates and poor survival. The sensitivity of AML blasts to drugs such as Ara-C can be increased by the down-regulation of bcl-2 expression by antisense oligonucleotides. All-trans retinoic acid (ATRA) has been reported to increase the sensitivity of AML cell lines to Ara-C and to induce differentiation in the HL60 promyelocytic cell line, with both effects being accompanied by a decrease in bcl-2 expression. Using flow cytometry and a monoclonal antibody to bcl-2, we have investigated the effects of ATRA (1 microM) on bcl-2 expression in the blast cells of 25 AML patients and the K562 cell line after incubation for 72 or 24 h, respectively. Using Kolmogorov-Smirnov statistical analysis where a D value of > 0.12 was statistically significant, we found that in 8/25 AML samples and the K562 cells there was a significant decrease in bcl-2 protein expression after incubation with ATRA (D value range 0.14-0.44). The mean peak fluorescence (MPF) values for the bcl-2 levels of the ATRA responders (n = 8) was reduced to 35.5 +/- 6.9 following incubation with ATRA compared to 47.6 +/- 8.2 (mean +/- SEM) for control samples incubated in the absence of ATRA (P = 0.014). There was no significant difference between the baseline bcl-2 molecules of equivalent soluble fluorochrome (MESF) levels in the ATRA responders (48.9 +/- 5.7, n = 8) and the non-responders (41.3 +/- 3.9, n = 17) (mean +/- SEM) (P = 0.28). The down-regulation of bcl-2 expression by ATRA was particularly associated with CD34-negative AML and of the eight AML patients' cells that responded to ATRA by down-regulating bcl-2, seven were CD34 negative (P < 0.05). Our data suggest that the addition of ATRA to combination chemotherapy would increase the chemosensitivity of some patients with AML, particularly CD34-negative AML, due to down-regulation of bcl-2 expression.
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PMID:Down-regulation of bcl-2 in AML blasts by all-trans retinoic acid and its relationship to CD34 antigen expression. 882 91

Irregular menstrual bleeding in users of hormonal contraception represents the single major reason for discontinuation of these contraceptive methods. The mechanisms which underlie these bleeding disturbances are poorly understood, but appear to be associated with changes in the endometrial microvasculature following abnormal patterns of sex steroid exposure. Endometrial microvascular density is known to be increased in users of the low-dose levonorgestrel contraceptive implant, Norplant. This study explores microvascular density in other conditions of spontaneous (post-menopausal) and induced (danazol and goserelin) endometrial atrophy. Endometrial biopsies were fixed, paraffin-embedded and sections were immunostained using anti-CD34 antibody to identify vascular endothelial cells. The mean microvascular density (+/-SEM) for control samples was 186 +/- 8 vessels/mm2. There were no statistically significant changes in vascular density observed across the menstrual cycle. Mean microvascular density in spontaneous and induced endometrial atrophy did not differ significantly from that observed in the control population. The mean microvascular density was 230 +/- 17 vessels/mm2 in 31 postmenopausal women, 269 +/- 67 vessels/mm2 in 25 subjects treated with danazol was and 191 +/- 45 vessels/mm2 in nine subjects treated with goserelin. These findings suggest that the mechanisms controlling microvascular density in conditions of endometrial atrophy may vary according to the nature of the atrophic stimulus.
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PMID:Microvascular density in conditions of endometrial atrophy. 892 Oct 81


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