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Erythropoietin (EPO) gene transcription is activated in kidney cells in vivo and in Hep3B cells exposed to hypoxia or cobalt chloride. Hypoxia- inducible factor 1 (HIF-1) is a nuclear factor that binds to the hypoxia-inducible enhancer of the EPO gene at a site that is required for transcriptional activation. HIF-1 DNA-binding activity is induced by hypoxia or cobalt chloride treatment of Hep3B cells. We report that treatment of Hep3B cells with desferrioxamine (DFX) induced HIF-1 activity and EPO RNA expression with kinetics similar to the induction of HIF-1 by hypoxia or cobalt chloride. Induction by each of these stimuli was inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. DFX appears to induce HIF-1 by chelating iron as induction was inhibited by coadministration of ferrous ammonium sulfate. DFX administration to mice transiently increased EPO RNA levels in the kidney. As previously shown for hypoxia and cobalt treatment, DFX also induced HIF-1 activity in non-EPO-producing cells, suggesting the existence of a common hypoxia signal-transduction pathway leading to HIF-1 induction in different cell types.

Clinical manifestations of arterial and venous thrombosis in a family with protein C deficiency was associated with two mutations in the light chain of protein C: Glu20-->Ala and Val34-->Met. Further studies showed that the mutation Glu20-->Ala which eliminated a gamma- carboxylation site was exclusively responsible for the anticoagulant defect of activated protein C (APC). Membrane-bound human factor Va is inactivated by APC after two sequential cleavages of the heavy chain at Arg506 and Arg306. Human factor Va inactivation by human recombinant APC (rAPC) and a mutant molecule with an alanine instead of a glutamic acid at position 20 (rAPC(gamma 20A)) was investigated in the presence and absence of phospholipid vesicles. During a 2-hour incubation period of the cofactor with either rAPC or rAPC(gamma 20A). In the absence of a membrane surface, factor Va is cleaved quantitatively at Arg506 and retains approximately 60% of its initial cofactor activity. After a 2- hour incubation period with rAPC membrane-bound factor Va has no cofactor activity, whereas in the presence of a membrane surface and rAPC(gamma 20A) factor Va retains 60% of its initial cofactor activity. The completed loss in factor Va cofactor activity upon incubation of the membrane-bound cofactor with phospholipid vesicles and rAPC is associated with cleavages at Arg506 and Arg306, whereas membrane-bound factor Va cleavage at Arg306 by rAPC(gamma 20A) is impaired, resulting in a cofactor that is cleaved at Arg506. Slow cleavage at Arg306 occurs when membrane-bound factor Va is incubated with rAPC(gamma 20A) and only small amounts of fragments of M(r) = 45,000 and 30,000 are noticed. Our data show that the genetic defect which leads to the absence of a gamma-carboxylation site at Glu20 impairs membrane binding of human APC, which in turn is required for cleavage of factor Va at Arg306 and inactivation of the cofactor. The consequence of impaired membrane-dependent cleavage at Arg306 is manifested in vivo by venous and arterial thrombosis.

Peripheral blood cells (PBCs) collected by leukapheresis after progenitor mobilization with chemotherapy and growth factors have been used successfully to replace marrow autografts in protocols requiring stem-cell support. Moreover, such transplants are often associated with more rapid recovery of blood cell counts than is routinely achieved with bone marrow. While conditions that mobilize colony-forming cells (CFCs) into the circulation are becoming increasingly well characterized, little information is available as to how these or other mobilizing treatments may influence the release of more primitive cells into the peripheral blood. To quantitate the peripheral blood content of such cells, we used the long-term culture-initiating cell (LTC-IC) assay, which detects a cell type that is able to produce progeny CFCs after a minimum of 5 weeks in cultures containing marrow fibroblasts. In this report, we present the findings on 21 patients who were transplanted over a 7-year period at our institution with PBCs alone. PBCs were collected in steady-state (n = 6) or during the recovery phase after high-dose cyclophosphamide (Cy; n = 15, nine with and six without additional growth factor administration). PBCs collected from another 11 patients given granulocyte colony-stimulating factor (G-CSF) were transplanted together with autologous marrow. Time-course studies of nine patients after Cy +/- granulocyte-macrophage CSF (GM-CSF) showed that CD34+ cells, CFCs, and LTC-ICs fell from normal to undetectable levels after Cy, and increased at the time of white blood cell (WBC) recovery: LTC-ICs to a mean of sixfold and CFCs to a mean of 26-fold higher than normal. The mean number of CD34+ cells, CFCs, and LTC-ICs present in the PBC harvest was twofold to 10-fold higher after mobilization than in steady-state collections; however, more than 2-log interpatient variability was observed. After PBC transplantation, the median time to a WBC count more than 10(9)/L was 12 days; polymorphonuclear leukocyte (PMN) count more than 0.5 x 10(9)/L, 15 days; and platelet count more than 20 x 10(9)/L, 17 days, although patients who received fewer than 1.5 x 10(5) CFCs/kg had a more than 50% chance of delayed count recovery (> 28 days). Patients who received Cy + GM-CSF-stimulated PBCs had more rapid and consistent platelet recoveries as compared with other groups receiving Cy mobilized or steady-state PBCs alone, and a rapid WBC recovery after Cy predicted a rapid WBC recovery after transplantation.

After the intravenous infusion of N-hydroxysuccinimido biotin into dogs, 80.6% +/- 9.7% (n = 5) of platelets were covalently labeled with biotin. The in vivo survival of the biotinylated platelets was monitored by flow cytometry and was normal as compared with previous reports for dog platelets. The ability of the biotinylated platelets to be activated was analyzed by measuring the expression of cell-surface P- selectin after incubation with graded concentrations of thrombin. When P-selectin expression was examined 3 hours after labeling, biotinylated platelets were indistinguishable from the nonlabeled population of platelets, indicating that biotinylation did not adversely affect the cells. On consecutive days after biotinylation, the thrombin dose- response curves for biotinylated and nonbiotinylated platelets were repeated, and as the biotinylated-platelets aged, they became less responsive to thrombin. On days 3, 4, and 5, the thrombin EC50 for the aged, biotinylated platelets as compared with the total population of platelets was 136%, 150%, and 178%, respectively. Increasing age clearly impairs the reactivity of platelets towards thrombin as quantitated by the expression of cell-surface P-selectin.

We have previously observed that ionizing radiation induces tyrosine phosphorylation in human B-lymphocyte precursors by stimulation of unidentified tyrosine kinases and this phosphorylation is substantially augmented by vanadate. Ionizing radiation generates reactive oxygen intermediates (ROI). Because H2O2 is a potent ROI generator that readily crosses the plasma membrane, we used H2O2 to examine the effects of ROI on signal transduction. We now provide evidence that the tyrosine kinase inhibitor herbimycin A and the free radical scavenger N- acetyl-cysteine inhibit both radiation-induced and H2O2-induced activation of NF-kappa B, indicating that activation triggered by ROI is dependent on tyrosine kinase activity. H2O2 was found to stimulate Ins-1,4,5-P3 production in a tyrosine kinase-dependent manner and to induce calcium signals that were greatly augmented by vanadate. The synergistic induction of tyrosine phosphorylation by H2O2 plus vanadate included physiologically relevant proteins such as PLC gamma 1. Although treatment of cells with H2O2 alone did not affect the activity of src family kinases, treatment with H2O2 plus vanadate led to activation of the p56lck and p59fyn tyrosine kinases. The combined inhibition of phosphatases and activation of kinases provides a potent mechanism for the synergistic effects of H2O2 plus vanadate. Induction of tyrosine phosphorylation by ROI may thus lead to many of the pleiotropic effects of ROI in lymphoid cells, including downstream activation of PLC gamma 1 and NF-kappa B.

Recent data have shown that the PML-RAR alpha fusion gene resulting from translocation t(15;17) is a highly reliable molecular marker of acute promyelocytic leukemia (APL). In this study performed on 97 Chinese patients with APL, the retrotranscriptase/polymerase chain reaction (RT/PCR) was used to evaluate the clinical relevance of the long (L) or short (S) PML-RAR alpha fusion mRNA isoforms and to study minimal residual disease during clinical remission (CR). There were more early deaths during the all-trans retinoic acid (ATRA) induction treatment and more relapses within 2 years of CR in the S-type (6 of 19 cases) than in the L-type group (2 of 33 cases) (P < .025). Among 12 cases analyzed before and after the ATRA-induced CR, 9 cases (75%) showed positive RT/PCR, whereas only 3 cases showed a negative result, justifying the need for chemotherapy after ATRA-induced CR. Eleven of 62 APL patients in CR, after ATRA-induced CR and chemotherapy consolidation (follow-up, from 3 to 72 months), showed positive RT/PCR. Five of them relapsed within 1 to 6 months after the positive test; one converted to negative after further chemotherapy; and 5 remained in CR status without further PCR data. However, the latter 5 cases all received further intensive consolidation therapy after the PCR positivity. These results show that a positive RT/PCR of PML-RAR alpha is a sensitive predictor of relapse in APL.

There have been several new developments in the field of autoimmune neutropenia over the past decade. Neutropenia caused by antibodies directed against granulocyte precursor cells, the oligoclonal nature of antineutrophil antibodies, and the expanding knowledge of neutrophil antigens, particularly in relationship to autoantibodies, are exciting new areas of investigation. Knowledge has also been advanced in the effector mechanisms of neutrophil autoantibodies and the effect of autoantibodies on the neutrophil function. In addition, some clinical syndromes of immune neutropenia have been better defined over the past decade, such as autoimmune neutropenia of infancy and chronic idiopathic neutropenia in adults. The past decade also saw interesting developments in the treatment of immune neutropenia, particularly in the use of gammaglobulin preparations and more recently in the advent of hematopoietic growth factors. This review focuses on these newer aspects of autoimmune neutropenia.

Hematopoietic growth factors not only modulate blood progenitor cell activity but also alter the function of mature phagocytes. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 1 ng/mL for 60 min) did not stimulate luminol-enhanced chemiluminescence of polymorphonuclear leukocytes (PMNs) in suspension but primed PMN for as much as a 15-fold increase in chemiluminescence in response to f-met- leu-phe (fMLP). Mixed mononuclear leukocytes (monocytes [approximately 20%] and lymphocytes [approximately 80%]; MNL) chemiluminescence was very low even after rhGM-CSF priming, but MNLs added to the PMNs (PMN- MNL) resulted in near doubling of rhGM-CSF-primed PMN fMLP-stimulated chemiluminescence. The enhancing factor(s) from MNLs were inherent rather than induced by the GM-CSF, and purified lymphocytes increased GM-CSF-primed PMN chemiluminescence equal to mixed MNLs. We could not detect cell-free “enhancing factor(s),” but cell-to-cell contact further enhanced rhGM-CSF-primed fMLP-stimulated PMN-MNL oxidative activity by 40%. Polyclonal rabbit anti-tumor necrosis factor (TNF) (but not preimmune serum) decreased both fMLP-stimulated rhGM-CSF- primed PMNs and PMN-MNL chemiluminescence, suggesting that TNF on the PMN surface is enhancing GM-CSF-primed chemiluminescence. GM-CSF priming markedly increased PMN superoxide release (sevenfold), but PMN superoxide release was not further enhanced by the presence of MNLs. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) and interleukin-3 (rhIL-3) displayed much smaller effects on pure PMNs and mixed PMN-MNL chemiluminescence and superoxide release than rhGM-CSF. rhGM-CSF primes PMNs for increased oxidative activity more than rhG-CSF and rhIL-3. Maximal oxidative activity was observed when mixed PMN-MNL were primed with GM-CSF in a cell pellet-promoting cell-to-cell contact. This enhanced activity can be attributed, in part, to both inherent enhancing factor(s) on lymphocytes and PMN-associated TNF induced by GM-CSF.

To determine the susceptibility of rabbits to experimental infection with human T-cell lymphotropic virus type-II (HTLV-II), four separate groups of four weanling rabbits each were inoculated intravenously with lethally irradiated HTLV-II-infected human cell lines Mo-T (HTLV-IIMo- infected T cells), WIL-NRA (an Epstein-Barr virus [EBV]-transformed B- lymphoblastoid cell line infected with HTLV-IINRA), 729pH6neo (an EBV- transformed lymphoblastoid cell line transfected with a molecular clone of HTLV-IIMo), or G12.1 (HTLV-II-infected T cells from a Panamanian Guaymi Indian). Two additional groups of four rabbits each were similarly inoculated with control uninfected 729 or HuT 78 cells. Early and persistent seroconversion to HTLV-II core antigen p24, as determined by Western immunoblot, occurred in all HTLV-II-inoculated rabbits and was most intense in rabbits inoculated with G12.1 cells; seroreactivity to other HTLV-II gag or env antigens occurred later, with less intensity, or not in all inoculated rabbits. Peripheral blood mononuclear cells (PBMC) and other lymphoid cells from HTLV-II- inoculated rabbits produced minimal p24 in vitro, as determined by enzyme immunosorbent capture assay. Virus was more readily detected by polymerase chain reaction amplification of HTLV-II pol sequences; this occurred most frequently in rabbits inoculated with Mo-T cells, and most frequently in PBMC as compared with other tissues tested (bone marrow, brain, and liver). No evidence of disease occurred in HTLV-II- inoculated rabbits observed for as long as 24 weeks. All control rabbits remained negative for evidence of HTLV-II infection, as determined by the same procedures. These results provide the first evidence of HTLV-II infection in a species other than humans, and demonstrate the usefulness of the rabbit as an animal model to study the biologic response to different isolates of this human retrovirus.

In situ hybridization using antisense RNA probes was used to localize cells that produce erythropoietin (EPO) in the livers of anemic transgenic mice expressing the human EPO gene and in livers of anemic nontransgenic mice. In transgenic mice bled from a hematocrit of 55% to one of 10%, hepatocytes surrounding central veins synthesized large amounts of human EPO mRNA. EPO-producing cells were very rare in the area of portal triads. In transgenic mice bled to a hematocrit of 20%, a similar number and distribution of cells contained human EPO mRNA as was found with a 10% hematocrit, but the cells were less heavily labeled, indicating increased EPO production per cell at 10% hematocrit as compared with 20% hematocrit. No human EPO mRNA was detected in the kidneys of anemic transgenic mice, although endogenous murine EPO mRNA was strongly expressed in cortical interstitial cells. In sections of livers from nontransgenic mice bled from a hematocrit of 45% to one of 10%, only isolated cells produced EPO. When the types of cells could clearly be identified, approximately 80% of these cells were hepatocytes, while 20% had a nonepithelial morphology and were located in or adjacent to the sinusoidal spaces. When the sense strand was used as the RNA probe for in situ hybridization, no labeled cells were seen in normal or anemic livers. These results demonstrate that hepatocytes are responsible for production of EPO in both transgenic and nontransgenic mice and that a second cell type that is similar in morphology to EPO-producing interstitial cells in the kidney also produces EPO in the livers of nontransgenic mice.

The outcome of treatment for a first relapse of Hodgkin's disease after primary chemotherapy was analyzed in 80 patients. They were divided into four groups: group 1 (n = 24) had initially been treated with three cycles of (mechlorethamine, vincristine, prednisone, and procarbazine [MOPP]) and wide-field irradiation therapy; group 2 (n = 25) had six cycles of MOPP; group 3 (n = 15) and group 4 (n = 16) both initially received MOPP/ABVD (MOPP plus doxorubicin, bleomycin, vinblastine, and dacarbazine) or MOPP/ABV hybrid, but group 3 received conventional salvage regimens whereas group 4 was treated with high- dose chemotherapy and autologous bone marrow transplantation as salvage therapy (n = 16). Freedom from second failure (FF2F) was used as the major endpoint. Actuarial FF2F for all patients was 38% after a median follow-up of 75 months for patients who were alive. Risk factor analysis was performed on the 71 patients who had been treated with curative intent. The presence or absence of any one of three risk factors had a strong negative impact on outcome: stage IV disease at primary diagnosis, B symptoms at relapse, or a time from primary treatment to relapse of less than 1 year. Actuarial FF2F at 5 years was 17% in the group of patients with one or more of these three factors present (n = 49). If none of these factors was present, FF2F was 82% (n = 22) (P less than .001). Even high-dose chemotherapy and autologous bone marrow transplantation were not able to overcome the negative impact of one or more risk factors (FF2F = 19%, n = 12). The outcome of salvage treatments depends most on the presence or absence of these three risk factors and less on the type of salvage treatment. Patients with none of these risk factors present have an excellent outcome if they are treated with non-cross-resistant chemotherapy, or radiotherapy, or both. Novel approaches are needed for patients with one or more of these factors present. Reports on salvage treatments for Hodgkin's disease in first relapse after primary chemotherapy should include data on the proportion of patients having stage IV disease at diagnosis, B symptoms at relapse, and a time from primary treatment to relapse of less than 1 year.

Genomic DNA samples from 12 protein S-deficient families with hereditary thrombophilia were analyzed by Southern hybridization using protein S cDNA probes. Protein S-deficient members of families A and B possessed identical restriction fragment length polymorphisms, which suggest the absence of 5.3 kb from one of their protein S alpha alleles. The abnormal alleles from individuals A7 and B1 were amplified by the polymerase chain reaction using a forward primer in intron K and a reverse primer in exon XIV. The amplified DNA was cloned and sequenced. Sequence comparison with the normal protein S alpha gene showed that most of intron L (roughly 4.7 kb), the entire exon XIII (151 bp), and about a quarter of intron M (407 bp) were missing from both the A7 and B1 clones. Exon XIII contains all three potential N- glycosylation sites in human protein S. This deletion may result in RNA transcripts in which exon XII is spliced to exon XIV. Such an arrangement would generate a stop codon at position 463 and consequently produce a nonglycosylated protein S molecule truncated by 173 amino acids.

Philadelphia (Ph1) chromosome-positive clonogenic progenitors usually disappear within 4 to 6 weeks in long-term cultures established from the marrow of patients with chronic myeloid leukemia (CML). In contrast, coexisting chromosomally normal hematopoietic cells are relatively well maintained. Thus, even though normal cells are initially undetectable, they may become the predominant population. Recently, we have begun to explore the potential of such cultures as a strategy for preparing CML marrow for autografting, and based on cytogenetic studies of the differential kinetics of Ph1-positive and Ph1-negative clonogenic cells, have chosen a 10-day period in culture to obtain maximal numbers of selectively enriched normal stem cells. Here we present the results of molecular analyses of the cells regenerated in vivo for the initial three CML patients to be treated using this approach by comparison with the differentiated cells generated by continued maintenance of an aliquot of the autograft in vitro (using a slightly modified culture feeding procedure to enhance the production and release of cells into the nonadherent fraction after 4 weeks) for the one patient whose genotype made molecular analysis of clonality status also possible. These analyses showed that cells with a rearranged breakpoint cluster region (BCR) gene were not detectable by Southern blotting in either in vitro or in vivo populations of mature cells that might be assumed to represent the progeny of primitive cells present at the end of the initial 10 days in culture. Production of BCR- negative cells was also shown to be temporally correlated with the appearance of nonclonal hematopoietic cells both in culture and in vivo. These findings provide support for the view that prolonged maintenance of CML marrow cells in long-term culture may allow molecular characterization of both the BCR-genotype and clonality status of cells with in vivo regenerative potential.

Sulfation of human coagulation factor V was investigated by biosynthetically labeling the products of HepG2 cells with [35S]sulfate. There was abundant incorporation of the sulfate label into a product identified as factor V by immunoprecipitation, lability to proteases, affinity for the lectin jacalin, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two or more sites in factor V incorporated sulfate as indicated by labeling of different peptide chains of factor Va. The 150-Kd activation fragment of factor Va incorporated the greatest amounts of sulfate. This fragment of factor Va was bound selectively by jacalin-agarose, reflecting its content of O-linked oligosaccharides. Analysis of an alkaline hydrolysate of sulfate-labeled factor Va by anion-exchange chromatography showed that the sulfate occurred partly in tyrosine sulfate residues and partly in alkaline-labile linkages. Sulfate groups are potentially important structural and functional elements in factor V, and labeling with [35S]sulfate provides a useful approach for examining the biosynthesis and processing of this protein. The hypothesis is advanced that sites of sulfation in factor V and several other plasma proteins contribute to the affinity and specificity of thrombin for these molecules, just as it does for the interaction of thrombin with the potent inhibitor hirudin from leeches.

Acute steroid-resistant graft-versus-host disease (AGVHD) after allogeneic bone marrow transplantation is frequently fatal. A new treatment for this T-lymphocyte-mediated condition uses an immunotoxin, H65-RTA, comprised of a monoclonal antibody that recognizes the CD5 lymphocyte differentiation antigen coupled to ricin A chain, a cytotoxic enzyme that inhibits protein synthesis. The safety and efficacy of this lymphocyte-targeted immunotoxin was evaluated in patients with severe AGVHD in a phase I-II dose escalation study with group expansion at the two middle doses. Thirty-four patients received up to 14 daily intravenous infusions of the immunotoxin. The principal side effects were constitutional symptoms such as fatigue and myalgias, and hypoalbuminemia with weight gain was seen at all doses. Thirty-two patients were evaluated for improvement or resolution of disease. Durable complete or partial responses were not dose-related and were seen in 16 patients. Skin GVHD had the highest incidence of response (73%), although improvement or resolution in gastrointestinal tract (45%) and liver (28%) GVHD was also noted. Survival in responding patients was significantly prolonged at all times as compared with those with no response (P = .03). Treatment was associated with a rapid decrease in peripheral blood T lymphocytes, which persisted for greater than 1 month after therapy. Anti-immunotoxin antibodies were seen in 6 of the 23 patients tested; these were of low titer and did not block immunotoxin binding to T cells. Results of this study indicate that anti-T-lymphocyte immunotoxins may form a new class of immunosuppressive agents useful in T-lymphocyte-mediated diseases.

In humans with B-cell malignancies, the presence of monoclonal B lymphocytes (clonal proliferation) can be detected by comparing the fluorescence intensity distributions of lymphocytes stained with anti- kappa and anti-lambda reagents. The sensitivity of previously described single-color immunofluorescence techniques to low levels of clonal excess is limited by background from cytophilic immunoglobulins on non- B cells and by the low proportion of circulating B cells in individuals with minimal disease. We have used two-color immunofluorescence and B- cell gating to develop an improved assay that avoids false positives due to non-B cells, without requiring restrictive light scatter gates that may exclude true positives. This method is sensitive to 0.2% monoclonal B cells admixed with fresh normal lymphocytes, to 0.6% monoclonal B cells admixed with normal lymphocytes that have been stored for up to 72 hours, and readily detects 1% monoclonal cells in patient specimens. The two color B-cell gated assay offers sensitivity equivalent to the single-color assay and improved specificity for detection of low levels of clonal excess.

Factor V (FV) present in platelet alpha-granules has a significant but incompletely understood role in hemostasis. This report demonstrates that a fraction of platelets express very high levels of surface-bound, alpha-granule FV on simultaneous activation with 2 agonists, thrombin and convulxin, an activator of the collagen receptor glycoprotein VI. This subpopulation of activated platelets represents 30.7% +/- 4.7% of the total population and is referred to as convulxin and thrombin-induced-FV (COAT-FV) platelets. COAT-FV platelets are also observed on activation with thrombin plus collagen types I, V, or VI, but not with type III. No single agonist examined was able to produce COAT-FV platelets, although ionophore A23187 in conjunction with either thrombin or convulxin did generate this population. COAT-FV platelets bound annexin-V, indicating exposure of aminophospholipids and were enriched in young platelets as identified by the binding of thiazole orange. The functional significance of COAT-FV platelets was investigated by demonstrating that factor Xa preferentially bound to COAT-FV platelets, that COAT-FV platelets had more FV activity than either thrombin or A23187-activated platelets, and that COAT-FV platelets were capable of generating more prothrombinase activity than any other physiologic agonist examined. Microparticle production by dual stimulation with thrombin and convulxin was less than that observed with A23187, indicating that microparticles were not responsible for all the activities observed. These data demonstrate a new procoagulant component produced from dual stimulation of platelets with thrombin and collagen. COAT-FV platelets may explain the unique role of alpha-granule FV and the hemostatic effectiveness of young platelets. (Blood. 2000;95:1694-1702)

Knowledge of the stromal microenvironment is crucial for understanding the hematopoietic system. We took advantage of an assay that permits analysis of primary stroma-initiating cells (SICs) on the clonal level, and further characterized SICs and the factors that regulate SICs. Stroma formation in this assay is dependent on a high-molecular-weight factor secreted by the stromal cell line AC3.U. Here we show that this factor is identical to macrophage colony-stimulating factor (M-CSF), and that purified M-CSF is sufficient for induction of stroma formation. M-CSF, isolated from the line AC3.U, as well as from L929 cells and COS cells transfected with an expression vector encoding M-CSF, migrated in two peaks as 160- and 650-kD species after gel filtration. These molecular-weight species encompassed all stroma-inducing activity, and both stimulated macrophage colony formation. Affinity chromatography and blocking studies with antibodies specific for M-CSF and c-fms confirmed M-CSF as the sole factor in the supernatant of the stromal cell line AC3.U that promotes stroma formation. Culture of marrow, for as little as 1 week, depleted M-CSF-dependent SIC while increasing the incidence of replatable, factor-independent SIC. This suggests that culture changes the properties of SICs, perhaps by inducing differentiation into mature stromal cells. Thus, our results show a novel function of M-CSF as an important modulator of stroma formation.

Chronic myelogenous leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome in clonally derived hematopoietic precursors and their progeny. The Ph chromosome arises from a translocation that deregulates the c-ABL protein tyrosine kinase, giving it transforming potential and increased kinase activity. We observed a unique 39-kD tyrosine phosphoprotein (pp39), previously reported in blastic CML cell lines, in neutrophils from 50 cases of chronic phase CML. This protein was prominently and constitutively tyrosine-phosphorylated in CML neutrophils and was not phosphorylated in normal neutrophils. Stimulation of normal neutrophils with cytokines and agonists did not induce tyrosine phosphorylation of proteins migrating in the region of pp39, and the phosphorylation state of pp39 in CML neutrophils was not affected by kinase inhibitors known to downregulate the ABL kinase. The pp39 was not phosphorylated in hematopoietic cells from healthy donors or from patients with Ph chromosome-negative myeloproliferative disorders. Using micro amino acid sequencing of purified preparations of pp39, we identified pp39 as CRKL protein, which is consistent with recent immunologic studies in the blastic K562 cell line. Immunoblotting with anti-CRKL antibodies showed the presence of CRKL protein in CML cells and cell lines as well as in antiphosphotyrosine immunoprecipitates from CML cells. Our results suggest that pp39 CRKL in CML neutrophils may be stably tyrosine-phosphorylated by the BCR/ABL kinase at an early stage of myeloid differentiation when the ABL kinase is active. CRK, CRKL, and other SH2 (SRC homology domain)/SH3-containing proteins function as adaptor molecules in nonreceptor tyrosine kinase signalling pathways. Although the CRKL protein is present in normal neutrophils, it is not tyrosine-phosphorylated, and the inability to induce such phosphorylation in normal neutrophils suggests a special role of this phosphoprotein in the pathogenesis of CML. Constitutive phosphorylation of CRKL is unique to CML, indicating that it may be a useful target for therapeutic intervention.

Peripheral blood cells (PBCs) collected by leukapheresis after progenitor mobilization with chemotherapy and growth factors have been used successfully to replace marrow autografts in protocols requiring stem-cell support. Moreover, such transplants are often associated with more rapid recovery of blood cell counts than is routinely achieved with bone marrow. While conditions that mobilize colony-forming cells (CFCs) into the circulation are becoming increasingly well characterized, little information is available as to how these or other mobilizing treatments may influence the release of more primitive cells into the peripheral blood. To quantitate the peripheral blood content of such cells, we used the long-term culture-initiating cell (LTC-IC) assay, which detects a cell type that is able to produce progeny CFCs after a minimum of 5 weeks in cultures containing marrow fibroblasts. In this report, we present the findings on 21 patients who were transplanted over a 7-year period at our institution with PBCs alone. PBCs were collected in steady-state (n = 6) or during the recovery phase after high-dose cyclophosphamide (Cy; n = 15, nine with and six without additional growth factor administration). PBCs collected from another 11 patients given granulocyte colony-stimulating factor (G-CSF) were transplanted together with autologous marrow. Time-course studies of nine patients after Cy +/- granulocyte-macrophage CSF (GM-CSF) showed that CD34+ cells, CFCs, and LTC-ICs fell from normal to undetectable levels after Cy, and increased at the time of white blood cell (WBC) recovery: LTC-ICs to a mean of sixfold and CFCs to a mean of 26-fold higher than normal. The mean number of CD34+ cells, CFCs, and LTC-ICs present in the PBC harvest was twofold to 10-fold higher after mobilization than in steady-state collections; however, more than 2-log interpatient variability was observed. After PBC transplantation, the median time to a WBC count more than 10(9)/L was 12 days; polymorphonuclear leukocyte (PMN) count more than 0.5 x 10(9)/L, 15 days; and platelet count more than 20 x 10(9)/L, 17 days, although patients who received fewer than 1.5 x 10(5) CFCs/kg had a more than 50% chance of delayed count recovery (28 days). Patients who received Cy + GM-CSF-stimulated PBCs had more rapid and consistent platelet recoveries as compared with other groups receiving Cy mobilized or steady-state PBCs alone, and a rapid WBC recovery after Cy predicted a rapid WBC recovery after transplantation.(ABSTRACT TRUNCATED AT 400 WORDS)

We have recently shown that a short course of high-dose interleukin-2 (IL-2) can markedly inhibit the graft-versus-host disease (GVHD)- promoting activity of donor CD4+ T cells. The difficulty in dissociating GVHD-promoting from graft-versus-leukemia (GVL) effects of alloreactive donor T cells currently prevents clinical bone marrow transplantation (BMT) from fulfilling its full potential. To test the capacity of IL-2 treatment to promote such a dissociation, we have developed a new murine transplantable acute myelogenous leukemia model using a class II major histocompatibility complex-positive BALB/c Moloney murine leukemia virus-induced promonocytic leukemia, 2B-4–2. BALB/c mice receiving 2.5 x 10(5) 2B-4–2 cells intravenously 1 week before irradiation and syngeneic BMT died from leukemia within 2 to 4 weeks after BMT. Administration of syngeneic spleen cells and/or a 2.5- day course of IL-2 treatment alone did not inhibit leukemic mortality. In contrast, administration of non-T-cell-depleted fully allogeneic B10 (H-2b) spleen cells and T-cell-depleted B10 marrow led to a significant delay in leukemic mortality in IL-2-treated mice. In these animals GVHD was inhibited by IL-2 treatment. GVL effects were mediated entirely by donor CD4+ and CD8+ T cells. Remarkably, IL-2 administration did not diminish the magnitude of the GVL effect of either T-cell subset. This was surprising, because CD4-mediated GVHD was inhibited in the same animals in which CD4-mediated GVL effects were not reduced by IL-2 treatment. These results suggest a novel mechanism by which GVHD and GVL effects of a single unprimed alloreactive T-cell subset can be dissociated; different CD4 activities promote GVHD and GVL effects, and the former, but not the latter activities are inhibited by treatment with IL-2.

To determine if interleukin-6 (IL-6) administration influences platelet function, platelet activation was analyzed sequentially in IL-6-treated (80 micrograms/kg/d) and control dogs. Platelet activation was determined in whole blood by flow cytometry by quantitating the binding of a monoclonal antibody to platelet surface P-selectin after stimulation with graded doses of thrombin. Administration of IL-6 resulted in a twofold decrease in the thrombin concentration required for induction of half-maximal P-selectin expression (ED50) compared with control animals. The ED50 returned to normal after cessation of IL- 6 administration. As measured by P-selectin expression, enhanced responsiveness to the strong agonist platelet activating factor (PAF) was also observed in the IL-6-treated dogs. IL-6 had no effect on the susceptibility of platelets to thrombin activation when incubated with anticoagulated dog blood. The data show that, in addition to augmenting the platelet count in normal dogs, IL-6 enhances the sensitivity of platelets to activation in response to thrombin and PAF.

Interleukin-8 (IL-8) is a neutrophil chemotactic and activating cytokine that is produced in response to several stimuli. Because monocytic cells are important producers of IL-8, we investigated whether interferon-gamma (IFN-gamma), a potent inducer of activation and differentiation of mononuclear phagocytes, affected IL-8 expression in this cell lineage. We found a low constitutive level of IL-8 mRNA expression that was upregulated by IFN-gamma in a dose- and time- dependent manner and via a protein-synthesis-dependent process in the human monocytic cell line U937. IL-8 protein secretion was also stimulated by IFN-gamma. Nuclear run-on experiments showed that the IL- 8 gene was transcriptionally active in control cells and that IFN-gamma did not enhance the transcriptional activity. The increase in IL-8 mRNA by IFN-gamma was concomitant with the stabilization of the mRNA and, therefore, controlled primarily at a posttranscriptional level. These results represent the first evidence that IFN-gamma upregulates IL-8 gene expression in cells of the monocytic lineage, and show the involvement of posttranscriptional mechanisms in the induction of IL-8 mRNA.

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.

The rheologic properties of senescent erythrocytes have been examined using two models of red blood cell (RBC) aging. In the rabbit, aged erythrocytes were isolated after biotinylation, in vivo aging, and subsequent recovery on an avidin support. Aged RBCs from the mouse were obtained using the Ganzoni hypertransfusion model that suppresses erythropoiesis for prolonged periods of time allowing preexisting cells to age in vivo. In both cases, the aged erythrocytes were found by ektacytometry to have decreased deformability due to diminished surface area and cellular dehydration. The aged rabbit erythrocytes were further characterized by micropipette methods that documented an average surface area decrease of 10.5% and a volume decrease of 8.4% for the cells that were 50 days old. Because both the surface area and volume decreased with cell age, there was little change in surface-to- volume ratio (sphericity) during aging. The aged cells were found to have normal membrane elasticity. In addition, human RBCs were fractionated over Stractan density gradients and the most dense cells were found to have rheologic properties similar to those reported for the aged RBCs from rabbits and mice, although the absolute magnitude of the changes in surface area and volume were considerably greater for the human cells. Thus, stringent density fractionation protocols that result in isolation of the most dense 1% of cells can produce a population of human cells with rheologic properties similar to senescent cells obtained in other species. The data indicate that progressive loss of cell area and cell dehydration are characteristic features of cell aging.

Hemoglobin (Hb) Constant Spring is an alpha-thalassemic hemoglobinopathy that is a major cause of severe alpha-thalassemia in Southeast Asians. The difficulty of diagnosing Hb Constant Spring using standard electrophoretic methods has led to interest in DNA-dependent diagnostic methods. The methods developed have had to contend with the high degree of homology of the alpha 2-globin gene (the site of the Hb Constant Spring mutation) and the alpha 1-globin gene. We have developed a single reaction polymerase chain reaction-based method that uses asymmetric priming and a temperature shift to accomplish dual ends, selective amplification of alpha 2 but not alpha 1 DNA and discrimination of normal and Hb Constant Spring alpha 2 genes by allele- specific fluorescence polymerase chain reaction. Advantages of this method over previous approaches include avoiding radioisotopes, precluding the need for electrophoresis, and serving as its own control for successful amplification. It is readily applicable to routine diagnosis, population screening, and prenatal diagnosis.

Seventy consecutive adult patients with acute myelogenous leukemia (AML), median age 44 years, received high-dose cytarabine (3 g/m2 every 12 hours for 12 doses) followed by daunorubicin (45 mg/m2 daily for three doses) for remission induction. A single, identical course was planned for postremission therapy. Complete remission (CR) was achieved in 63 patients (90%, 95% confidence interval [CI] 83% to 97%), 60 after a single course. Eight patients were selected to undergo elective bone marrow transplantation (BMT) during first CR. Of the remaining 55 patients, 40 (73%) underwent planned post-CR therapy; 15 patients did not, owing to early relapse, excessive toxicity from the induction chemotherapy, or refusal. Nineteen patients, including 13 who received planned post-CR therapy, remain in continuous CR at a median follow-up of 5.2 years (range 3.0 to 7.1 years). The 5-year actuarial leukemia- free survival was 30% (95% Cl, 19% to 42%) for all patients achieving CR and 32% (95% Cl, 19% to 47%) for the 40 patients who received the planned post-CR chemotherapy. Analysis of various putative prognostic factors for CR and overall and leukemia-free survival showed significance for a previous history of myelodysplasia, higher initial leukocyte counts, certain French-American-British (FAB) types, and certain abnormal karyotypes. None of these factors was consistently significant regarding the above parameters, although small patient numbers in certain analyses may have obscured significant associations. Myelosuppression was occasionally prolonged after remission induction and especially post-CR therapy. Severe cerebellar toxicity was observed in 13 patients; in 11 cases, this toxicity was fully reversible. Other serious complications were infrequent. Intensive chemotherapy with high- dose cytarabine and daunorubicin has substantial antileukemic activity in adult AML, and may represent an improvement over conventional therapy. Relapses were common, however, even in patients who received planned therapy, and substantial toxicity was observed. The optimum use of this regimen in AML remains to be determined.

The biochemical processes that determine the lifespan of mammalian erythrocytes are unknown; one prominent theory suggests that antibody binding to the senescent red blood cell identifies it for removal from the circulation. To address this question, we have used a newly developed procedure for the isolation of aged erythrocytes that involves the biotinylation of rabbit red blood cells, in vivo aging of these cells, and the eventual in vitro recovery of the aged red blood cells by their affinity for an avidin support. Erythrocytes isolated with this method were found to have near-normal levels of cell- associated Ig throughout their 60-day lifespan. These data suggest that IgG accumulation is not part of the normal senescence process for erythrocytes in rabbits.

In humans with B-cell malignancies, the presence of monoclonal B lymphocytes (clonal proliferation) can be detected by comparing the fluorescence intensity distributions of lymphocytes stained with anti- kappa and anti-lambda reagents. The sensitivity of previously described single-color immunofluorescence techniques to low levels of clonal excess is limited by background from cytophilic immunoglobulins on non- B cells and by the low proportion of circulating B cells in individuals with minimal disease. We have used two-color immunofluorescence and B- cell gating to develop an improved assay that avoids false positives due to non-B cells, without requiring restrictive light scatter gates that may exclude true positives. This method is sensitive to 0.2% monoclonal B cells admixed with fresh normal lymphocytes, to 0.6% monoclonal B cells admixed with normal lymphocytes that have been stored for up to 72 hours, and readily detects 1% monoclonal cells in patient specimens. The two color B-cell gated assay offers sensitivity equivalent to the single-color assay and improved specificity for detection of low levels of clonal excess.

In a recent article, Bishop et al’ state that high-dose cytarabine has not previously been used as induction for untreated acute myeloid leukemia (AML). In fact, high-dose cytarabine has been used by itself: with am~acrine,~ and with daunorubicin4 in this role. Additionally, doses of cytarabine significantly above those considered standard had previously been used by other groups for initial induction’ with daunorubicin. The report by Bishop et all builds on these previous experiences and clearly supports a role for high-dose cytarabine as initial therapy, as we and other groups have reported.