Optimum methods to mobilize stem cells.
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Cross-Over Studies↗
Biomedical subjects
L Akard
Publications and source records attributed to L Akard.
Induction of apoptosis by arachidonic acid in chronic myeloid leukemia cells.
The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is associated with transforming ability and an intrinsic resistance to induction of apoptosis by genotoxic agents. Arachidonic acid (AA), a biologically active fatty acid, plays a crucial role as a mediator of signaling pathways involved in cell proliferation and survival. In this study, we investigated the potential role of AA as a proapoptotic agent in CML. Pretreatment of human CML isolated progenitor cells with AA (100 microM for 18 h) induced 71-75% inhibition of in vitro colony formation of granulocyte-macrophage colony-forming units, multilineage colony-forming units, and erythroid burst-forming units. This inhibition was significantly greater than the effect on normal progenitor cells (19-39% growth inhibition of erythroid burst-forming units, multilineage colony-forming units, and granulocyte-macrophage colony-forming units). AA also inhibited growth of the bcr-abl-transformed cell line H7.bcr-abl A54. In contrast, a minimal effect of AA on inhibition of cell growth was observed in the parental nontransformed NSF/N1.H7 cell line. The antiproliferative effect of AA was associated with apoptosis. Gamma-linolenic acid, a precursor of AA, also inhibited cell growth, whereas other unsaturated and saturated fatty acids had no effect. Pharmacological inhibition of cyclooxygenase, lipooxygenase, and cytochrome P450 monooxygenase enzymes prior to exposure to AA did not rescue cells from the inhibitory effect of AA. Moreover, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable arachidonate analogue, also inhibited cell growth, suggesting that the effect of AA did not require further metabolism. Treatment with antioxidants prior to stimulation with AA was also ineffective in preventing its antiproliferative effect. Thus, AA inhibited proliferation of CML cells by inducing apoptotic cell death. The signaling mechanisms of AA-induced inhibition of cell growth appeared to be independent of its conversion into eicosanoids or free radical generation.
5,8,11,14-Eicosatetraynoic Acid↗
Comparison of the distribution of progenitor cells in G-CSF-mobilized peripheral blood and steady-state bone marrow after counterflow centrifugal elutriation.
Blood-derived progenitor cells obtained following mobilization with granulocyte colony-stimulating factor (MoPBSC) are increasingly being used as an alternative to bone marrow (BM) in allogeneic stem cell transplantation. The higher numbers of mature T lymphocytes in MoPBSC grafts may increase the risk of (chronic) graft-vs.-host disease. Counterflow centrifugal elutriation (CCE) is an effective method for T-cell depletion of BM grafts. The elutriation characteristics of steady-state BM and MoPBSC were compared using a CCE procedure in which fractions were obtained after small incremental increases in flow rate with constant centrifugal force. Counterflow centrifugal elutriation experiments with MoPBSC from six healthy volunteers showed that 54% of all cells collected were recovered in the < or = 15 mL/minute fractions, whereas experiments with mononuclear BM cells from five healthy volunteers resulted in recovery of 52% of collected cells from the > or = 19 mL/minute fractions. The peak concentrations of CD34+ cells were found in the same fraction (18 mL/minute), but more CD34+ cells from MoPBSC were recovered from the small (< or = 16 mL/minute) fractions (54% for MoPBSC, 26% for BM; p = 0.08). The small CD34+ cells from BM were more frequently lacking CD38 and human leucocyte antigen-DR expression than the small CD34+ cells from MoPBSC. Mature T-cells (CD3+) in BM and MoPBSC samples had similar CCE features, as did early (long-term culture initiating cells, high-proliferative potential colony-forming cells) and more mature (colony-forming units granulocyte/macrophage, BFU-e) hematopoietic progenitor cells. The results of this study suggest that T-cell depletion by CCE of MoPBSC as compared to BM products, may lead to a greater loss of CD34+ cells, but not of immature hematopoietic progenitor cells.
Adult↗
Counterflow centrifugal elutriation as a method of T cell depletion may cause loss of immature CD34+ cells.
Counterflow centrifugal elutriation (CCE) is capable of separating cells on the basis of size. CCE has been used successfully to deplete allogeneic bone marrow (BM) grafts of T lymphocytes to decrease the risk of acute graft-versus-host disease. Previous studies have shown that more immature CD34+ cells in human BM tend to be smaller than more mature CD34+ cells. Human BM was subjected to CCE with the 4 ml standard chamber at constant rotor speed (2300 r.p.m.) and increasing flow-rate (14-23 ml/min, rotor-off). The eleven fractions collected were assayed for CD34+ and CD3+ cells, and for CFU-GM, HPP-CFC and long-term culture initiating cells (LTC-IC). The CD3+ T cells were enriched in the early (small-cell) fractions 14-17 ml/min. CD34+ cells were enriched in fractions 17-21 ml/min, and CFU-GM were concentrated in the same fractions. HPP-CFC and LTC-IC showed nearly identical CCE profiles, with enrichment in fractions 16-18 ml/min. When fraction < or = 17 ml/min was chosen as cut-off, the small-cell fraction contained 94.0% of all CD3+ cells, 44.4% of total cells, 33.2% of CD34+ cells and 34.7% of CFU-GM; however, 67.6% of HPP-CFC and 72.4% of LTC-IC were recovered in this small-cell fraction. These data suggest that T cell depletion through CCE as used by us, while losing only minor proportions of CD34+ cells and CFU-GM, carries the risk of losing the majority of more immature progenitor cells. This may lead to an increased risk of graft failure, in particular in HLA-mismatched transplants.
Antigens, CD34↗
Lineage commitment of HLA-DR/CD38-defined progenitor cell subpopulations in bone marrow and mobilized peripheral blood assessed by four-color immunofluorescence.
We used four-color fluorescence analysis to compare lineage antigen expression in relationship to CD38 and HLA-DR on CD34+ progenitor cells in adult human bone marrow and mobilized peripheral blood. Each of four progenitor cell subpopulations defined by HLA-DR and CD38 intensity (CD38-/HLA-DR-, CD38-/HLA-DR+, CD38+/HLA-DR+, and CD38+/HLA-DR-) were present in both progenitor cell sources in similar ratios. The most prevalent subpopulation consisted of cells that expressed both CD38 and HLA-DR. Virtually all progenitor cells that lacked CD38 also lacked lineage antigens regardless of their HLA-DR expression. In contrast, the majority of the cells within both CD38+ progenitor cell subpopulations possessed either lineage antigens or the proliferation-associated antigen, CD71. Furthermore, CD71 was expressed on three times the number of CD38+/HLA-DR- cells when compared with the CD38-/HLA-DR- subpopulation. Within CD34+ progenitor cell subpopulations defined by the expression of CD38 and HLA-DR, the CD38+/HLA-DR- component appears to be the most mature, based on the expression of CD71 and various lineage-associated antigens, including representative markers characterizing early lymphoid, myeloid, and erythroid precursors. Thus, selection of the most immature CD34+ progenitor cells based solely on the lack of HLA-DR expression results in isolation of two distinct cell populations with markedly different maturation status and resultant growth characteristics.
ADP-ribosyl Cyclase↗
Differences in CD34+ cell subpopulations between human bone marrow and "mobilized" peripheral blood as determined with counterflow centrifugal elutriation.
Engineering of hematopoietic progenitor cells (HPCs) from bone marrow (BM) or "mobilized" peripheral blood (MoPB) is becoming increasingly important. Counterflow centrifugal elutriation (CCE) has been used to separate cells on the basis of their size. In this study, CCE was applied to evaluate BM and MoPB for differences in their HPC populations. Using a standard 4-mL elutriation chamber at 2300 rpm, CD34+ cells from BM peaked at a flow rate of 19 mL/minute, with 85% of all CD34+ cells recovered from fractions 15-22 mL/minute. The CD34+ cells from MoPB, mobilized with chemotherapy and granulocyte colony-stimulating factor (G-CSF), peaked at 22 mL/minute, with 90% of all CD34+ cells recovered from fraction 19-26 mL/minute. Colony-forming cells (colony-forming units granulocyte/macrophage [CFU-GM] + burst-forming unit-erythroid [BFU-E] + multipotent colony-forming units [CFU-GEMMs]) followed the distribution of CD34+ cells very closely, also with a shift to higher flow-rates for MoPB compared with BM. The lower flow-rate fractions of both BM and MoPB contained an increased proportion of CD34+ cells that did not express HLA-DR and/or CD38 on their surface, suggesting that the earliest CD34+ cells were enriched in the low-flow rate fractions. Although CFU-GMs, BFU-Es, and CFU-GEMMs from BM all peaked in the same fraction (19 mL/minute), high-proliferative potential colony-forming cells (HPP-CFCs) were concentrated in fraction 17 mL/minute, indicating that these earlier progenitor cells were slightly smaller. With MoPB, HPP-CFCs did not appear to be smaller than BFU-Es or CFU-GEMMs. CCE appears to be an attractive method for separating HPCs from BM or MoPB into populations of different maturity. Differences in CD34+ cell populations between BM and MoPB may help explain the differences in repopulation kinetics observed after transplantation.
Antigens, CD34↗
Phorbol ester-induced priming of superoxide generation by phosphatidic acid-stimulated neutrophils and granule-free neutrophil cytoplasts.
This study was undertaken to examine the mechanisms involved in polymorphonuclear leukocyte superoxide release stimulated by exogenous phosphatidic acid (PA). Unlike the immediate burst of superoxide release affected by membrane-permeable dioctanoylglycerol (DiC8-DAG), dioctanoyl phosphatidic acid (DiC8-PA) induced superoxide release after a lag period of 5-20 min. This period was considerably reduced or eliminated when cells were primed by substimulatory levels of phorbol myristate acetate (PMA). Granule-depleted neutrophil cytoplasts also responded to DiC8-PA with a burst of superoxide generation. Activation of the cytoplast superoxide generating system in response to DiC8-PA was also significantly faster after cells had been preexposed to substimulatory levels of PMA, indicating that at least a portion of the priming mechanism was independent of PMA-induced degranulation. To further examine the potential mechanism of PMA priming of responses to PA, we evaluated the activity of neutrophil ecto-phosphatidic acid phosphohydrolase (ecto-PA phosphohydrolase), which generates diacylglycerol from exogenous PA. PMA priming had no discernable effect on the activity of this enzyme. In addition, propranolol, an inhibitor of PA phosphohydrolase, did not selectively inhibit PMA priming of neutrophil responses to DiC8-PA, indicating that priming did not result from acceleration of DiC8-PA hydrolysis. We therefore investigated the possibility that activation of protein kinase C was the basis of the primed response. Several semiselective protein kinase C inhibitors (calphostin C, H-7, and acylmethylglycerol) inhibited DiC8-DAG- and DiC8-PA-induced superoxide release as well as PMA-primed responses to approximately the same extent. These results are consistent with the hypothesis that neutrophil responses to phosphatidate are mediated by diglyceride generated by the action of ecto-PA phosphohydrolase. PMA priming does not result from increased catalytic activity of ecto-PA phosphohydrolase but rather seems to result from potentiation of an intermediate involved in the cells' response to multiple stimuli.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Maturation of mobilized peripheral blood progenitor cells: preclinical and phase I clinical studies.
The use of mobilized peripheral blood progenitor cells (PBPC) after high-dose chemotherapy has markedly decreased the period of severe neutropenia. In an attempt to further decrease the duration of neutropenia, the potential of PBPC to mature during in vitro culture was assessed, with special attention being paid to culture medium, growth factors, and cell concentration. Concentrations of 10(6) PBPC/mL resulted in better recovery than 10(7)/mL as far as total cells, CFU-GM, and granulocytes were concerned. The combination of IL-3 + GM-CSF+G-CSF appeared to be better than any of these growth factors alone. Simple media, such as Medium 199, gave poorer cell recovery than more complex media, such as IMDM. With 10(6)/mL nonenriched PBPC in IMDM with IL-3/GM-CSF/G-CSF, on day 15 CFU-GM reached 450% of the initial level. At that point, granulocytes had increased 15-fold. A small phase I study was performed to assess the toxicity of infusing 1000-2000 mL of PBPC cultured for 3 days at 3-10 x 10(6)/mL with IL-3/GM-CSF/G-CSF in LifeCell bags. Although no clear decrease in the duration of neutropenia was observed, the infusions were uncomplicated in 5 of the 6 patients and had minor side effects in the sixth patient. These data suggest that in vitro differentiation of nonenriched PBPC is possible. However, to develop a clinically applicable method, several logistical problems will have to be overcome.
Antineoplastic Combined Chemotherapy Protocols↗
Selective T cell depletion with CD8-conjugated magnetic beads in the prevention of graft-versus-host disease after allogeneic bone marrow transplantation.
The effects of a new immunomagnetic method of selectively depleting CD8+ lymphocytes from donor bone marrow were studied in 29 patients undergoing transplantation from HLA-identical sibling (n = 20) or alternative (n = 9) donors. The direct immunomagnetic depletion method consistently removed > 95% of CD8+ cells and the non-specific loss of other cell subsets was only about 15%. Recovery of CFU-GM and BFU-e was on average > 100%. The final graft contained 0.9 +/- 0.6 x 10(8)/kg nucleated cells and 1.4 +/- 2.7 x 10(5)/kg CD8+ cells. Patients also received cyclosporine starting day -1. Engraftment occurred in 28 patients (97%), including three patients who received a non-TBI conditioning regimen. One patient receiving an unrelated transplant failed to engraft. Median time to ANC > 500 x 10(6)/L was 17 (12-23) days. Four of 20 patients receiving grafts from HLA-identical siblings (20%) developed acute GVHD grade > or = II. However, five of eight patients with grafts from alternative donors (63%) had grade > or = II GVHD. Nearly all patients developed fever around day 7, accompanied by fluid overload, mild skin rash and shortness of breath. This syndrome necessitated treatment with steroids. Immunomagnetic CD8 depletion is a simple and reproducible method of selective T cell depletion. In combination with cyclosporine it appears to be effective in the prevention of severe acute GVHD in HLA-identical sibling transplants, but not in transplants from less perfectly matched donors.
Adult↗
Regulation of neutrophil responses by phosphotyrosine phosphatase.
By using immunofluorescent flow cytometry, we observed a profound up-regulation of CD45 on the plasma membrane of neutrophils exposed to low levels of a culture supernatant of the Gram-negative pathogen, Fusobacterium nucleatum (FN). Plasma membranes of neutrophils freshly prepared form human blood possessed little enzymatically active phosphotyrosine phosphatase. The activity of this enzyme was markedly potentiated in plasma membranes prepared from cells preexposed to the FN culture supernatant. This activity was vanadate sensitive and could be immunoprecipitated with anti-CD45 Ab. Cells preexposed to the FN culture supernatant were inhibited in their ability to release superoxide when challenged with the bacterial chemotactic factor, FMLP, but not PMA. The tyrosine kinase inhibitor, genistein, likewise inhibited FMLP but not PMA-induced superoxide release. Pretreatment of neutrophils with vanadate reversed FN-mediated inhibition of FMLP-triggered superoxide release but had no effect on genistein-mediated inhibition of FMLP-induced superoxide release. Of several proteins tyrosine phosphorylated in response to treatment of neutrophils with FMLP, Western analysis revealed one (m.w. approximately 93,000) that was lost when FMLP-stimulated cells were exposed to FN. This effect was inhibited when the cells were preexposed to vanadate. These results are consistent with the hypothesis that plasma membrane tyrosine phosphatase modulates FMLP-induced superoxide release by reversing the effects of tyrosine kinases activated in the initial phases of cell stimulation.
Cell Membrane↗
Infection prevention in severely myelosuppressed patients: a comparison between ciprofloxacin and a regimen of selective antibiotic modulation of the intestinal flora.
PURPOSE: To study whether oral ciprofloxacin would be as effective in preventing bacterial infections in severely myelosuppressed patients as selective antibiotic modulation of the gut flora with neomycin/polymyxin B sulfate/nalidixic acid (NPN). PATIENTS AND METHODS: One hundred and five patients undergoing allogeneic or autologous bone marrow transplant, or induction therapy for acute leukemia in 1988 and 1989 were studied. Patients were stratified according to the type of therapy, and randomized in a ratio of 2:1 to either oral ciprofloxacin 500 mg BID, or a combination of oral neomycin 250 mg QID, polymyxin-B 100 mg QID, and oral nalidixic acid 1,000 mg BID. Treatment began on admission and continued until the absolute granulocyte count was greater than 500/mm3 for 3 consecutive days. RESULTS: The 96 evaluable patients were evenly distributed over the 3 treatment groups; 63 patients received ciprofloxacin and 33 received NPN. Fever developed in 92% of patients on ciprofloxacin and in 97% of patients on NPN. (P = 0.66), 6.6 +/- 5.8 and 7.2 +/- 5.3 days from the start of prophylaxis, respectively. Twenty-five patients on ciprofloxacin developed 29 microbiologically documented infections, fewer than the 26 infections in the 22 patients on NPN (P = 0.02). Patients on ciprofloxacin had fewer bacteremias (33%) than did the NPN patients (55%) (P = 0.05). Gram-negative bacteremias were very rare (2 cases; no Enterobacteriaceae), but streptococcal bacteremias were frequent in both arms (27 cases). Side effects were not significantly different, but compliance with ciprofloxacin was better. CONCLUSIONS: Ciprofloxacin is at least as effective as the combination of neomycin/polymyxin/nalidixic acid in the prophylaxis of bacterial infections in myelosuppressed patients, and is better tolerated. Additional agents to prevent streptococcal infections are needed.
Adolescent↗
Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes.
Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg(2+)-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase D-dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergent availability and cation sensitivity on the apparent distribution of PAPH in neutrophil subcellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzymes was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.
Cell Membrane↗
Cytokine-stimulated up-regulation of protein tyrosine phosphatase in neutrophil plasma membranes.
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Cell Membrane↗
Parallel up-regulation of CD11B and CD45 on neutrophilic leukocytes exposed to soluble factors of oral pathogens.
We tested culture supernatants from a battery of oral bacterial strains for their ability to influence the expression of CD11b and CD45 on the neutrophil plasma membrane. Several bacterial extracts stimulated the up-regulation of both CD11b and CD45 simultaneously. Two supernatants in particular (a clinical isolate of A. actinomycetemcomitans and F. nucleatum ATCC25586) potently stimulated the deployment of CD11b and CD45 from their intracellular storage site to the plasma membrane. Both supernatants inhibited superoxide release stimulated by exposure of neutrophils to formyl methionyl leucyl phenylalanine (FMLP) but had variable effects on superoxide release stimulated by phorbol myristate acetate (PMA). The ability of products of oral bacteria to modulate neutrophil plasma membrane antigen composition may regulate functional reactivity and thus be an important factor in the pathogenesis of periodontal infection and inflammation.
Antigens, CD↗
Immunomagnetic depletion of CD8+ lymphocytes from marrow grafts.
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Adolescent↗
Characteristics of engraftment after repeated autologous bone marrow transplantation.
The rate of engraftment after autologous bone marrow transplantation (ABMT) is extremely variable and largely unpredictable. To identify factors influencing engraftment, we studied 35 patients with refractory germ cell tumors undergoing high-dose chemotherapy with carboplatin (900-2000 mg/m2) and etoposide (1200 mg/m2) with bone marrow rescue. Prior to the initiation of chemotherapy, bone marrow sufficient for two marrow infusions was harvested (range 0.86-4.82 x 10(8) nucleated cells per kg). All 35 patients received half of the collected bone marrow 3 days after the last dose of chemotherapy; 23 responders received a second round of the same chemotherapy followed by infusion of the second half of the bone marrow. Eighteen patients could be compared for the two transplant episodes. The "rate of engraftment" was defined as the unweighted mean of four parameters: 1) the number of days until the absolute granulocyte count surpassed 0.2 x 10(9)/liter, 2) the number of days until the absolute granulocyte count surpassed 0.5 x 10(9)/liter, 3) the number of days until the last platelet transfusion, and 4) the number of days until the reticulocyte count surpassed 25 x 10(9)/liter. No significant correlation was found between rate of engraftment and such factors as the number of nucleated cells per kg infused, the dose of chemotherapy, extent of prior chemotherapy, tumor response to the high-dose chemotherapy, age of the patient, or the days of granulocytopenic fever (all p greater than 0.20). In contrast, a close correlation was found for the number of units of platelets (p = 0.005) and red blood cells (p = 0.006) transfused following each of the two transplants. There was no significant difference between rate of engraftment after first and second transplantation. Comparison of these data with the results obtained in reported ABMT with separate harvests suggests that the characteristics of the infused marrow determine the rate of engraftment after ABMT. This model of repeated transplantation could provide an important tool for assessing the therapeutic efficacy of hematopoietic growth factors.
Bone Marrow Transplantation↗
Treatment of refractory lymphoma with high dose cytarabine, cyclophosphamide and either TBI or VP-16 followed by autologous bone marrow transplantation.
This study was designed to test the efficacy and toxicity of combining high-dose cytarabine (3 g/m2 every 12 h x 8 doses day -7 to day -4, total dose 24 g/m2), methyl prednisolone (0.5 mg/kg every 4 h day -7 to day -1), and cyclophosphamide (CY) (60 mg/kg day -3 and day -2) with either total body irradiation (TBI) (900 cGy in a single fraction on day -1) or VP-16 (600 mg/m2/days -7, -5, and -3) in patients not eligible for TBI secondary to prior radiotherapy. We treated 14 patients (eight male, six female) with either non-Hodgkin's lymphoma (n = 5) or Hodgkin's disease (n = 9). All patients had failed prior conventional chemotherapy (median two regimens range 1-5). Five patients were treated with TBI and nine with VP-16. There were eight complete remissions, two partial remissions, four were inevaluable for response due to early death. Overall survival is 21% (3/14) and relapse-free survival is 7% (1/14) with the sole disease-free survivor now 40 months from transplant. Very significantly, among patients receiving TBI, there were no survivors (median survival 24 days, range 17-330 days) and 4/5 had pulmonary complications. Median DLCO in these four patients was 61% (range 50-67) prior to transplant and none had an infectious etiology established by bronchoalveolar lavage. Median time to an absolute granulocyte count of 500 x 10(6)/l was 16 days (range 10-37 days) and to a platelet count of 20 x 10(9)/l was 12 days (range 7-22 days). In conclusion, the addition of high-dose cytarabine (24 g/m2) to CY and single-dose TBI or VP-16, while being very active, produced excessive pulmonary toxicity in this group of patients with lymphoma.
Adolescent↗