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Recombinant human erythropoietin (rHuEPO) stimulates erythropoietic bone marrow cells and increases erythrocyte production. This prospective study was designed to evaluate the effects of rHuEPO on regeneration of erythropoiesis after allogeneic or autologous bone marrow transplantation (BMT). Seventeen centers participated in this randomized, double-blind, placebo-controlled multicenter trial. The randomization was performed centrally for each center and stratified according to allogeneic or autologous BMT and major ABO-blood group incompatibility. One hundred and six patients received rHuEPO after allogeneic BMT and 109 patients received placebo. After autologous BMT, 57 patients were treated with rHuEPO and 57 with placebo. Patients received either 150 IU/kg/day C127 mouse-cell-derived rHuEPO or placebo as continuous intravenous infusion. Therapy started after bone marrow infusion and lasted until independence from erythrocyte transfusions for 7 consecutive days with stable hemoglobin levels greater than or equal to 9 g/100 mL or until day 41. After allogeneic BMT, the reticulocyte counts were significantly higher with rHuEPO from day 21 to day 42 after BMT. The median time (95% confidence intervals) to erythrocyte transfusion independence was 19 days (range, 16.3 to 21.6) with rHuEPO and 27 days (range, 22.3 to > 42) with placebo (P < .003). The mean (+/-SD) numbers of erythrocyte transfusions until day 20 after BMT were 6.6 +/- 4.8 with rHuEPO and 6.0 +/- 3.8 with placebo. However, from day 21 to day 41, the rHuEPO-treated patients received 1.4 +/- 2.5 (median, 0) transfusions and the control group received 2.7 +/- 4.0 (median, 2) transfusions (P = .004). In the follow-up period from day 42 up to day 100, 2.4 +/- 5.6 transfusions were required with rHuEPO and 4.5 +/- 9.6 were required with placebo (P =.075). A multivariate analysis (ANOVA) showed that acute graft-versus-host disease (GVHD), major ABO-blood group incompatibility, age greater than 35 years, and hemorrhage significantly increased the number of transfusions. However, after day 20, rHuEPO significantly reduced the number of erythrocyte transfusions in these patient groups, as well as reducing incompatibility in the major ABO-blood group. For the whole study period, rHuEPO reduced the transfusion requirements in GVHD III and IV from 18.4 +/- 8.6 to 8.5 +/- 6.8 U (P = .05). After autologous BMT, there was no difference in the time to independence from erythrocyte transfusions and in the regeneration of reticulocytes. Marrow purging strongly increased the requirement for transfusions as well as the time to transfusion independence. rHuEPO had no relevant effect on regeneration of thrombopoiesis after allogeneic or autologous BMT. Overall, no major differences in side effects or complications between rHuEPO-treated and placebo-treated patients occurred. After allogeneic BMT, rHuEPO significantly accelerates the reconstitution of erythropoiesis and reduces the number of erythrocyte transfusions after day 20. The strongest effect occurs in patients with acute GVHD. After autologous BMT, rHuEPO has no clinically relevant effect on regeneration of erythropoiesis. (C) 1994 by The American Society of Hematology.

Myeloblasts derived from the peripheral blood of a patient with acute myelogenous leukemia (ORL47) were found to represent the malignant counterpart of the newly elucidated monocyte-dendritic cell colony- forming unit (mono-DC-CFU). The specific cytokine conditions require to achieve intermediate and terminal maturation of DCs and monocytes from these progenitors were defined. With tumor necrosis factor (TNF) + granulocyte-macrophage colony-stimulating factor (GM-CSF) + stem cell factor treatment numerous colony-like clusters developed. In contrast with normal DC development, further advancement of mono-DC-CFU and terminal DC maturation from the leukemic cells were dependent on the addition of interleukin-6. Functional and phenotypic analysis showed that the capacity to differentiate was maintained fully in the DC compartment, but only partially in the monocyte compartment, as judged by the lack of CD14 surface expression. Cells found at intermediate stages of DC development were potent stimulators of a mixed leukocyte reaction, a function usually attributed to mature DCs. As previously shown for normal DC development, antibodies to TNF alpha and GM-CSF blocked proliferative responses and DC growth. The importance of these observations in the classification of leukemias, normal DC development, and potential clinical strategies is discussed.

We analyzed the mutations in patients from 10 Polish kindreds with a bleeding diathesis due to factor VII deficiency. Patients from eight families had plasma levels of factor VII coagulant activity (VII:C) and factor VII antigen (VII:Ag) that were less than 4% of normal. The coding sequence of the factor VII gene was amplified from genomic DNA by polymerase chain reaction (PCR). Sequencing demonstrated a C to T transition at position 10798 resulting in Ala294Val, a G to A transition at 10976 resulting in Arg353Gln, and a single bp deletion at 11125 to 11128 causing a frameshift mutation in the triplet encoding amino acid 404. Homozygosity for the three sequence alterations was confirmed with the restriction enzymes AvaII and MspI and allele specific PCR, respectively. A homozygous patient from a ninth family with levels of VII:C and VII:Ag of 4% and 17%, respectively, had Ala294Val and the frameshift mutation, but not Arg353Gln. Investigation of a homozygous patient from a tenth kindred with VII:C and VII:Ag of 11% and 47%, respectively, demonstrated Ala294Val and Arg353Gln, but not the frameshift mutation. Based on the above data, we conclude that the frameshift mutation in the codon for amino acid 404 is associated with marked reductions in VII:C, Arg353Gln can decrease plasma levels of factor VII in the presence of other mutations in the factor VII gene, and Ala294Val results in a dysfunctional factor VII molecule.

We have investigated the question of erythropoietin (Epo) hypersensitivity versus Epo independence as the basis for the endogenous erythroid bursts (EEBs) that develop in cultures without added Epo from hematopoietic cells of polycythemia vera (PV) patients. Using an improved serum-free (SF) medium containing interleukin (IL)-3, but no insulin-like growth factor-1 (IGF-1), and devoid of contaminants that influence erythropoiesis, we compared circulating normal and PV early erythroid progenitors (BFU-E) with respect to their responses in vitro to recombinant human (rHu) Epo. Cultures were seeded with Ficoll- Hypaque density-separated peripheral blood (PB) mononuclear cells (MNCs), and erythroid bursts, together with their component colonies of > or = 50 cells, were scored in situ at 13 to 16 days of culture. The Epo dose-response curve of BFU-E from PV patients was found to be statistically indistinguishable from that of normal subjects. This observation provides compelling evidence against the Epo- hypersensitivity hypothesis. In the complete SF medium minus Epo, the sensitivity of BFU-E to IGF-1 was much greater in PV than in normals, the dose-response curve being shifted to the left by at least 2 orders of magnitude. These data show that the erythroid progenitor cell response in PV is hypersensitive to IGF-1, and independent of Epo. The data also emphasize the importance of truly SF medium conditions for assessment of progenitor cell sensitivities to recombinant growth factors. Depletion of adherent cells totally prevented erythroid burst formation by normal circulating progenitors, but did not prevent the hypersensitive response to IGF-1 of such cells from PV patients. Hence, again unlike its normal counterpart, the progenitor cell response in PV appears to be independent of adherent cell control.

Although peripheral blood stem cell collections (PBSC) are thought to have less tumor involvement than bone marrow (BM), the incidence of circulating tumor cells in patients with breast cancer has not been widely investigated. We prospectively investigated the incidence and viability of tumor cell involvement in PBSC and BM collections from breast cancer patients undergoing high-dose chemotherapy/hematopoietic stem cell transplantation. Paired samples of PBSC and BM from 48 patients were analyzed using an immunocytochemical technique that detects one epithelial-derived tumor cell per 5 x 10(5) mononuclear cells. Immunostained tumor cells were detected in 9.8% (13/133) PBSC specimens from 9/48 (18.7%) patients and in 62.3% (38/61) BM specimens from 32/48 (66.7%) patients, a significantly higher rate than in PBSC (P < .005). The geometric mean concentration of tumor cells in contaminated PBSC specimens was 0.8/10(5) mononuclear cells (range 0.33 to 2.0/10(5)) compared with 22.9/10(5) mononuclear cells in BM (range 1 to 3,000/10(5), P < .0001). In culture experiments, clonogenic tumor colonies grew in 21/26 immunocytochemically positive specimens. No tumor colony growth was detected in 30/32 immunocytochemically negative specimens. Immunocytochemical detection of tumor involvement in BM and PBSC correlated significantly with in vitro clonogenic growth (P < .0001). We conclude that PBSC contain fewer tumor cells than paired BM specimens from patients with advanced breast cancer and that these tumor cells appear to be capable of clonogenic growth in vitro.

The effect of human fibrinogen on the proliferation of purified SBA- CD34+ human bone marrow progenitors was investigated in clonal cultures. Fibrinogen alone or in combination with erythropoietin had no significant effect. However, in the presence of recombinant human interleukin-3 (IL-3), fibrinogen increased significantly in a dose- dependent manner the number of mixed and burst-forming unit-ethrocyte-- derived colonies, whereas the number of other colonies did not significantly change. In the presence of fibrinogen, low concentrations of IL-3 (0.17 U/mL) produced three times more mixed colonies than without fibrinogen, reaching the number of colonies obtained with optimal concentrations of IL-3 (1.67 U/mL). Fibrinogen fragment D had the same effect in the presence of IL-3 as intact fibrinogen, whereas fibrinogen fragment E and human collagen IV did not. This effect was not mediated by integrins, because peptides or monoclonal antibodies that block fibrinogen binding on integrins alpha IIb beta 3, alpha v beta 3 (RGD-peptides), alpha m beta 2 (OKM-1), and alpha x beta 2 (HC1/1) did not affect the observed mitogenic effect. The mitogenic effect of fibrinogen and its D fragment was not mediated by induction of IL-6 or granulocyte--colony-stimulating factor secretion, because it was not inhibited by blocking antisera against these two growth factors. Our results indicate that fibrinogen potentiates the effect of IL-3 on primitive hematopoietic progenitors and suggest that the mitogenic effect of fibrinogen could be mediated via a specific mitogenic receptor that does not belong to the integrin family.

Blast colony assays were performed on freshly obtained bone marrow samples from 19 newly diagnosed or relapsed children with acute lymphoblastic leukemia (ALL) of B lineage to determine the effect of added granulocyte-monocyte colony-stimulating factor (GM-CSF). Of the 19 marrow samples tested, 7 responded to GM-CSF with a mean increase in ALL blast colonies of 346%. Blast cells from one of the responders chosen for flow cytometric study showed expression of GM-CSF receptors on 38% of cells. These findings prompted us to establish five ALL cell lines of diverse phenotypes to examine the expression of GM-CSF and GM- CSF receptor genes in human leukemia, and to determine the role of GM- CSF in autocrine and paracrine growth control of ALL cells. One line, termed G2, manifested a GM-CSF-mediated autocrine pattern of cell growth with the following features: G2 blast colony growth in a serum- free system without added growth factor was density dependent; exogenous GM-CSF augmented G2 colony formation when the cells were seeded at low density; G2 cells constitutively expressed mRNA for GM- CSF and GM-CSF receptor; G2 cells also produced and secreted measurable amounts of GM-CSF into cell culture supernatant; and, monoclonal anti- GM-CSF antibodies abolished G2 colony growth when added to cultures with cells seeded at low density without growth factors. Of the other four ALL cell lines, three expressed mRNA for GM-CSF receptor and responded in vitro to added GM-CSF with increased blast colony growth; however, none of these four cell lines expressed mRNA for constitutive production of GM-CSF. A fifth ALL cell line lacked receptors for GM-CSF and did not respond in clonogenic assays to added GM-CSF. Thus, a bioregulator of normal hematopoiesis plays a central role in autocrine growth control of G2 ALL cells, and an important paracrine growth- promoting role in three of four other ALL cell lines.

To determine whether an association exists between the presence of antiphospholipid antibodies and pregnancy loss, a cross-sectional study was performed. Consecutive women who were referred to three outpatient rheumatology clinics and who had systemic lupus erythematosus (SLE) and a history of one or more pregnancies were evaluated. Patients were interviewed to determine outcomes of all previous pregnancies. Blood was taken on two separate occasions at least 3 months apart to test for the presence of the lupus anticoagulant and anticardiolipin antibodies; on both occasions, five tests of the lupus anticoagulant, with well- defined normal ranges, and an enzyme-linked immunosorbent assay to measure IgG anticardiolipin antibodies were performed. Patients were considered to be positive for the lupus anticoagulant if one or more tests was abnormal on both occasions and positive for anticardiolipin antibodies if the test was abnormal on both occasions. Forty-two women were studied. Statistically significant associations were shown between lupus anticoagulant positivity and previous pregnancy loss (odds ratio [OR], 4.8; 95% confidence intervals [CI], 1.0 to 23.6; P = .05) and between anticardiolipin antibody positivity and previous pregnancy loss (OR, 20.0; 95% CI, 1.3 to 97.0; P = .01). All seven women with multiple episodes of pregnancy loss were lupus anticoagulant positive and four of these were also anticardiolipin antibody positive. If patients who are transiently positive for lupus anticoagulant and/or anticardiolipin antibodies are considered to be test positive, the associations with pregnancy loss are no longer statistically significant. Within the group of lupus anticoagulant-positive patients, we observed stronger associations between the presence of six or more positive tests and pregnancy loss than between the presence of two to five positive tests and pregnancy loss. No single test for the lupus anticoagulant provides a statistically significant association with pregnancy loss. The results of our study show that by performing multiple lupus anticoagulant tests and by repeating testing for lupus anticoagulant and anticardiolipin antibodies on more than one occasion, significant associations between the presence of antiphospholipid antibodies and previous pregnancy loss can be shown in patients with SLE.

There is substantial evidence that the volume of medical procedures in a hospital has an inverse relationship with mortality. We analyzed data for 1313 recipients of HLA-identical sibling bone marrow transplants for early leukemia (acute leukemia in first remission or chronic myelogenous leukemia in first chronic phase) to determine whether transplant outcome differed in small and large centers. Transplants were performed in 86 bone marrow transplant centers active between the years 1983 and 1988, which participated in the International Bone Marrow Transplant Registry. Twenty-one (24%) centers performed five or fewer allogeneic transplants per year during the study period; five (6%) performed more than 40 per year. After adjustment for differences in patient and disease characteristics, the relative risks of treatment- related mortality (1.53, P less than .01) and treatment failure (1.38, P less than .04) were higher among patients who received transplants at centers doing five or fewer transplants per year than among those at larger centers. Among patients receiving transplants in centers performing more than five transplants a year, there was no statistically significant correlation between number of transplants and outcome.

Recent evidence indicates that Reed-Sternberg (RS) cells from many cases of Hodgkin's disease have features of activated lymphocytes and that lymphokines from activated lymphocytes induce proliferation of L- 428 RS cells. It is shown here that a lymphokine similar to a lymphokine secreted by activated lymphocytes is secreted by L-428 cells. This lymphokine has a molecular weight approximately equal to 68,000 daltons, identical to glycosylated recombinant interleukin-4 (rIL-4), and cross-reacts with monoclonal anti-IL-4 in Western immunoblotting. This Hodgkin's cell growth factor (HCGF) is 100% neutralized by polyclonal anti-IL-4 antibodies and competes for the IL- 4 receptor. After acid-elution, the L-428 RS cell has been shown to have 3,396 +/- 120 high-affinity receptor sites/cell. HCGF competes with rIL-4 for this receptor and L-428 cells contain mRNA for IL-4. Although all evidence indicates that IL-4 is an important secreted autocrine growth factor for L-428 RS cells, anti-IL-4 has no effect on the sustained serum-free growth of these Hodgkin's cells, suggesting that either the IL-4 receptor and the IL-4 receptor-growth factor complex are protected from antibody inhibition or other mechanisms are responsible for the sustained proliferation of L-428 RS cells.

Glycoprotein (GP) IIb-IIIa serves as the platelet fibrinogen receptor. Studies of the tertiary structure of GPIIIa have shown that the protein has a large loop structure of at least 325 amino acids in length. To further characterize this loop structure, intact platelets were digested with alpha-chymotrypsin. Digestion products were examined using the anti-GPIIIa monoclonal antibodies (MoAbs) AP3, D3GP3, and C5GP3, as well as the human alloantibody, anti-PLA1. AP3 recognized GPIIIa digestion products of 109, 95, and 68 Kd. D3GP3 and C5GP3 recognized an additional band of 51 Kd. Time course digestions demonstrated that the 51-Kd fragment was generated by proteolysis of the 68-Kd peptide. Sequence analysis of the reduced 51-Kd peptide showed that this fragment began at amino acid 422. The nonreduced 51-Kd peptide was reactive with antibodies directed against the first 13 amino acids of GPIIIa, demonstrating the presence of a covalently attached N-terminal peptide. These data suggest that: (1) the minimum length of the loop structure is at least 384 amino acids; (2) the AP3 epitope is formed at least in part by a determinant contained within residues 348 to 421; and (3) the D3GP3 and C5GP3 epitopes are contained within amino acids 422 to 692 of GPIIIa, a region that may be flexible and involved in conformational changes that occur after ligand binding.

Several culture media for the growth of human circulating erythroid burst-forming units (BFU-E) that have been claimed to be “serum-free” (“SF”) have actually included albumin preparations known to be contaminated with an undefined burst-promoting activity (BPA); a BPA has also been found in the preparations of other “SF” medium components. This has precluded reliable investigation of the growth factor (GF) requirements of these progenitors. Using a defatted, BPA- free bovine serum albumin (BSA) and the recombinant human growth factors (GFs) erythropoietin (rHu Epo), insulinlike growth factor 1 (rHu IGF-1), and interleukin-3 (rHu IL-3), we have developed an improved serum-free (SF) medium for the production of erythroid bursts from normal adult human peripheral blood mononuclear cells (PBMNC), which requires both hemin and retinyl acetate for its optimal performance. In the presence of BSA without IL-3 or Epo, no burst or colony formation was observed. With IL-3 and Epo alone, only a small number of day 14 erythroid colonies was obtained (12 +/- 1/10(5) PBMNC). Addition of hemin (0.1 mmol/L) allowed the direct scoring of day 14 hemoglobinized colonies and increased their number sevenfold (86 +/- 5). Inclusion of retinyl acetate at physiologic concentrations further augmented the number of colonies threefold to fourfold. Under these apparently optimal conditions, we found that IGF-I could entirely replace Epo. However, IGF-I required a 100-fold higher molar concentration than that of Epo to reach maximal stimulation. The combined effect of Epo and IGF-I was found to be less than the sum of their individual effects, suggesting an overlap in the sensitivities of erythroid progenitors to these GFs. The colony-forming efficiencies of erythroid progenitors in the improved SF medium was very high: 700 single, day 14 erythroid colonies/10(5) PB MNC (at 0.25 mmol/L hemin) distributed as 126 clusters (bursts), with a mean of 5.6 component colonies per burst. These findings show that IGF-I has an Epo-like activity that targets circulating early erythroid progenitors or their progeny, providing strong evidence for the existence of an Epo- independent pathway for normal human adult erythropoiesis, possibly operative when Epo levels are low.

In contrast to other neoplasms, antigen-specific autologous cytolytic T cells have not been detected in patients with human pre-B-cell leukemias. The absence of efficient B7 family (B7-1/CD80;B7-2/CD86)-mediated costimulation has been shown to be a major defect in tumor cells' capacity to function as antigen-presenting cells. We show here the generation of autologous anti-pre-B-cell leukemia-specific cytolytic T-cell lines from the marrows of 10 of 15 patients with pre-B-cell malignancies. T-cell costimulation via CD28 is an absolute requirement for the generation of these autologous cytolytic T cells (CTL). Although costimulation could be delivered by either bystander B7 transfectants or professional antigen-presenting cells (indirect costimulation), optimal priming and CTL expansion required that the costimulatory signal was expressed by the tumor cell (direct costimulatory. These anti-pre-B-cell leukemia-specific CTL lysed both unstimulated and CD40-stimulated tumor cells from each patient studied but did not lyse either K562 or CD40-stimulated allogeneic B cells. Cytolysis was mediated by the induction of tumor cell apoptosis by CD8(+) T cells via the perforin-granzyme pathway. Although we were able to generate anti-leukemia-specific CTL from the bone marrow, we were unable to generate such CTL from the peripheral blood of these patients. These studies show that antigen-specific CTL can be generated from the bone marrow of patients with pre-B-cell leukemias and these findings should facilitate the design of adoptive T-cell-mediated immunotherapy trials for the treatment of patients with B-cell precursor malignancies. (C) 1997 by The American Society of Hematology.

Inflammation may contribute to the pathogenesis of atherosclerosis. On the basis of previous reports that human atherosclerotic lesions contain alpha-defensins, a class of cationic proteins released by activated neutrophils, the study was designed to ask whether defensins modulate the binding and catabolism of low-density lipoprotein (LDL) by human vascular cells. The results of the study demonstrated that defensin stimulated the binding of (125)I-LDL to cultured human umbilical vein endothelial cells, smooth muscle cells, and fibroblasts approximately 5-fold in a dose-dependent and saturable manner. Defensin and LDL formed stable complexes in solution and on cell surfaces. Stimulation of LDL binding by defensin was not inhibited by antibodies against the LDL-receptor (LDL-R), or by recombinant receptor-associated protein, which blocks binding of ligands to the alpha(2)-macroglobulin receptor/LDL-R-related protein and other LDL-R family members. Furthermore, defensin stimulated the binding, endocytosis, and degradation of LDL by fibroblasts lacking LDL-R. Stimulation of LDL degradation by defensin was inhibited approximately 75% by low concentrations of heparin (0.2 units/mL) and was similarly reduced in CHO cells lacking heparan-sulfate-containing proteoglycans. The effect of defensin was substantially increased in cells overexpressing the core protein of the syndecan-1 heparan sulfate proteoglycan. The alpha-defensins released from activated neutrophils may provide a link between inflammation and atherosclerosis by changing the pattern of LDL catabolism from LDL-R to the less efficient LDL-R-independent, proteoglycan-dependent pathway. (Blood. 2000;96:1393-1398)

Integrin-mediated adhesion influences cell survival and may prevent programmed cell death. Little is known about how drug-sensitive tumor cell lines survive initial exposures to cytotoxic drugs and eventually select for drug-resistant populations. Factors that allow for cell survival following acute cytotoxic drug exposure may differ from drug resistance mechanisms selected for by chronic drug exposure. We show here that drug-sensitive 8226 human myeloma cells, demonstrated to express both VLA-4 (alpha4beta1) and VLA-5 (alpha5beta1) integrin fibronectin (FN) receptors, are relatively resistant to the apoptotic effects of doxorubicin and melphalan when pre-adhered to FN and compared with cells grown in suspension. This cell adhesion mediated drug resistance, or CAM-DR, was not due to reduced drug accumulation or upregulation of anti-apoptotic Bcl-2 family members. As determined by flow cytometry, myeloma cell lines selected for drug resistance, with either doxorubicin or melphalan, overexpress VLA-4. Functional assays revealed a significant increase in alpha4-mediated cell adhesion in both drug-resistant variants compared with the drug-sensitive parent line. When removed from selection pressure, drug-resistant cell lines reverted to a drug sensitive and alpha4-low phenotype. Whether VLA-4-mediated FN adhesion offers a survival advantage over VLA-5-mediated adhesion remains to be determined. In conclusion, we have demonstrated that FN-mediated adhesion confers a survival advantage for myeloma cells acutely exposed to cytotoxic drugs by inhibiting drug-induced apoptosis. This finding may explain how some cells survive initial drug exposure and eventually express classical mechanisms of drug resistance such as MDR1 overexpression.

We investigated the mechanisms responsible for severe factor VII (FVII) deficiency in homozygous Italian patients with either Gly97Cys or Gln100Arg mutations in the second epidermal growth factor domain of FVII. Transient expression of complementary DNA coding for the mutations in COS-1 cells showed impaired secretion of the mutant molecules. Using stably transfected Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling studies, immunohistochemistry, and experiments with inhibitors of protein degradation, showing that FVII-Cys97 did not accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII-Arg100, the level of intracellular FVII was not increased by several inhibitors of protein degradation, but FVII-Arg100 was retained in the endoplasmic reticulum for a longer period of time than wild-type FVII. FVII-Arg100 had a lower apparent molecular weight than did wild-type FVII under nondenaturing conditions, which is attributable to misfolding due to abnormal disulfide bond formation.

Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34(+) cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45(+) cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions.

The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF-mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF-containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF-mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF-driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.

The ob gene product, leptin, has been shown in several studies to be involved in weight control and recombinant leptin recently has entered clinical trials to treat obesity. The leptin receptor (OB-R/B219) is expressed in a variety of protein isoforms not only in the central nervous system, but also in reproductive, and hematopoietic tissues. We reported recently that the OB-R/B219 was associated with a variety of hematopoietic lineages as well as the small fraction of cells containing the long-term reconstituting hematopoietic stem cells. Herein we report that leptin significantly stimulates the proliferation and differentiation of yolk sac cells and fetal liver cells and stimulates directly hematopoietic precursors. Leptin alone can increase the number of macrophage and granulocyte colonies, and leptin plus erythropoietin act synergistically to increase erythroid development. These data show that leptin has a significant, direct effect on early hematopoietic development and can stimulate the differentiation of lineage-restricted precursors of the erythrocytic and myelopoietic lineages. These observations along with a recent report strongly support our previous hypothesis that leptin has an unanticipated important role in hematopoietic and immune system development.

Analysis of a variant translocation t(11;17) in a case of acute promyelocytic leukemia (APL) led to discovery of a novel zinc finger gene, PLZF, fused to the retinoic acid receptor-alpha (RAR alpha) gene. We reviewed the clinical and molecular features of five additional patients with t(11;17)-associated APL. The clinical course of three patients was characterized by early death and three experienced disseminated intravascular coagulation. Morphologically all of the patients fell in a unusual morphologic spectrum of APL, with features intermediate between M2 and M3 AML. All six patients had PLZF-RAR alpha gene fusion as detected by reverse transcription/polymerase chain reaction assay, Southern blotting, or pulsed-field gel electrophoresis. Five of the six patients failed to achieve complete remission after initial chemotherapy or differentiation therapy with all-trans retinoic acid (ATRA). A sixth patient responded to initial chemotherapy, but on relapse failed to respond to ATRA. When tested in vitro, cultured cells from three of the patients failed to differentiate in response to ATRA. APL associated with t(11;17) and fusion of the PLZF and RAR alpha genes is a discrete clinico-pathologic syndrome with a distinctly worse prognosis than t(15;17) APL.

Abstract 3379 Background: The BCR-ABL tyrosine kinase inhibitor imatinib is highly effective for chronic myeloid leukemia (CML). However, some patients gradually develop resistance to imatinib, resulting in therapeutic failure. Metabonomic and genomic profiling of patients' responses to drug interventions can provide novel information about the in vivo metabolism of low-molecular-weight compounds and extend our insight into the mechanism of drug resistance. Based on a multi-platform of high-throughput metabonomics, SNP array analysis, karyotype and mutation of Abl kinase domain, the metabolic phenotypes and genomic polymorphisms of CML patients and their diverse responses to imatinib were characterized. Methods: We identified 26 untreated CML patients (UCML), 33 patients treated with imatinib at daily doses of 300–800 mg, and 18 healthy volunteers. 14 patients were resistant to imatinib. Routine cytogenetic analysis was performed in patients. In the resistant patients, ABL kinase domain mutations were detected. High-quality genomic DNA was processed in accordance with the genomic mapping 250K NspI protocol and hybridized to 250K NspI SNP arrays according to the manufacturer's instructions in 9 patients treated with imatinib. Gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) was utilized for the measurement of the small molecular weight endogenous compounds. The metabolic phenotypes of CML patients, and the responses of CML to imatinib were characterized by means of GC/TOFMS based metabonomic technique. Results: Mutations were detected in only 1 BC patient (L232P, F336L, and C349R). A total of 44 deletions, 2 duplication, and 7 regions of loss of heterozygosity (LOH) were identified by SNP array analysis. In addition to sex chromosome, four of 6 CP RCML patients did not show other abnormal genome. Deletions, duplication and LOH on chromosome 17, 9, 22, 5 and 19 were identified in several important chromosomal regions of BC patients. The untreated CML patients (UCML) showed different metabolic pattern from those of healthy controls, and the discriminatory metabolites suggested the perturbed metabolism of the urea cycle, tricarboxylic acid cycle, lipid metabolism, and amino acid turnover in UCML. Some amino acids, such as glutamate, ornithine, glycine, and pyroglutamate, were found at higher levels in UCML compared with those in HC, which indicates a cellular requirement for a higher turnover of structural proteins. After imatinib treatment, patients sensitive to imatinib (SCML) and patients resistant to imatinib (RCML) had similar metabolic phenotypes to those of healthy controls and UCML, respectively. SCML showed a significant metabolic response to imatinib, with marked restoration of the perturbed metabolism. Most of the metabolites characterizing CML were adjusted to normal levels, including the intermediates of the urea cycle and tricarboxylic acid cycle (TCA). In contrast, neither cytogenetic nor metabonomic analysis indicated any positive response to imatinib in RCML. Taken together, it strongly suggested that metabolic variation between BC and CP patients were closely related to genomic alterations. Conclusion: We report for the first time the associated genetic and metabonomic responses of CML patients to imatinib and show that the perturbed in vivo metabolism of UCML is independent of imatinib treatment in resistant patients. Thus, metabonomics can potentially characterize patients sensitivity or resistance to drug intervention. Disclosures: No relevant conflicts of interest to declare.

About 30% of adults with acute lymphoblastic leukemia (ALL) and 20% to 40% of children and adults with acute myelogenous leukemia (AML) never achieve remission, even with intensive chemotherapy. Most die of resistant leukemia, often within 6 months or less. In this study of 126 patients with resistant ALL or AML, allogeneic bone marrow transplants from HLA-identical siblings produced remissions in 113 of 115 (98%) evaluable patients. The 3-year probability of leukemia-free survival was 21% (95% confidence interval, 15% to 29%). Leukemia-free survival was similar in ALL (23%, 12% to 40%) and AML (21%, 14% to 31%). Only 3 of 27 patients at risk relapsed more than 2 years posttransplant.

Trisomy 12 is the most common cytogenetic abnormality in chronic lymphocytic leukemia (CLL), and a number of studies have suggested that it may be an adverse prognostic indicator. We have evaluated the usefulness of fluorescence in situ hybridization with a chromosome 12- specific probe as a simple means for detecting trisomy 12 in interphase cells. Forty cases of B-cell CLL previously studied with conventional cytogenetic techniques were analyzed with a biotinylated probe to the centromeric region of chromosome 12. Thirty of these retrospective cases could be reevaluated with in situ hybridization. Our analysis showed three hybridization signals (ie, trisomy 12) in interphase cells from seven of seven cases found previously to have trisomy 12. Trisomy 12 was also detected in five additional cases: in one case thought to have a normal karyotype, in two cases that had been inadequate for routine cytogenetic analysis, and in two cases that had been found to have an abnormal karyotype without trisomy 12. In a prospective series of 20 newly accrued CLL cases, all cases were analyzed successfully by in situ hybridization and six (30%) showed trisomy 12. We were able to perform the analysis on routinely prepared and previously Wright- stained peripheral blood smears. We conclude that fluorescence in situ hybridization is a simple means for the detection of trisomy 12 in CLL. The technique is more sensitive than conventional cytogenetic analysis and would be a useful tool in clinical studies.

Graft rejection, mixed chimerism, graft-versus-host disease (GVHD), leukemia relapse, and tolerance are interrelated manifestations of immunologic reactivity between donor and host cells that significantly affect survival after allogeneic bone marrow transplantation (BMT). In this report, a mouse model of BMT, in which the donor and host were compatible at the major histocompatibility complex (MHC), was used (1) to examine the interrelationship of pretransplant conditioning and T- cell content of donor BM with regard to lymphoid chimerism and GVHD and (2) to determine how these factors affected graft-versus-leukemia (GVL) reactivity and donor-host-tolerance. AKR (H-2k) host mice were administered optimal or suboptimal total body irradiation (TBI) as pretransplant conditioning followed by administration of BM cells from B10.BR (H-2k) donor mice with or without added spleen cells as a source of T lymphocytes. Transplanted mice were injected with a supralethal dose of AKR leukemia cells 20 and 45 days post-BMT to assess GVL reactivity in vivo. The pretransplant conditioning of the host and T- cell content of the donor marrow affected the extent of donor T-cell chimerism and the severity of GVH disease. GVL reactivity was dependent on transplantation of mature donor T cells and occurred only in complete chimeras. Transplantation of T-cell-deficient BM resulted in the persistence of host T cells, ie, incomplete donor T-cell chimerism, even when lethal TBI was used. Mixed chimerism was associated with a lack of GVL reactivity, despite the fact that similar numbers of donor T cells were present in the spleens of mixed and complete chimeras. In this model, moderate numbers of donor T cells facilitated complete donor T-cell engraftment, caused only mild GVHD, and provided a significant GVL effect without preventing the subsequent development of tolerance after conditioning with suboptimal TBI. In contrast, severe, often lethal, GVHD developed when the dose of TBI was increased, whereas tolerance and no GVH/GVL reactivity developed when the T-cell content of the marrow was decreased.

Ultraviolet irradiation inhibits alloreactive and mitogen-induced responses and might reduce both graft-versus-host and host-versus-graft reactions after bone marrow transplantation (BMT). We have studied proliferative responses to mitogens and reactivity in mixed lymphocyte culture after irradiation with ultraviolet (UV)-B light using splenocytes from Balb/c (H-2d) and CBA (H-2k) mice. Response to mitogens and in MLC was strongly inhibited by 20 J/m2 and abolished at 50 J/m2. Clonogenic cell recovery (CFU-GM; CFU-S) after UV-B irradiation was also reduced. When bone marrow and spleen cells were transplanted from parent (Balb/c) animals into F1 hybrid (Balb/c X CBA) recipients, all animals died with features indicative of graft-versus- host disease (GVHD) in 34 days. If the grafts were first irradiated with 100 J/m2 of UV-B at a mean wavelength of 310 nm, then 76% survived to day 80 when they were killed and shown to have normal marrow cellularity. The remainder died in marrow aplasia or of GVHD. H-2 typing in a group of surviving recipients showed either donor hematopoiesis only (8 of 15), mixed allogeneic chimerism (5 of 15), or recipient type hematopoiesis (2 of 15). Higher doses (200 to 300 J/m2) were detrimental to survival with 88% of recipients dying in marrow aplasia. Syngeneic BMT in Balb/c mice showed slower hematopoietic reconstitution when the grafts were first irradiated with 100 J/m2. After BMT from Balb/c to CBA mice all recipients of unirradiated grafts died within 54 days. By contrast, after graft irradiation with 100 J/m2 survival of recipient animals to day 80 was 59%. If these grafts were treated with 50 J/m2 survival was only 26% with an increase in deaths due to GVHD. Hematopoiesis at day 80 in a group of survivors studied by Ig heavy chain allotyping indicated donor type hematopoiesis in 6 of 10 (50 J/m2) and 2 of 9 (100 J/m2). These data indicate that UV-B irradiation inhibits lymphocyte reactivity and can prevent GVHD. However, there is clear in vitro and in vivo evidence of stem cell damage, such that autologous marrow recovery was demonstrated in a proportion of recipients. In parent----F1 UV-irradiated transplants, sustained hematopoietic recovery was effected in the majority by donor stem cells.

Monocytes activated by lipopolysaccharide (LPS) and interferon gamma (IFN gamma) rapidly secrete a number of monokines with different functional properties. Interleukin–4 (IL–4), a T-cell derived cytokine, has been shown to reduce the production of monokines with cytostatic activity for tumor cells, chemotactic activity for monocytes, and factors that stimulate thymocyte proliferation. This latter activity is mediated by a number of monokines like IL–1, tumor necrosis factor alpha (TNF alpha), and IL–6. To elucidate which cytokines produced by monocytes are controlled by IL–4, we tested the effect of IL–4 on the secretion of IL–1 alpha, IL–1 beta, TNF alpha, and IL–6 induced by LPS or IFN gamma. IL–4 was found to inhibit the secretion of IL–1 beta and TNF alpha by activated monocytes almost 100%. The secretion of IL–6 was found to be reduced 70% to 85% in the presence of IL–4, whereas there was no effect on the secretion of IL–1 alpha (IL–1 alpha is mainly cell- associated). Time-course experiments demonstrate that IL–4 reduces the secretion of monokines for a prolonged period of time (greater than 40 hours). The reduced secretion of IL–1 beta and TNF alpha was specifically induced by IL–4 because anti-IL–4 antiserum completely restored normal monokine production. These data suggest that IL–4 plays a role in the regulation of immune responses by reducing the production of functionally important monokines.

Chronic graft-versus-host disease (GVHD) is an important complication of bone marrow transplantation. We analyzed risk factors for chronic GVHD in 2,534 recipients of HLA-identical sibling transplants surviving at least 90 days after transplantation. The actuarial probability of developing chronic GVHD within three years posttransplant was 46% +/- 3% (95% confidence interval). The most important risk factor for chronic GVHD was acute GVHD. The 3-year probabilities of chronic GVHD were 28% +/- 3%, 49% +/- 5%, 59% +/- 6%, 80% +/- 9%, and 85% +/- 15% for persons with grades 0, I, II, III, and IV acute GVHD, respectively (P less than .0001). Among patients with no or grade I acute GVHD, recipient age greater than 20 years, use of non-T-cell depleted bone marrow, and alloimmune female donors for male recipients predicted a higher risk of chronic GVHD. When all three adverse risk factors were present, the probability of chronic GVHD was 62% among persons with no prior acute GVHD and 85% among those with grade I acute GVHD. Among patients with grade II through IV acute GVHD, no other risk factor predicted chronic GVHD. These data identify individuals who might benefit from treatment strategies aimed at changing the incidence of chronic GVHD.

To determine whether graft-versus-leukemia (GVL) reactions are important in preventing leukemia recurrence after bone marrow transplantation, we studied 2,254 persons receiving HLA-identical sibling bone marrow transplants for acute myelogenous leukemia (AML) in first remission, acute lymphoblastic leukemia (ALL) in first remission, and chronic myelogenous leukemia (CML) in first chronic phase. Four groups were investigated in detail: recipients of non--T-cell depleted allografts without graft-versus-host disease (GVHD), recipients of non-- T-cell depleted allografts with GVHD, recipients of T-cell depleted allografts, and recipients of genetically identical twin transplants. Decreased relapse was observed in recipients of non--T-cell depleted allografts with acute (relative risk 0.68, P = .03), chronic (relative risk 0.43, P = .01), and both acute and chronic GVDH (relative risk 0.33, P = .0001) as compared with recipients of non--T-cell depleted allografts without GVHD. These data support an antileukemia effect of GVHD. AML patients who received identical twin transplants had an increased probability of relapse (relative risk 2.58, P = .008) compared with allograft recipients without GVHD. These data support an antileukemia effect of allogeneic grafts independent of GVHD. CML patients who received T-cell depleted transplants with or without GVHD had higher probabilities of relapse (relative risks 4.45 and 6.91, respectively, P = .0001) than recipients of non--T-cell depleted allografts without GVHD. These data support an antileukemia effect independent of GVHD that is altered by T-cell depletion. These results explain the efficacy of allogeneic bone marrow transplantation in eradicating leukemia, provide evidence for a role of the immune system in controlling human cancers, and suggest future directions to improve leukemia therapy.

Hydroxyurea is a successful and cost effective drug therapy for sickle cell disease. Treatment with hydroxyurea is associated with a significant decrease in sickle cell complications, hospitalizations and transfusion requirements by about 50% and mortality reduction by 40% in clinical studies. The drug is unfortunately underutilized in sickle cell disease in the United States despite clear efficacy data and management experience. There is no data on the utilization of hydroxyurea in Africa, a part of the world with the highest global burden of sickle cell disease. We prospectively interviewed 206 consecutive adults and pediatric sickle cell patients as part of the Nigerian pulmonary hypertension screening study and reviewed over 1000 patients followed longitudinally at Ahmadu Bello university teaching hospital in Zaria, Nigeria. We also interviewed 10 hematologists (3 specialists and 7 hematologists in training) at the same university hospital. 65% of the 206 prospectively evaluated patients met the Multicenter Study of Hydroxyurea clinical indications for hydroxyurea treatment. No patient (zero percent) was on hydroxyurea therapy. All hematologists (100%) reported their discomfort with instituting hudroxyurea. Barriers to hydroxyurea utilization identified by practitioners included safety and toxicity profile (100%), patient compliance (100%), effective follow up (100%), drug availability (100%), affordability (100%) and specifically concern for reactivation of latent tuberculosis (50%) and carcinogenesis (100%) and teratogenicity (100%). Only 5% of patients had been informed of or were aware of hydroxyurea as a treatment option in sickle cell disease. Patient related barriers to hydroxyurea identified include lack of awareness (95%), cost (100%), availability (100%), need for frequent follow up (90%), pregnancy restrictions and need for concomitant contraceptive use (98%) and risk of infections (98%). Our study indicates the absolute lack of hydroxyurea utilization in a major health care center in Nigeria. Nigeria has the highest incidence of sickle cell disease in the world with about 150,000 children born with the disease annually. Sickle cell disease accounts for about 9 –16% of under-five mortality rates in the country. The sickle cell disease related morbidity, mortality and health systems financial burden remains very high in Nigeria and most of Africa. Local health care provider education and support and patient counseling and education are needed for the successful introduction of hydroxyurea in Nigeria. Clinical studies designed to assess the safety and efficacy of hydroxyurea in unique African settings is needed to facilitate the introduction and utilization of hydroxyurea in Nigeria and other parts of Africa.