Slow platelet recovery after PBPC transplantation from unrelated donors.

The effects of the composition of PBPC grafts from matched related donors (MRDs) and matched unrelated donors (MUDs) have not been compared. In a single-center study, the compositions of 55 MRD PBPC grafts and 33 MUD grafts were studied for their effect on the rate of engraftment in patients who had evidence of donor cell engraftment on day +28. The MUD grafts came more frequently from young male donors and contained more CD34(+) cells but similar numbers of colony-forming units granulocyte-macrophage (CFU-GM) and burst forming units-erythroid. The recovery of neutrophils to >500/mm(3) was equally fast in both groups, but recovery of platelets to >20,000/mm(3) was significantly delayed in the MUD group (P<0.001). The MUD group also required more transfusions of platelets and red cells. Patients receiving grafts containing low numbers of CFU-GM had markedly delayed platelet recovery. The patients with the slowest engraftment tended to have prolonged transportation times. Storage experiments suggested a major loss of viable CD34(+) cells and CFU-GM when undiluted PBPC products are stored at room temperature. The data suggest that a fraction of the MUD grafts suffer during transportation. In vitro proliferation assays should be part of the validation and auditing of transportation of MUD grafts.

Longitudinal study of adaptation to the stress of bone marrow transplantation.

PURPOSE: This prospective longitudinal study of adaptation to bone marrow transplantation (BMT) addressed three questions: (1) When during BMT do individuals experience the greatest distress? (2) What factors are associated with this distress? (3) Are there variables that could be potential clinical indicators of persons in greatest need of preventive intervention? PATIENTS AND METHODS: One hundred one participants undergoing either an autologous or allogeneic BMT completed questionnaires before hospitalization, before bone marrow infusion, 7 days and 14 days after transplantation, and then 1 month, 3 months, and 12 months after hospitalization. Adaptation was indicated by the degree of emotional distress. Independent variables were personal control, social support from specific sources, cognitive response, self-perception, and coping strategies, controlling for symptomatology. RESULTS: The greatest emotional distress occurred after admission to the hospital and before the bone marrow infusion. Anxiety and depression decreased 1 week after the transplant, although symptomatology increased during this time. The periods of least emotional distress were 3 months and 1 year after transplantation. Factors that accounted for the greatest variance in emotional distress/adaptation were the degree of emotional distress at baseline, personal control, cognitive response, and symptomatology. CONCLUSION: According to this longitudinal study, which includes pretransplant data, data from in-hospital transplantation, and posttransplant data, (1) psychosocial vulnerability of these BMT recipients was greatest during hospitalization before the transplant, (2) perceived personal control may be a potential indicator of vulnerability to secondary psychosocial morbidity, and (3) the demonstrated significance of psychosocial well-being before BMT indicates the importance of obtaining prospective data for both research and clinical purposes.

Matched-pair analysis of hematopoietic progenitor cell mobilization using G-CSF vs. cyclophosphamide, etoposide, and G-CSF: enhanced CD34+ cell collections are not necessarily cost-effective.

Using matched-pair analysis, we compared two popular methods of stem cell mobilization in 24 advanced-stage breast cancer patients who underwent two consecutive mobilizing procedures as part of a tandem transplant protocol. For the first cycle, 10 microg/kg/day granulocyte colony-stimulating factor (G-CSF) was given and apheresis commenced on day 4 and continued for < or =5 days (median 3 days). One week after the first cycle of apheresis, 4000 mg/m2 cyclophosphamide, 400 mg/m2 etoposide, and 10 microg/kg G-CSF were administered for < or =16 days (cycle 2). Apheresis was initiated when the white blood cell (WBC) count exceeded 5000 cells/microL and continued for < or =5 days (median 3 days). Mean values of peripheral blood WBC (31,700+/-3200 vs. 30,700+/-3300/microL) were not significantly different between cycles 1 and 2. Mean number of mononuclear cells (MNC) collected per day was slightly greater with G-CSF mobilization than with the combination of chemotherapy and G-CSF (2.5+/-0.21x10(8) vs. 1.8+/-0.19x10(8) cells/kg). Mean daily CD34+ cell yield, however, was nearly six times higher (12.9+/-4.4 vs. 2.2+/-0.5x10(6)/kg; p = 0.01) with chemotherapy plus G-CSF. With G-CSF alone, 13% of aphereses reached the target dose of 5x10(6) CD34+ cells/kg in one collection vs. 57% with chemotherapy plus G-CSF. Transfusions of red blood cells or platelets were necessary in 18 of 24 patients in cycle 2. Three patients were hospitalized with fever for a median of 3 days after cycle 2. No patients received transfusions or required hospitalization during mobilization with G-CSF alone. Resource utilization (cost of drugs, aphereses, cryopreservation, transfusions, hospitalization) was calculated comparing the median number of collections to obtain a target CD34+ cell dose of 5x10(6) cells/kg: four using G-CSF vs. one using the combination in this data set. Resources for G-CSF mobilization cost $7326 vs. $8693 for the combination, even though more apheresis procedures were performed using G-CSF mobilization. The cost of chemotherapy administration, more doses of G-CSF, transfusions, and hospitalizations caused cyclophosphamide, etoposide, and G-CSF to be more expensive than G-CSF alone. A less toxic and less expensive treatment than cyclophosphamide, etoposide, and G-CSF is needed to be more cost-effective than G-CSF alone for peripheral blood progenitor cell mobilization.

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.

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.

Hematopoietic growth factor after autologous peripheral blood transplantation: comparison of G-CSF and GM-CSF.

Autologous peripheral blood stem cell (PBSC) transplantation results in rapid hematologic recovery when sufficient numbers of CD34+ cells/kg are infused. Recent studies suggest that filgrastim (G-CSF) administration following transplantation leads to more rapid neutrophil recovery and lower total transplant costs. This study compares the use of G-CSF (5 microg/kg/day) with sargramostim (GM-CSF) 500 microg/day from day 0 until neutrophil recovery (ANC >1500/mm3) in patients with breast cancer or myeloma who had PBSC mobilized with the combination of cyclophosphamide, etoposide, and G-CSF. Twenty patients (13 breast cancer and seven myeloma) received GM-CSF and 26 patients (14 breast cancer and 12 myeloma) received G-CSF. The patients were comparable for age and stage of disease, and received stem cell grafts that were not significantly different (CD34+ x 10(6)/kg was 12.5 +/- 11.1 (mean +/- s.d.) for GM-CSF and 19.8 +/- 18.5 for G-CSF; P = 0.10). The use of red cells (2.8 vs 2.3 units), and platelet transfusions (2.5 vs 3.1) was similar for the two groups, as was the use of intravenous antibiotics (4.3 vs 4.6 days) and the number of days with temperature >38.3 degrees C (2.3 vs 1.8). Platelet recovery was also similar in both groups (platelets >50,000/mm3 reached after 11.8 vs 14.9 days). The recovery of neutrophils, however, was faster using G-CSF. ANC >500/mm3 and >1000/mm3 were reached in the GM-CSF group at 10.5 +/- 1.5 and 11.0 +/- 1.7 days, respectively, whereas with G-CSF only 8.8 +/- 1.2 and 8.9 +/- 2.2 days were required (P < 0.001). As a result, patients given G-CSF received fewer injections than the GM-CSF patients (10.9 vs 12.3). Resource utilization immediately attributable to the use of growth factors and the duration of pancytopenia, excluding hospitalization, were similar for the two groups. This study suggests that neutrophil recovery occurs more quickly following autologous PBSC transplant using G-CSF in comparison to GM-CSF, but the difference is not extensive enough to result in lower total cost.

Characterization and partial purification of CD34+ progenitor cell ecto-phosphatidic acid phosphohydrolase.

Phosphatidic acid and its hydrolysis product, diacylglycerol, play potentially vital roles as extracellular messengers in numerous cellular systems and may play a key role in regulating hematopoiesis. In this study, we describe an ecto-phosphatidic acid phosphohydrolase that potentially regulates cellular responses to phosphatidic acid on bone marrow derived human hematopoietic progenitors. We partially purified hematopoietic progenitor ecto-PAPase using a novel in-gel phosphatase assay and then characterized the enzyme on phenotypically defined subpopulations of hematopoietic CD34+ progenitors isolated by flow cytometry. The most pronounced PAPase activity was confined to uncommitted CD34+/CD38+ hematopoietic progenitors, which lacked the expression of other lineage-associated antigens. We conclude that hematopoietic progenitor cells at various stages of maturation possess a potent ecto-PAPase, an enzyme well positioned to regulate progenitor cell growth and differentiation induced by phosphatidic acid and related lipids.

Chemotactic migration triggers IL-8 generation in neutrophilic leukocytes.

Neutrophils recovered from inflammatory exudates possess increased levels of IL-8, but exposure of neutrophils to chemoattractants results in only a modest stimulation of IL-8 generation. This study was undertaken to explore the hypothesis that IL-8 generation in these cells is dependent upon the process of migration. Neutrophils synthesized up to 30 times as much IL-8 during migration in response to a gradient of diverse chemoattractants than they did when stimulated directly by the attractants in the absence of a gradient. This IL-8 response was dependent on migration since it was not observed in cells exposed to concentration gradients of chemoattractants under conditions that prevented cell movement. While actinomycin-D (1 microg/ml) had little influence on the generation of IL-8 during chemotaxis, the protein synthesis inhibitor cycloheximide (10 microg/ml) markedly blunted the accumulation of cell-associated IL-8, suggesting that new protein synthesis from preexisting mRNA was responsible for the effect. Consistent with this interpretation, migrating cells incorporated over 10 times as much [3H]leucine into IL-8 as did nonmotile neutrophils exposed to chemoattractants. A substantial portion of the IL-8 generated during chemotaxis was released upon subsequent metabolic stimulation. Thus, the IL-8 synthesized during chemotaxis is uniquely positioned to exert a regulatory influence on inflammatory processes governed by neutrophilic leukocytes responding to inflammatory and infectious stimuli.

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.

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.

Characterization and purification of neutrophil ecto-phosphatidic acid phosphohydrolase.

Phosphatidic acid and its derivatives play potentially important roles as extracellular messengers in biological systems. An ecto-phosphatidic acid phosphohydrolase (ecto-PAPase) has been identified which effectively regulates neutrophil responses to exogenous phosphatidic acid by converting the substrate to diacylglycerol. The present study was undertaken to characterize this ecto-enzyme on intact cells and to isolate the enzyme from solubilized neutrophil extracts. In the absence of detergent, short chain phosphatidic acids were hydrolysed most effectively by neutrophil plasma membrane ecto-PAPase; both saturated and unsaturated long chain phosphatidic acids were relatively resistant to hydrolysis. Both long (C18:1) and short (C8) chain lyso-phosphatidic acids were hydrolysed at rates comparable with those observed for short chain (diC8) phosphatidic acid. Activity of the ecto-enzyme accounted for essentially all of the N-ethylmaleimide-insensitive, Mg2+-independent PAPase activity recovered from disrupted neutrophils. At 37 degrees C and pH7.2, the apparent Km for dioctanoyl phosphatidic acid (diC8PA) was 1. 4x10(-3) M. Other phosphatidic acids and lysophosphatidic acids inhibited hydrolysis of [32P]diC8PA in a rank order that correlated with competitor solubility, lysophosphatidic acids and unsaturated phosphatidic acids being much more effective inhibitors than long chain saturated phosphatidic acids. Dioleoyl (C18:1) phosphatidic acid was an unexpectedly strong inhibitor of activity, in comparison with its ability to act as a direct substrate in the absence of detergent. Other inhibitors of neutrophil ecto-PAPase included sphingosine, dimethyl- and dihydro-sphingosine, propranolol, NaF and MgCl2. Of several leucocyte populations isolated from human blood by FACS, including T cells, B cells, NK lymphocytes and monocytes, ecto-PAPase was most prevalent on neutrophils; erythrocytes were essentially devoid of activity. A non-hydrolysable, phosphonate analogue of phosphatidic acid, phosphonate 1, efficiently solubilized catalytic activity from intact neutrophils without causing cell disruption or increasing permeability. Enzyme activity in solubilized extracts was purified in the absence of detergent by successive heparin-Sepharose, gel filtration and anion exchange chromatography. By assaying activity in renatured SDS/polyacrylamide gel slices, the molecular mass of neutrophil ecto-PAPase was estimated to be between 45 and 52 kDa, similar to the molecular mass of previously purified plasma membrane PAPases. Since a large portion of neutrophil plasma membrane PAPase is available for hydrolysis of exogenous substrates, ecto-PAPase may play an important role in regulating inflammatory cell responses to extracellular phosphatidic acid in biological systems.

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.

Impaired stem cell collection by consecutive courses of high-dose mobilizing chemotherapy using cyclophosphamide, etoposide, and G-CSF.

Tandem cycles of myeloablative chemotherapy can increase dose intensity and total dose of chemotherapy, but sufficient numbers of progenitor cells must be collected to ensure hematologic recovery after each treatment. This study was undertaken to determine if two courses of mobilizing chemotherapy given 4 weeks apart using cyclophosphamide 4000 mg/m2 and etoposide 400 mg/m2, combined with G-CSF 5-10 mg/kg on days 3-16 could each provide sufficient numbers of peripheral blood progenitor cells to support tandem cycles of myeloablative chemotherapy in 20 patients with stage IV breast cancer. Leukapheresis of blood with WBC > 1000/mm3 was performed daily for up to five collections (days 12-16), and mononuclear cells, CFU-GM, and CD34+ cells were compared between the first and second collections. The second course of mobilizing treatment resulted in similar numbers of mononuclear cells collected but far fewer CFU-GM and CD34+ progenitor cells. This prevented using the second collection of progenitor cells as the sole source for the second transplant. The data suggest that a second course of cyclophosphamide, etoposide, and G-CSF given 4 weeks after the first leads to progenitor cell depletion, and efforts to increase the yield of blood-derived progenitors should focus on the initial mobilizing procedure.

Growth factor induction of cytosolic protein tyrosine kinase activity in human haemopoietic progenitor cells isolated by flow cytometry.

We employed a highly sensitive method to assay protein tyrosine kinase activity in extracts of subpopulations of CD34+ bone marrow progenitor cells isolated by fluorescence activated cell sorting in an attempt to better define how growth-factor induction of enzymatic activity relates to progenitor cell maturation. FACS analysis confirmed that, under the conditions employed, essentially all of the CD34+ cells in adult human marrow that lacked the CD38 antigen were devoid of the myeloid maturation marker CD33 as well as the lineage antigens: CD10, 13, 14, 15, 16, 19, 71 and glycophorin A. A variable portion (50-90%) of these CD34+, CD38- progenitor cells expressed HLA-DR. CD34+, CD38- cells that did not express HLA-DR were found to lack detectable levels of either membrane or cytosolic tyrosine kinase activity. HLA-DR+ progenitor cells that lacked CD38 possessed elevated levels of cytosolic tyrosine kinase activity but only low levels of plasma membrane activity. In contrast, CD34+ cells that expressed CD38 (and HLA-DR) possessed high levels of membrane-associated tyrosine kinase activity. A cocktail of haemopoietic growth factors that included IL-3, IL-6 and stem cell factor effectively induced tyrosine kinase activity in CD34+, CD38-, HLA-DR- progenitor cells. Growth factor induction of tyrosine kinase activity in these cells was not inhibited by actinomycin D or cyclohexamide. Most of the tyrosine kinase activity induced by these growth factors was recovered from the cytosolic fraction of disrupted cells. Thus, induction of cytosolic tyrosine kinase activity is an early event in the response of uncommitted haemopoietic cells to haemopoietic growth factors. Subsequent activation of membrane tyrosine kinases may initiate key transduction processes as these cells begin to differentiate.

Immunomagnetic CD4+ and CD8+ cell depletion for patients at high risk for severe acute GVHD.

Acute GVHD remains a major problem in allogeneic BMT, in particular when donors other than HLA-identical siblings are used. To determine the efficacy of an immunomagnetic method for depletion of CD4+ and CD8+ lymphocytes from the marrow graft, a series of 15 patients was studied. Thirteen patients had matched unrelated donors, and two patients had related donors. Cyclosporine was used as GVHD prophylaxis in combination with CD4+ and CD8+ depletion, which removed 94.1 +/- 3.2%, 97.0 +/- 5.1%, and 96.7 +/- 3.1% of CD3+, CD4+ and CD8+ cells, respectively. All patients engrafted promptly with AGC > 500/mm3 after a median of 16 days post-BMT. Acute GVHD grade II-IV developed in 0/2 related transplants and 4/13 MUD transplants; only one patient had grade III-IV acute GVHD. No late graft failure was observed. Three patients relapsed; two had advanced disease at the time of BMT. Seven patients are alive and in CCR after a median of 497 days; actuarial survival is 39% at 24 months. The fever syndrome observed with selective CD8+ cell depletion was not seen with the combined CD4+ and CD8+ cell depletion. Immunomagnetic CD4+ and CD8+ cell depletion of marrow grafts, in combination with in vivo cyclosporine, is a simple, reproducible and effective method to decrease the incidence and severity of acute GVHD in patients at high risk for this complication after allogeneic BMT.

Cytosolic inactivation of translocated neutrophil plasma membrane protein tyrosine phosphatase.

Phosphotyrosine phosphatases (PTPases) regulate cellular metabolic activation by reversing the effects of tyrosine kinases activated earlier in intracellular signaling pathways. We coupled fluorescence-activated cell sorter analysis using anti-CD45 monoclonal antibody with direct measurements of enzyme activity in resolved subcellular fractions to define mechanisms that potentially regulate the availability and activity of CD45-PTPase on neutrophil plasma membranes. Neutrophils in freshly obtained blood as well as neutrophils freshly isolated from blood were found to possess detectable levels of plasma membrane CD45 as assessed by immunofluorescence. However, plasma membranes from these cells were essentially devoid of PTPase catalytic activity, which was largely confined to the specific granules. Granulocyte-macrophage colony-stimulating factor (GM-CSF) upregulated both the catalytic and antigenic components of CD45-PTPase on the plasma membrane of these cells. Upregulation was associated with a shift in the particulate subcellular PTPase catalytic activity from the specific granule fraction to the plasma membrane fraction. The tyrosine kinase inhibitor genistein abrogated GM-CSF-promoted upregulation of plasma membrane CD45 PTPase but did not prevent the GM-CSF-dependent decrease in specific granule catalytic activity. Anti-CD45 antibody immunoprecipitated PTPase activity from both specific granules of resting cells and plasma membranes of GM-CSF-treated cells. However, antiphosphotyrosine immunoprecipitated only activity that had translocated to the plasma membrane, suggesting a role for CD45 phosphorylation in translocation. Western analysis confirmed the tyrosine phosphorylation of CD45 in plasma membranes of GM-CSF-treated neutrophils. Preincubation of plasma membranes of GM-CSF-stimulated neutrophils with cytosol from resting cells resulted in a time- and temperature-dependent loss in membrane PTPase as a consequence of the effects of a cytosolic inactivator. Cytosol obtained from stimulated neutrophils possessed substantially reduced levels of this PTPase inactivator. We conclude that activity of the catalytic component of membrane PTPase in circulating neutrophils is regulated by a cytosolic inactivator. Upon stimulation, intact CD45 PTPase is incorporated into the plasma membrane by a process that requires tyrosine phosphorylation. As a result of inhibition of the cytosolic inactivator, the translocated PTPase expresses full activity, thereby amplifying the potential regulatory influence of the enzyme on the cells' functional response.

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.

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.