scholarly journals Combination treatment in vitro with Nutlin, a small-molecule antagonist of MDM2, and pegylated interferon-α 2a specifically targets JAK2V617F-positive polycythemia vera cells

Blood ◽  
2012 ◽  
Vol 120 (15) ◽  
pp. 3098-3105 ◽  
Author(s):  
Min Lu ◽  
Xiaoli Wang ◽  
Yan Li ◽  
Joseph Tripodi ◽  
Goar Mosoyan ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Combination Treatment ◽  
Colony Formation ◽  
P38 Map Kinase ◽  
Hematopoietic Progenitor Cells ◽  
Interferon Α ◽  
Low Doses ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells

Abstract Interferon (IFN-α) is effective therapy for polycythemia vera (PV) patients, but it is frequently interrupted because of adverse events. To permit the long-term use of IFN, we propose combining low doses of IFN with Nutlin-3, an antagonist of MDM2, which is also capable of promoting PV CD34+ cell apoptosis. Combination treatment with subtherapeutic doses of Peg IFN-α 2a and Nutlin-3 inhibited PV CD34+ cell proliferation by 50% while inhibiting normal CD34+ cells by 30%. Combination treatment with Nutlin-3 and Peg IFN-α 2a inhibited PV colony formation by 55%-90% while inhibiting normal colony formation by 22%-30%. The combination of these agents also decreased the proportion of JAK2V617F-positive hematopoietic progenitor cells in 6 PV patients studied. Treatment with low doses of Peg IFN-α 2a combined with Nutlin-3 increased phospho-p53 and p21 protein levels in PV CD34+ cells and increased the degree of apoptosis. These 2 reagents affect the tumor suppressor p53 through different pathways with Peg IFN-α 2a activating p38 MAP kinase and STAT1, leading to increased p53 transcription, whereas Nutlin-3 prevents the degradation of p53. These data suggest that treatment with low doses of both Nutlin-3 combined with Peg IFN-α 2a can target PV hematopoietic progenitor cells, eliminating the numbers of malignant hematopoietic progenitor cells.

Treatment with Pegylated Interferon Alpha 2a in Combination with the Bcl-Xl Inhibitor ABT-737 Specifically Targets JAK2V617F Positive Hematopoietic Progenitor Cells From Patients with Polycythemia Vera.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3916-3916 ◽  
Author(s):  
Min Lu ◽  
Wei Zhang ◽  
Daniel Yoo ◽  
Dmitriy Berenzon ◽  
Yan Li ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Interferon Alpha ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Hematopoietic Progenitor Cells ◽  
Low Doses ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells

Abstract Abstract 3916 Poster Board III-852 Polycythemia vera (PV) is a Philadelphia chromosome negative chronic myeloproliferative neoplasm (MPN) which is characterized by acquisition of a mutation in JAK2 (JAK2V617F). The administration of a pegylated form of interferon-alpha-2a (Peg IFNa-2a) to patients with PV has recently been reported to lead to hematological remissions and a reduction of the JAK2V617F allele burden in most patients receiving this modality of therapy. The mechanism underlying this profound clinical response of PV patients to Peg IFNa-2a has been the subject of a great deal of speculation. In order to evaluate the mechanism by which Peg IFNa-2a affects hematopoiesis in PV patients, CD34+ cells isolated from cord blood and the peripheral blood of patients with PV were cultured in semisolid media in the presence and absence of 200 and 500 U of Peg IFNa-2a. These relatively low doses of Peg IFNa-2a did not alter hematopoietic colony formation by CB CD34+ cells but inhibited PV CFU-GM colony formation by 35% and 50%, and BFU-E colony formation by 60% and 80%, respectively. Furthermore, the hematopietic colonies that formed in the presence of Peg IFNa-2a were composed of far fewer cells than those cultured in the presence of cytokines alone. In addition, individual hematopoietic colonies were plucked and the JAK2 genotype was assessed by nested allele-specific PCR assay. Exposure of PV CD34+ cells to Peg-IFNa-2a (500U) resulted in a reduction in the proportion of JAK2V617F-positive hematopoietic progenitor cells from 81.7±16.3% to 50.3±27.6% (p=0.004). Samples from 81.9% of the PV patients (9 of 11 samples) responded in this fashions to Peg IFNa 2a treatment. We then showed that incubation of PV CD34+ cells but not CB CD 34+ cells with 200 and 500U of Peg IFNa-2a resulted in increased rates of apoptosis by 4.3% and 15.3%, respectively. Erythroblasts and megakayoctes from patients with PV have been previously shown to be characterized by over-expression of the anti-apoptotic proteins Bcl-xL. We then examined if the effects of IFNa-2a could be enhanced by addition of the Bcl-xL inhibitor-ABT-737. After 2 days of treatment, Peg IFNa 2a plus ABT-737 induced significantly greater degree of apoptosis (∼50%) of a JAK2V617F positive erythroleukemia cell line (HEL cells) as compared to treatment with each agent alone, (Peg-IFNa-2a, <5%; ABT-737, 20%). PV CD34+ cells were incubated with Peg IFNa 2a (500 U) alone, ABT-737 (0.25 uM) alone or ABT-737 plus Peg IFNa 2a for 4 days and the numbers of cells were decreased by 35%, 40% and 65 %, respectively; and the corresponding percentage of apoptotic cells was 20%, 15% and 60%, respectively. Western blot analysis showed that the Bcl-xL protein level in PV but not CB mononuclear cells was reduced by treatments with ABT-737 alone or in combination with Peg IFNa 2a. Furthermore, treatment of PV CD34+ cells with ABT-737 plus Peg IFNa 2a (200U) lead to the appearance of a smaller proportion of JAK2 V617F-positive (46.7±26%) hematopoietic progenitor cells as compared to cells incubated with cytokines alone (81.7±17%) or cytokines plus Peg IFNa 2a (69±20%). These data suggest that low doses of Peg IFNa 2a selectively and directly eliminate Jak2V617F hematopietic progenitor cells which likely accounts for the therapeutic responses that have been observed with the use of this agent in the clinic. The enhanced elimination of JAK2V617F hematopoietic progenitor cells observed with the combination of ABT-737 and Peg-IFNa-2a suggests that this strategy might be an even more optimal approach for the treatment of JAK2V617F positive MPN which merits further testing in the clinic. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Treatment with the Bcl-xL inhibitor ABT-737 in combination with interferon α specifically targets JAK2V617F-positive polycythemia vera hematopoietic progenitor cells

Blood ◽  
2010 ◽  
Vol 116 (20) ◽  
pp. 4284-4287 ◽  
Author(s):  
Min Lu ◽  
Jiapeng Wang ◽  
Yan Li ◽  
Dmitriy Berenzon ◽  
Xiaoli Wang ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Hematopoietic Progenitor Cells ◽  
Interferon Α ◽  
Low Doses ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Inhibitory Activities ◽  
Higher Doses ◽  
Bh3 Mimetic ◽  
Modest Effect

Abstract Polycythemia vera (PV) treatment with interferon α (IFNα) is frequently limited by dose-related toxicity. PV CD34+ cells are characterized by overexpression of Bcl-xL, which can be antagonized by ABT-737 leading to apoptosis. We explored the effects of ABT-737 and IFNα on PV hematopoiesis. Both IFNα and ABT-737 alone or in combination had a modest effect on normal hematopoiesis but each individually were able to markedly induce PV CD34+ cell apoptosis and suppress hematopoietic colony formation. The inhibitory activities of these agents in combination were greater against PV hematopoiesis than either agent alone. The exposure of PV CD34+ cells to low doses of IFNα and ABT-737 in combination resulted in the reduction of the proportion of JAK2V617F+ colonies similar to that observed with higher doses of IFNα. These data provide the rationale for combination therapy with low doses of IFNα and a BH3 mimetic for patients with PV.

The Transcriptional Program of Polycythemia Vera Revelas Altered Expression of Multiple Putative Tumor Suppressor Genes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 796-796 ◽  
Author(s):  
Windy D. Berkofsk-Fessler ◽  
Jonathan D. Licht ◽  
Melanie-Jane McConnell ◽  
Donna S. Neuberg ◽  
Timothy S. Bowler ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Tumor Suppressor Genes ◽  
Hematopoietic Progenitor Cells ◽  
Multiple Testing Correction ◽  
Hematopoietic Progenitor ◽  
Suppressor Genes ◽  
Putative Tumor Suppressor ◽  
Cd34 Cells

Abstract Polycythemia vera (PV) is a myeloproliferative disease characterized by accumulation of erythrocytes and cells of the myeloid and megakaryocyte lineages. Although genes like PRV-1 and PTP-MEG2 have been implicated in the pathology of PV, there is no consensus on their importance in the disease process. Progenitor cells from PV patients can grow in the absence of erythropoietin, and are hypersensitive to a variety of other growth factors. This suggests that polycythemic hematopoietic progenitor cells possess a significantly different genetic program. We tested this idea by molecular profiling hematopoietic progenitor cells (CD34+) from PV specimens and normal donors. We purified CD34+ cells from the marrow of 10 PV patients and harvested total RNA. Biotinylated cRNA was made through two rounds of linear amplification, and hybridized to Affymetrix HGU133A genechips. CD34+ cells from marrow mononuclear cells of 5 normal controls were processed similarly. The resulting datasets were normalized to the median across chips and across genes. Unsupervised hierarchical clustering showed that PV samples had a distinct gene expression profile from the controls. We then performed supervised clustering using a non-parametric t-test (Wilcoxon rank sum test) using the Benjamini and Hochberg multiple testing correction held to a p-value of 0.01 to determine genes that were significantly different between disease and normal samples. Using these stringent criteria, there were 331 genes that reached significance. Strikingly most of these were decreased in expression compared with control CD34+ cells and only 34 genes were upregulated in PV. A 35 gene predictor set was discovered through the use of a k-nearest neighbor metric. This set was 100% accurate for the prediction of PV in a leave one out cross-validation approach. Among these genes are EVI1, a known oncogene and one of only two genes upregulated in PV on this list, and the putative tumor suppressor genes TUSC4 (NPR2), NDRG1 and KLF4. Also among the predictor genes is BAALC, a gene expressed in normal CD34+ cells and known to be a prognostic indicator gene for acute myeloid leukemia.

Selected Members of the Angiopoietin-Like Family of Human Proteins Enhance Survival and Replating Capacity of Immature Human Hematopoietic Progenitor Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3367-3367
Author(s):  
Hal E. Broxmeyer ◽  
Edward F. Srour ◽  
Scott Cooper ◽  
Carrie T. Wallace ◽  
Giao Hangoc ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Gm Csf ◽  
Cd34 Cells

Abstract Angiopoietin-like (ANGPTL) molecules are a family of secreted proteins which have characteristic structures of angiopoietins. This includes a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils (CC), a short linker peptide, and a globular fibrinogen-like domain (FLD). Zhang et. al. (Nat. Med., 12(2):240–245, 2006) reported that human ANGPTL-2, 3, 3CC, 5 and 7, but not ANGPTL4, enhanced ex-vivo expansion of highly enriched mouse bone marrow (BM) long term competitive repopulating hematopoietic stem cells in serum-free culture with SCF, TPO, IGF-2, and FGF-1. To the present, there have not been publications describing effects of human ANGPTL molecules on hematopoietic progenitor cells (HPC) or on human hematopoietic cells. Thus, we evaluated purified recombinant human ANGPTL-2CC, 3, 3CC, 3FLD, 4, 4CC, 5, 6 and 7 (AdipoGen, Inc, Seoul, Korea) for effects on proliferation and survival of HPC from human cord blood (CB). No endotoxin was detected in the ANGPTL molecule preparations (<0.1 EU/ug endotoxin per LAL method). None of the ANGPTL molecules at up to 500ng/ml stimulated HPC colony formation by themselves, or enhanced or inhibited HPC colony formation of low density (LD) or CD34+ human cord blood (CB) cells stimulated by GM-CSF, GM-CSF plus SCF, Epo plus SCF, or the combination of Epo, SCF, IL-3 and GM-CSF. However, ANGPTL-2CC, 3, and 3CC at 200 and 100, but not 10ng/ml significantly enhanced the survival of human LD and CD34+ HPC (CFU-GM, BFU-E, CFU-GEMM) subjected to delayed addition of growth factors (Epo, SCF, IL-3, GM-CSF). Survival is a measure of anti-apoptosis for the hematopoietic progenitor cells in this context. The other ANGPTL molecules were not active at up to 500ng/ml. The survival enhancing effects of ANGPTL-3 was neutralized by purified rabbit anti-ANGPTL-3 IgG, but not by anti-ANGPTL-4, -6, or -7. Replating of HPC colonies offers an estimate of the self-renewal capabilities of HPC. We found that ANGPTL-3, but not -4, -6, or -7 enhanced the replating capacity of single CFU-GEMM colonies by greater than 2 fold. Thus far, we have not detected significant effects of the ANGPTL molecules on ex-vivo expansion of human CB CD34+ cells, alone, or in combination with SCF, TPO, Flt3-ligand, with or without IL-3, after assessing output of HPC, % and numbers of CD34+ cells, or cell cycle status of produced cells. In summary, we have implicated a few members of the ANGPTL family of proteins in functional effects on human HPC survival and replating/”self-renewal” activity, effects requiring the CC domain of the ANGPTL molecules. This information may be of relevance to regulation of HPC, and of use for protocols to use these cells for transplantation.

Combination Treatment with RG7112 and Pegylated Interferon α 2a Specifically Targets JAK2V617F Positive Progenitor Cells From Patients with Myeloproliferative Neoplasms.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2865-2865
Author(s):  
Min Lu ◽  
Xiaoli Wang ◽  
Goar Mosoyan ◽  
Yan Li ◽  
Ronald Hoffman
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Combination Treatment ◽  
Colony Formation ◽  
Combined Treatment ◽  
Therapeutic Effects ◽  
Low Doses ◽  
Cd34 Cells ◽  
Long Term Treatment ◽  
Cord Blood Cd34

Abstract Abstract 2865 We have recently reported that MDM2 levels are increased in PV CD34+ cells as a consequence of JAK2617F, while the p53 levels are reduced in CD34+ cells from both patients with PV and PMF. These observations led us to explore therapeutic approaches by which to up-regulate p53 as a strategy to treat MPN patients. We previously reported that combination treatment with low doses of Peg IFNα 2a and Nutlin-3, an antagonist of MDM2, induced PV CD34+ apoptosis and inhibited PV colony formation significantly. The combination of these agents also decreased the proportion of JAK2V617F-positive HPCs. We demonstrated that these two drugs affect p53 through different pathways with Peg IFNα 2a activating p38 MAP kinase and STAT1 leading to increased p53 transcription while nutlin-3 prevents the degradation of p53 (Lu et al, Blood, 2012; In Press). RG7112 is an orally bioactivity small molecule inhibitor of p53-MDM2 binding which activates the p53 pathway. RG7112 is currently being evaluated in several phase 1 cancer trials. Based on our previous data, we hypothesized that RG7112 would be an effective drug to combine with low doses of IFN alpha to treat MPN patients. In order to evaluate the therapeutic effects of RG7112 alone or in combination with Peg IFNα 2a on hematopoietic progenitor cells from MPN patients, CD34+ cells were isolated from patients with PV and PMF patients and were treated with varying of doses of RG7112 alone or/and in combination with a low dose of Peg IFNα 2a and its effects on PV CD34+ cell proliferation, apoptosis and colony formation was evaluated. The effects of this drug on MPN CD34+ cells was compared to its effects on cord blood CD34+ cells After treatment with RG7112 at doses ranging from 100 nM to 5000 nM for 4 days, the numbers of PV CD34+ cells were reduced to a far greater degree (about 60%) than that observed with cord blood CD34+ cells (90% and 80%) at the doses between 200 and 500 nM indicating that low doses of RG7112 selectively affects MPN HPCs. 200nM of RG7112 and 200 U/ml of Peg-IFNα 2a alone induced cell cycle arrest of PV CD34+ cells, and combination treatment significantly promoted apoptosis of MPN CD34+ cells (2.5 folds). This observation leads us to examine the possibility that low doses of RG7112 might be used in combination with Peg-IFNα 2a to treat MPN patients. Treatment with 200 nM of RG7112 alone decreased PV CFU-GM and BFU-E-derived colony formation by 20% while combination treatment with 200 U/ml of Peg-IFNα 2a decreased PV CFU-GM and BFU-E-derived colony formation by 60% and 70%, respectively. Similarly, treatment with 200 nM of RG7112 alone decreased PMF CFU-GM and BFU-E-derived colony formation by 20% and 40%, respectively, while combination treatment with 200 U/ml of Peg-IFNα 2a decreased PMF CFU-GM and BFU-E-derived colony formation by 60% and 80%, respectively. We also found that treatment with both agents in combination also decreased the size of the hematopoietic colonies. By contrast, treatment with the same low doses of RG7112 and Peg-IFNα 2a alone or in combination did not significantly affect the ability of normal CD34+ cells to generate CFU-GM and BFU-E-derived colonies. Combined treatment with low doses of RG7112 and Peg-IFNα 2a decreased the total number of JAKV617F-positive PV HPC by 9% - 25% (p=0.014) leading to an increased proportion of colonies with wild type JAK2. In conclusion, these data demonstrate that IFNα plus RG7112 specifically target malignant MPN HPC by activating p53 in vitro, which we propose can be exploited therapeutically to eliminate JAK2V617F positive HPC in both PV and PMF patients. These results strongly suggest that combinations of low doses of IFNα and RG7112 might serve as a novel therapeutic strategy for the long term treatment of MPN patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Orderly Process of Sequential Cytokine Stimulation Is Required for Activation and Maximal Proliferation of Primitive Human Bone Marrow CD34+ Hematopoietic Progenitor Cells Residing in G0

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 658-668 ◽  
Author(s):  
Amy C. Ladd ◽  
Robert Pyatt ◽  
Andre Gothot ◽  
Susan Rice ◽  
Jon McMahel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Phase I ◽  
Progenitor Cells ◽  
Phase Ii ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Cytokine Stimulation ◽  
Phase Iv

Abstract Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+ ). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with &lt;20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0 /G1 , unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation.

scholarly journals Allogeneic blood stem cell transplantation: peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34+ Thy- 1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2842-2848 ◽  
Author(s):  
M Korbling ◽  
YO Huh ◽  
A Durett ◽  
N Mirza ◽  
P Miller ◽  
...  
Keyword(s):  
Body Weight ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Allogeneic Transplantation ◽  
Hematopoietic Progenitor Cells ◽  
Donor Blood ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
The Mean ◽  
Single Bone

Abstract Apheresis-derived hematopoietic progenitor cells have recently been used for allogeneic transplantation. Forty-one normal donors were studied to assess the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (12 micrograms/kg/d) on the peripheralization of hematopoietic progenitor cells and lymphoid subsets. The white blood cell, polymorphonuclear cell (PMNC), and lymphocyte concentrations at the peak of rhG-CSF effect in the donor's peripheral blood (PB) exceeded baseline by 6.4-, 8.0-, and 2.2-fold, respectively. Corresponding concentrations of PB CD34+ cells and primitive subsets such as CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells increased by 16.3-fold, 24.2-fold, and 23.2-fold, respectively in eight normal donors. The percentage of CD34+ Thy-1dim and CD34+ Thy- 1dim CD38- cells among CD34+ cells increased as well, suggesting an additional peripheralization effect of rhG-CSF on primitive CD34+ subsets. The preapheresis PB CD34+ and CD34+ Thy-1dim cell concentrations were predictive of their corresponding apheresis yield per liter of donor blood processed PB lymphoid subsets were not significantly affected by rhG-CSF treatment. The mean apheresis-derived yield of CD34+, CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells per kilogram of recipient body weight and per liter of donor blood processed was 48.9 x 10(4) (n = 41), 27.2 x 10(4) (n = 10), and 1.9 x 10(4) (n = 10), respectively. As compared with 43 single bone marrow (BM) harvest, the CD34+ cell yield of peripheral blood progenitor cell allografts of 41 normal donors exceeded that of BM allografts by 3.7- fold and that of lymphoid subsets by 16.1-fold (CD3+), 13.3-fold (CD4+), 27.4-fold (CD8+), 11.0-fold (CD19+), and 19.4-fold (CD56+CD3-). All PBPC allografts were cryopreserved before transplantation. The mean recovery of CD34+ cells after freezing, thawing, and washing out dimethylsulfoxide was 86.6% (n = 31) and the recovery of lymphoid subsets was 115.5% (CD3+), 121.4% (CD4+), 105.6% (CD8+), 118.1% (CD19+), and 102.4% (CD56+CD3-). All donors were related to patients: 39 sibling-to-sibling, 1 parent-to-child, and 1 child-to-parent transplant. Thirty-eight transplants were HLA fully identical, two transplants differed in one and two antigens. Engraftment occurred in 38 recipients; two patients died too early to be evaluated, and one patient did not engraft. The lowest CD34+ cell dose transplanted and resulting in complete and sustained engraftment was 2.5 x 10(6)/kg of recipient body weight.(ABSTRACT TRUNCATED AT 400 WORDS)

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