ALBERT

Description: Interferon (IFN) therapy has become widely used for the treatment of chronic myelogenous leukemia. Hematologic remissions can be induced in about 60% of patients. Moreover, in a small number of patients loss of the Philadelphia (Ph) chromosome and of the BCR-ABL rearrangement is observed. We have used genomic Southern blotting as well as a two-step polymerase chain reaction (PCR) method to score for BCR-ABL messenger RNA (mRNA) in patients with hematologic remission induced by treatment with IFN alpha-2b alone or in combination with IFN gamma. Concomitantly, cytogenetic analysis was performed. In 11 of 115 patients reported here, a complete loss of rearranged BCR fragments was observed in Southern blots of peripheral blood (PB) and/or bone marrow (BM) cell samples. Malignant marker bands disappeared first in the PB. In six patients, this genotype remained stable, whereas in five patients, low-intensity, rearranged bands reappeared despite continuation of treatment. The reappearance of the malignant marker was not accompanied by a clinical relapse. Ph-negative metaphases were observed in PB cells of four patients and in the PB and BM cells of two of these patients. In the samples of the other patients, residual Ph- positive cells were detected. By two-step PCR, residual BCR-ABL rearranged transcripts were found in samples of 10 patients.

Description: Between February 1986 and March 1990, 56 patients with relapsed Hodgkin's disease treated with high-dose cyclophosphamide, carmustine, and etoposide (CBV) received an autologous peripheral stem cell transplantation (PSCT) rather than an autologous bone marrow transplantation (ABMT) because each patient had a marrow abnormality, either hypocellularity or tumor involvement. At least 6.5 x 10(8) [corrected] mononuclear cells/kg patient weight were collected from the peripheral blood of each patient, cyropreserved, and returned intravenously following CBV administration. Three patients had an early death 2, 22, and 25 days after PSCT. The actuarial event-free survival for these 56 patients at 3 years was 37% and was at least as good as that reported for relapsed Hodgkin's disease patients treated with CBV and ABMT. The 30 patients who had no marrow metastases at the time of PSC harvesting had an actuarial event-free survival of 47%, while those 26 patients with marrow metastases had a significantly different actuarial event-free survival of 27% (P = .02). CBV and PSCT for patients with relapsed Hodgkin's diseases who have marrow hypocellularity in traditional harvest sites or histopathologic evidence of BM metastases can result in long-term event-free survival.

Description: Tissue factor (TF) is the first factor of the extrinsic pathway of coagulation. Normally, TF is not expressed on the surface of endothelial cells. However, expression of TF can be induced in these cells in response to stimulation by diverse inflammatory mediators such as interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF- alpha), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). We have studied the effect of these mediators on the kinetics of the induction of TF-related procoagulant activity (PCA) on human umbilical vein endothelial cells (HUVECs). PCA is transiently induced on HUVECs, attaining a peak some 4 to 8 hours after addition of inflammatory agents, with maximal accumulation of TF messenger RNA (mRNA) occurring 3 to 5 hours earlier. Because the expression of PCA by treated HUVECs returns to basal levels by 20 to 30 hours, we examined the response of these cells to a second inflammatory stimulus. Continuous incubation of cells with a single inflammatory agent for 24 to 48 hours induces a hyporesponsive state with respect to the reinduction of TF expression by the same agent (14% of the initial stimulation for IL-1 beta, 39% for TNF-alpha 30% for LPS, and 7% for PMA). Such a diminution in PCA was also observed in the levels of TF mRNA. By contrast, pretreatment of HUVECs with one agent did not dramatically affect the reinduction of TF by any of the three other factors. We subsequently focused our attention on the induction of the autologous refractory period by IL-1 beta. De novo protein synthesis was not required during the preincubation of ECs for hyporesponsiveness to be observed. The establishment of the refractory state did not depend on the downmodulation of IL-1 beta receptor affinity or expression. Moreover, pretreatment of HUVECs with IL-1 beta increased prostacyclin (PGI2) production in response to a second stimulation by IL-1 beta, although such cells were unable to reexpress TF under the same conditions. This result suggests that distinct secondary messenger pathways are involved in TF induction and PGI2 synthesis by IL-1 beta in HUVECs.

Description: The hematopoietic growth factors, granulocyte-macrophage colony- stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), enhance the effector functions of mature myeloid cells, including the interaction with vascular endothelium. We examined the direct effect of recombinant human GM-CSF (rhGM-CSF) and recombinant human G-CSF (rhG-CSF) on the growth and function of cultured human umbilical vein endothelial cells (HUVEC). Endothelial cell growth supplement (ECGS) increased the proliferation of passaged and primary cells by 305% +/- 45% (mean +/- SEM, n = 5, P less than .01) over control cells at 4 days; GM-CSF and G-CSF had no effect. Endothelial cell procoagulant activity was increased after 4-hour incubation with recombinant interleukin-1 beta (IL-1 beta) 10 U/mL and recombinant tumor necrosis factor (TNF) 10 U/mL to 1,721% +/- 376% (n = 7, P less than .005) and 247% +/- 71% (n = 4) of control levels, respectively. gamma-Interferon (gamma-IFN) 50 U/mL had no direct effect of its own but was able to prime the response to IL-1 beta. There was no direct or priming effect of GM-CSF (1 ng to 1 microgram/mL) on the expression of procoagulant activity in endothelial cells. GM-CSF and G-CSF (1 ng/mL to 1 microgram/mL) had no effect on the expression of either tissue plasminogen activator (tPA) or plasminogen activator inhibitor-1 (PAI- 1) by endothelial cells. The secretion of tPA by endothelial cells was increased, however, after 24-hour incubation with thrombin 4 U/mL (314% +/- 72% of control levels, n = 5, P less than .025). The production of PAI-1 was increased by TNF 200 U/mL (241% +/- 44% of control, n = 3, P less than .005), thrombin 4 U/mL (180% +/- 12% of control, n = 5, P less than .0005) and IL-1 beta 10 U/mL (275% +/- 44% of controls, n = 5, P less than .0005). In four experiments, endothelial cells showed no specific binding of 125I-GM-CSF, whereas peripheral blood (PB) neutrophils demonstrated the presence of 802 +/- 78 high-affinity receptors for GM-CSF. Thus, we found no effect of rhGM-CSF or rhG-CSF on the proliferation activities by these cells. These findings are in accordance with the lack of demonstrable receptors for GM-CSF on cultured HUVEC.

Description: Between April 1985 and March 1987 130 children and adolescents up to 18 years of age with first relapse of acute lymphoblastic leukemia (ALL) were registered on the stratified and randomized multicentric trial ALL- REZ BFM 85 designed for patients pretreated with intensive front-line therapies. Stratification criteria were time and site of relapse: bone marrow (BM) relapse on or up to 6 months after stopping front-line therapy (group A), BM relapse beyond 6 months after therapy (group B), and isolated extramedullary relapse at any time (group C). Treatment consisted of alternating courses of polychemotherapy including randomly administered high- or intermediate-dose methotrexate (HDMTX:12 g/m2 as 4-hour infusion; IDMTX: 1 g/m2 as 36-hour infusion). During maintenance therapy the patients received daily oral thioguanine and biweekly intravenous (IV) MTX. The overall second complete remission (CR) rate was 92% (groups A, B, and C: 88%, 92%, and 100%), and the probability of event-free survival (EFS) at 6 years is 0.31 +/- 0.04 (groups A, B, and C: 0.18 +/- 0.05, 0.30 +/- 0.07, and 0.72 +/- 0.11). HDMTX did not prove to be superior to IDMTX, which led to premature stopping of randomization. Risk factor analyses showed early relapse, particularly BM relapse within 18 months, and T-cell phenotype to be independent predictors of poor outcome. The incidence of central nervous system (CNS) relapses following BM relapse was 19%, indicating that reprophylaxis to the CNS with IV/intrathecal (IT) MTX was insufficient. For 17 children who received bone marrow transplantation in second CR from HLA-compatible siblings the EFS was 0.53 +/- 0.12 at 5 years. Their outcome was not influenced by the above-mentioned risk factors. With the proposed treatment regimen long-lasting second remissions can be achieved in about one third of patients even after intensive front- line treatment.

Description: The VLA-4 integrin (CD49d/CD29), initially discovered on lymphoid cells, is actually known to be highly expressed on T cells, B cells, monocytes, and derived cell lines. Unlike other VLA integrins, mainly involved in cell-matrix adhesive interactions, VLA-4 has also been implicated in several cellular interactions. Based on the published alpha 4 cDNA sequence, a 1,142-bp alpha 4 cDNA fragment was amplified using the polymerase chain reaction. This fragment was used to isolate three overlapping genomic clones from a phage library. By Southern analysis with the cDNA probe, and using the polymerase chain reaction on DNA isolated from a panel of human/mouse somatic cell hybrids, the alpha 4 gene was mapped to chromosome 2. Fluorescence in situ hybridization confirmed this assignment and allowed a more precise mapping to chromosome 2q31–32.

Description: We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.

Description: 4.1(-) hereditary elliptocytosis (HE) is a variety of elliptocytosis resulting from the reduction (heterozygosity) or the absence (homozygosity) of protein 4.1. It is nearly always encountered in its heterozygous form. It has been found among Caucasians and North Africans in a sporadic fashion. We report the study on nine family cases of 4.1(-) HE. They were recruited independently (to the exclusion of any other variety of HE) in a limited area around the city of Annecy (French Northern Alps). The mode of genetic transmission, as well as the clinical, morphologic, and protein phenotypes fully conformed to the classical description. Western blots ruled out the existence of any protein 4.1 species of abnormal size. No obvious DNA rearrangement was detectable in any of the nine families with three 4.1 cDNA probes covering the entire coding sequence and part of the flanking 5′ and 3′ untranslated sequences. On the basis of five polymorphic sites (Bgl II, 2; Pvu II, 3), we found five different haplotypes in normal members of the 4.1(-) families. 4.1(-) HE was associated with the most common haplotype in all the propositi. 4.1 mRNA was studied in four families. Dot-blot hybridization experiments and Northern blots failed to show any detectable change in three families. On the other hand, they showed a 2-kb deletion in the 4.1(-) messenger RNA 5′-moiety in one family. These findings emphasize the heterogeneity of 4.1(-) HE at the molecular level.

Description: Human erythrocyte membrane alpha and delta glycophorins (glycophorins A and B) carry the antigens for the M,N,S,s blood group system. Synthetic oligonucleotides spanning coding regions for M,N,S, and s epitopes were used to examine DNAs from 50 individuals selected at random and from individuals known to exhibit S(-)s(-)U- or S(-)s(-)U+ blood group phenotypes. We showed that M,N,S,s, blood group-specific sequences occur as multicopies in the human genome and reside within the alpha and delta glycophorin genes and also within the third glycophorin gene (glycophorin E gene). DNA typing with M- and N-epitope-specific probes showed distinct patterns that allowed correlation of the genotypes with the blood group phenotypes. The correlation using S- and s-specific probes was less definite owing to cross-hybridization. An Mspl restriction fragment length polymorphism (RFLP) residing in the E gene was detected in the black population. This RFLP is also carried by all individuals tested who exhibit the S(-)s(-)U- and S(-)s(-)U+ blood groups phenotypes, thereby serving as a useful marker for the S-s- alleles. The site of cleavage resulting in this RFLP was localized to the second intron of the E gene, and cleavage could occur through differential methylation of its two alleles.

Description: We have examined alpha/beta V gene segment usage of peripheral blood CD4+ and CD8+ T cells, respectively, from patients with multiple myeloma and monoclonal gammopathy of undetermined significance, by using T cell receptor (TCR) for antigen monoclonal antibodies (MoAbs). In 7 of 16 patients we found an increase in the usage of various TCR V gene segments. The expansion was confined to either the CD4+ or the CD8+ T-cell subset, except for one patient where an abnormal pattern was observed both within the CD4+ and CD8+ T-cell subsets. In one patient 47%, and in another patient 30% of the CD8+ lymphocytes reacted with alpha V12.1 and beta V6.7 antibodies, respectively. In two other patients 29% and 40% of the CD4+ lymphocytes reacted with beta V6.7 and beta V8.1 antibodies, respectively. We conclude that T cells with a predominant V gene usage is a frequent feature in patients with abnormal clonal B cells of malignant or benign types. T- and B-cell populations are normally clonally linked in regulatory circuits. An abnormal proliferation of B cells might therefore induce, or be regulated by, an expansion of clonal T cells, as suggested by the present results.

Description: We have isolated from an HEL cell cDNA library an alternatively spliced transcript for the platelet membrane glycoprotein IIb (GPIIb) that resulted from the deletion of the 34 amino acids of exon 28 of the GPIIb gene. Confirming an earlier report, we also detected this transcript in platelet mRNA. To determine the consequences of exon 28 deletion on the expression of the GPIIb/IIIa heterodimer, we expressed cDNA for GPIIb-28 in COS-1 cells, either individually or simultaneously with a cDNA for GPIIIa. When recombinant GPIIb-28 was expressed alone, it did not acquire resistance to the enzyme endo-beta-N- acetylglucosaminidase H, was not cleaved into heavy and light chains, and was not transported to the cell surface. However, when recombinant GPIIb-28 was coexpressed with recombinant GPIIIa, GPIIb/IIIa heterodimers were assembled. Nevertheless, these heterodimers failed to complete posttranslational processing and were degraded intracellularly. Exon 28 contains one site for Asn-linked glycosylation. To determine if loss of this glycosylation site was responsible for the effects of exon 28 deletion, we removed the site from the exon 28 of intact GPIIb by oligonucleotide-mediated mutagenesis. However, absence of the carbohydrate appended to exon 28 did not prevent normal GPIIb/IIIa heterodimer expression. Our studies indicate that absence of the amino acids encoded by GPIIb exon 28 sufficiently perturbs the quaternary configuration of the GPIIb/IIIa heterodimer to impair its subsequent intracellular transport and processing. They also indicate that this alternatively spliced form of GPIIb mRNA, although present in megakaryocytes, is unlikely to make a significant contribution to the GPIIb/IIIa complexes expressed on platelets.

Description: The effects of pertussis toxin (PT) on the growth and dimethylsulfoxide (Me2SO4)-induced differentiation of the HL-60 human promyelocytic leukemia cell line were tested. Cell growth was quantified by direct cell counts. Cell differentiation was estimated by measuring the expression of myeloid-specific cell-surface antigens (Mo-1 and fMet-Leu- Phe [fMLP] receptors), the ability of the cells to produce superoxide anions on stimulation with fMLP, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA), and by monitoring the level of expression of messenger RNA (mRNA) for tumor necrosis factor alpha (TNF alpha). By itself, PT did not affect the proliferation of HL-60 cells in serum-containing medium. In contrast, PT (but not its B-oligomer) dose-dependently inhibited the Me2SO4-induced expression of Mo-1, fMLP receptors, and the oxidative responses to the chemotactic factor and to A23187, but not to PMA. The addition of Me2SO4 induced a significant increase in the steady-state levels of TNF alpha mRNA, and this effect was strongly inhibited by PT. Finally, the bacterial toxin did not reverse the block of cell division that follows the addition of Me2SO4. These results provide evidence for the involvement of a PT substrate (presumably a guanine nucleotide-binding protein) in the regulation of the maturation of the excitation-response coupling sequence in human myeloid cell precursors and show that the regulation of cell division and maturation of HL-60 cells are under distinct sets of control mechanisms.

Description: Band 3 variants occur rather frequently in different populations. Based on sodium dodecyl sulfate (SDS)-polyacrylamide electrophoretic properties, a widespread polymorphism (band 3 Memphis) has been previously described. It corresponds to a protein that has been hypothesized to be elongated in its N-terminal cytoplasmic domain. Band 3 from a heterozygote subject for this polymorphism and that displays a normal reactivity towards stilbene disulfonates has been isolated and its primary structure determined by protein chemistry. Reverse-phase high performance liquid chromatography tryptic peptide mapping showed, as the only difference with controls, that the enzymatic cleavage between the two N-terminal peptides did not occur, yielding a 69 residue-long fragment. Further cleavages of this peptide (cyanogen bromide, V8 protease), amino acid composition, and sequence analyses demonstrated that the lysine at position 56 was replaced by a glutamic acid. Thus, surprisingly, a single amino acid change is responsible for the large difference in the electrophoretic behavior. This result suggests that single amino acid substitutions may similarly be involved in the structural modification of several other protein variants, described as elongated or shortened based only on SDS-polyacrylamide electrophoresis studies. When deletions/insertions were confirmed by sequence analysis, their extent was often different from that expected from electrophoresis.

Description: Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. Immunochemical and serologic studies have identified a number of glycophorin C variants that include the Yus, Gerbich, and Webb phenotypes. We report here the molecular characterization of these variants. Amplification of glycophorin C mRNA from the Yus phenotype, using two oligonucleotide primers that span the coding domain, generated a 338-bp fragment compared with a 395-bp fragment generated by amplification of normal glycophorin C mRNA. Sequencing of the mutant 338-bp fragment identified a 57-bp deletion that corresponds to exon 2 of the glycophorin C gene. Similar analysis showed deletion of 84-bp exon 3 in the Gerbich phenotype. In contrast to the generation of shorter than normal DNA fragments from mRNA amplification in the Yus and Gerbich phenotypes, amplification of mRNA from the Webb phenotype generated a normal-sized fragment. Sequencing of this DNA fragment showed an A----G substitution at nucleotide 23 of the coding sequence, resulting in the substitution of asparagine by serine. This modification accounts for the altered glycosylation of glycophorin C seen in this phenotype. These results have enabled us to characterize glycophorin C variants in three different phenotypes that involve deletions of exons 2 and 3 of the glycophorin C gene, as well as a point mutation in exon 1 that results in altered glycosylation of this protein.

Description: Eleven patients with plasma cell dyscrasias underwent high-dose chemoradiotherapy and anti-B-cell monoclonal antibody (MoAb)-treated autologous bone marrow transplantation (ABMT). The majority of patients had advanced Durie-Salmon stage myeloma at diagnosis, all were pretreated with chemotherapy, and six had received prior radiotherapy. At the time of ABMT, all patients demonstrated good performance status with Karnofsky score of 80% or greater and had less than 10% marrow tumor cells. Eight patients had residual monoclonal marrow plasma cells and 10 patients had paraprotein. Following high-dose melphalan and total body irradiation (TBI) there were seven complete responses, three partial responses, and one toxic death. Granulocytes greater than 500/mm3 were noted at a median of 21 (range 12 to 46) days posttransplant (PT) and untransfused platelets greater than 20,000/mm3 were noted at a median of 23 (12 to 53) days PT in 10 of the 11 patients. Natural killer cells and cytotoxic/suppressor T cells predominated early PT, with return of B cells at 3 months PT and normalization of T4:T8 ratio at 1 year PT. Less than 5% polyclonal marrow plasma cells were noted in all patients after transplant. Three of the seven complete responders have had return of paraprotein, two with myeloma, and have subsequently responded to alpha 2 interferon therapy. Eight patients are alive at 18.9 (8.9 to 43.1) months PT and four remain disease-free at 12.3, 17.5, 18.9, and 29 months PT. This preliminary study confirms that high-dose melphalan and TBI can achieve high response rates without unexpected toxicity in patients who have sensitive disease, and that MoAb-based purging techniques do not inhibit engraftment. Although the follow-up is short- and long-term outcome to be determined, relapses post-ABMT in these heavily pretreated patients suggest that ABMT or alternative treatment strategies should be evaluated earlier in the disease course.

Description: Glycoprotein IV (GPIV; CD36 or GPIIIb) is a cell surface glycoprotein that has been proposed as mediating a number of physiologically important processes such as the adhesion of platelets to thrombospondin (TSP) and collagen, the cytoadherence of Plasmodium falciparum-infected erythrocytes, and the TSP-dependent interaction of monocytes with platelets and macrophages. Because platelets of the Naka-negative phenotype have recently been shown to lack detectable GPIV, their availability offered the opportunity to test directly these hypotheses regarding its adhesive functions. It has been found that Naka-negative platelets and monocytes do not support cytoadherence of P falciparum- infected erythrocytes. Naka-negative platelets are deficient in the initial stages of their adhesion to fibrillar collagen and this defect is most marked under Mg(2+)-free conditions. Finally, the ability of Naka-negative platelets to bind TSP before or after activation is unimpaired as compared with normal controls. These results do not support a role for GPIV as the TSP receptor.

Description: Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.

Description: Anti-von Willebrand factor (vWF) monoclonal antibody NMC-4 completely inhibited vWF binding to platelet glycoprotein (GP) lb induced by either ristocetin or botrocetin at an IgG concentration of approximately 10 micrograms/mL, and also blocked binding of asialo-vWF to GP lb. NMC-4 coupled beads isolated a 97-Kd fragment (Fr) from a whole tryptic digest of vWF. The N-terminal sequencing of the nonreduced 97-Kd Fr, in combination with amino acid analysis, showed it to be a homodimer of residues 449 through 728 of the constituent subunit. Present data, together with the results obtained from previous studies, confirm the existence of one or three possible inter-subunit disulfide bonds between cysteine residues 459, 462, and 464. NMC-4 bound to reduced vWF Fr(s) more weakly than to nonreduced Fr(s), but it did not react with Fr III-T2 of vWF, a disulfide-linked twin heterodimer of residues 273 through 511 and 674 through 728 (Marti et al, Biochemistry 26:8099, 1987). Fr III-T2 completely inhibited ristocetin-induced vWF binding at a concentration of 100 mumol/L but had no effect on botrocetin-induced binding. In addition, both the N- and C-terminal polypeptides, residues 449 through 549 and 674 through 728, generated by subdigestion of the 52/48-Kd Fr (Fujimura et al, J Biol Chem 261:381, 1986), inhibited preferentially ristocetin-induced vWF binding without affecting to botrocetin-induced vWF binding. These findings suggest that amino acid residues 512 through 673 of the vWF subunit are involved in botrocetin-induced vWF binding.

Description: We have previously shown that human interferon-gamma (Hu-IFN-gamma) induces platelets to become efficient effector cells, capable of killing young larvae of the parasite Schistosoma mansoni. Recently, binding sites for IFN-gamma on platelets have been characterized. We show here the presence of high-affinity receptors for IFN-gamma on the surface of the human megakaryocytic Dami cell line. Scatchard analysis indicated the presence of about 11,000 binding sites per cell, with a kd of 3 +/- 0.5 x 10(-10) mol/L; the apparent molecular weight of the receptor was 90 Kd. Receptor-bound 125I Hu-recombinant IFN-gamma was rapidly internalized and degraded when the temperature was increased from 4 degrees C to 37 degrees C. The half-life of this receptor was about 7 hours, and pretreatment of cells with IFN-gamma or phorbol myristate acetate had very little effect on the surface receptor number and no detectable effect on IFN-gamma receptor messenger RNA (mRNA) expression. The receptor was functional, because 24 hours of treatment with IFN-gamma led to the increase of HLA class I mRNA expression and to the initiation of HLA class II mRNA expression. These effects were selective because platelet glycoprotein Ib, IIb, or IIIa mRNA expression and cell proliferation were unaffected.

Description: Bone marrow harvested from cancer patients for autologous bone marrow reinfusion (ABMR) after myeloablative treatment may be injured, in both its proliferating and stromal cell pools, by either previous treatment or manipulation at the time of harvest. We have examined the relative effects of seven covariates on hematologic recovery after ABMR in children with neuroblastoma (NBL) using univariate and step-up analysis. We measured recovery by times to achieve (1) white blood cell counts greater than 1,000/microL; (2) absolute neutrophil counts greater than 500/microL; and (3) platelet counts greater than 20,000/microL without transfusion. In univariate analysis, recovery was significantly associated with the amount of prior chemotherapy and the interval between last chemotherapy and marrow harvest. Patient sex, the number of granulocyte-macrophage colonies infused, harvest-to-freeze interval, and use of purging were marginally associated. After adjusting for potential confounders in a multivariate model, the amounts of chemotherapy and granulocyte-macrophage colonies infused were independently significant predictors of time to total white blood cell count recovery; chemotherapy courses and chemo-to-harvest interval were predictors of neutrophil count recovery; and sex, use of purging, and harvest-to-freeze interval were marginal predictors of platelet recovery. The speed of hematologic recovery after ABMR seems to depend mainly on pre-existing factors and marginally on manipulation of the marrow after harvest. These factors may affect both proliferating and stromal cell pools.

Description: Homozygous beta thalassemic mice received 50 U (1,660 U/kg) of recombinant human erythropoietin (rhEpo) 5 days a week for 2 weeks. Hemoglobin increased from 9.2 +/- 0.6 g/dL to 10.5 +/- 0.4 g/dL (P = .002) and hematocrit increased from 29.2% +/- 0.9% to 34.1% +/- 1.9% (P = .0014). The beta minor/alpha globin chain synthesis ratio increased slightly but significantly between day -4 (0.75 +/- 0.07) and day 4 (0.81 +/- 0.04) (P = .01) and reached a minimum ratio (0.67 +/- 0.03) on day 15 (P = .001), being parallel to reticulocyte counts and to the incorporated trichloracetic acid (TCA)-insoluble radioactivity, therefore parallel to the erythropoietic output in thalassemic mice, as in normal mice. Erythrocyte defects were improved in beta thalassemic mice treated by rhEpo: membrane-associated alpha globin was significantly decreased (P less than .01), thiol group reactivity of ankyrin was significantly improved (P less than .05), spectrin alterations were reduced, and deformability of mouse thalassemic red blood cells was normalized. These results provide experimental criteria for modulating globin chain imbalance necessary for the therapy of human beta thalassemia intermedia, and suggest that rhEpo might be of interest to improve the red blood cell mass and reduce erythrocyte alterations in this disease.

Description: Platelet-specific antigen Sib(a) has been highly implicated in the pathogenesis of refractoriness to human leukocyte antigen (HLA)-matched platelet transfusions in Japan. We provide evidence that the Sib(a) antigen is located on the glycoprotein (GP) Ib alpha and has a close association with the molecular weight (mol wt) polymorphism of GPIb. In modified antigen-capture ELISA (MACE), anti-Sib(a) antibody reacted only with GPIb/IX held by a murine anti-GPIb/IX monoclonal antibody (MoAb). The reactivity of anti-Sib(a) antibody to Sib(a)-positive (Sib(a+)) platelets was abolished after they were treated with Serratia marcescens protease. Platelets from 50 healthy volunteers were semiquantitatively phenotyped for Sib(a) antigen by MACE and divided into three distinct groups: strongly positive, positive, and negative. They were also analyzed by sodium dodeyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and periodic acid-silver staining for mol wt polymorphism of GPIb, phenotyped as A, B, C, or D. Without exception, Sib(a+) platelets showed larger phenotypes (A or B). Removal of sialic acid from Sib(a+) platelets did not reduce the binding of anti-Sib(a). Finally, anti-Sib(a) antibody specifically immunoprecipitated A and B phenotypes of GPIb from Sib(a+) platelets. Thus, Sib(a) antigen evidently is located in the region of glycocalicin that is present only on the A and B phenotypes of GPIb.

Description: Megakaryocytic maturation was analyzed in long-term bone marrow cultures in the absence of added growth factors. Megakaryocytes could be observed for periods of up to 13 weeks in both the supernatant and stromal layer of these cultures. Using acetylcholinesterase staining for enumeration and sizing of megakaryocytes, and a novel rat antimurine platelet monoclonal antibody (MoAb) that detects only megakaryocytes in bone marrow, the number, volume, and ploidy of these cells were assessed microscopically and by flow cytometry. Correlation of these measurements with ambient interleukin-6 (IL-6) levels showed no relationship between IL-6 bioactivity and megakaryocyte number. Conversely, the relatively high IL-6 bioactivity present during the first 2 weeks of culture was correlated with increased megakaryocytic size and ploidy, while the relatively lower IL-6 bioactivity present after week 3 corresponded to decreased megakaryocytic size and ploidy. Addition of neutralizing anti-IL-6 MoAb decreased megakaryocytic size and ploidy at times when ambient IL-6 levels were relatively high, while the addition of exogenous IL-6 increased size and ploidy at times when endogenous IL-6 concentrations were low. The data show that long- term bone marrow cultures can be used as a means to evaluate megakaryocytic maturation in vitro, and suggest that, to some extent, IL-6 plays a role in the maturation process in this system.

Description: Raji, a malignant B-lymphocyte cell line containing Epstein-Barr virus genomic elements, has been conditioned to proliferate optimally in transferrin (Tf)-free medium containing a very low concentration of an iron salt. We provide evidence that an Tf-independent iron uptake system is physiologically important for maintaining the growth of these cells. The data show that Raji cells take up iron from iron salts using a relatively high-capacity, low-affinity, temperature- and calcium- dependent uptake system. The apparent capacity of this system increases when: (1) cells are cultured in Tf-free medium containing high concentrations of iron salt as opposed to medium containing Tf; and (2) when the iron salt concentration of Tf-free medium is lowered to about 1.6 mumol/L. Cellular iron uptake also increases when a maximum number of cells are in S and G2 and M cell phases of the cell cycle. The cells are sensitive to growth inhibition by the addition of deferoxamine. This evidence supports the hypothesis that certain malignant lymphocytes, under iron deplete conditions, fulfill an iron requirement for proliferation by an adaptation such as Tf-independent iron uptake.

Description: We have examined whether transfected mouse erythroleukaemia (MEL) cells can be used to examine differential expression of human gamma- and beta- globin genes. These cells, which express only their adult globin genes, will transcribe the human adult beta gene but not the fetal gamma genes when they are introduced on an intact human chromosome 11 by cell fusion. However, MEL cells stably transfected with the human A gamma gene attached to one of the active elements (HS2) of the beta-globin locus control region (LCR) readily produce gamma-globin mRNA in amounts equivalent to those seen with a comparable beta gene insert. When both beta and gamma genes are attached to HS2, equal amounts of beta A gamma mRNAs are produced, irrespective of the gene order. Furthermore, when HS2 is inserted into the 5′ end of a 40-kb cosmid containing the G gamma A gamma-117 delta beta genes in their normal chromosomal organization (but with the Greek HPFH -117 A gamma gene mutation), it directs expression of readily detectable amounts of G gamma A gamma and beta-globin mRNAs in MEL cells. Therefore, under these circumstances we have observed no competition between beta and gamma genes for expression in MEL cells. These findings suggest that MEL cells are capable of perpetuating regulatory information involved in developmental control when it is provided by an intact chromosome, but are incapable of reconstructing such information on transfected DNA.

Description: In a series of 100 acute myeloid leukemia (AML) patients defined by cytochemistry and immunophenotyping, 20 expressed T-lymphocyte associated antigens on the surface of their blasts. While 15 expressed two or more T-cell antigens, five were found to express only CD7. All patients belonged to the French-American-British type M4, and four were under the age of 40. Despite intensive chemotherapy, four never obtained a complete remission and the fifth died of relapse after an allogenic bone marrow transplantation. While 12 randomly selected T- cell antigen negative AML patients showed only few rearrangements in Ig- or T-cell receptor (TCR) genes, such genetic alterations were demonstrated in four of five patients for the TCR delta gene and in all patients for the TCR beta gene. Interestingly, DNA fragments of similar size were demonstrated in three of five patients for both the beta and delta genes. These data suggest that the solitary presence of CD7 among T-cell antigens in otherwise clearcut AML cases identifies a group of patients with similarities in antigen receptor gene configuration as well as outcome.

Description: Very high concentrations of erythropoietin (epo), in clonogenic cultures, result in reduced production of neutrophils, and fetal progenitors are more sensitive to this effect of epo than are those of adults. However, the significance of this observation is unclear because no evidence of reduced neutrophil production has been presented following administration of recombinant epo to human or animal subjects. In the present study we injected newborn rats, beginning on the first day of life, with 20, 200, or 2,000 U epo/kg body weight, and measured serum epo concentrations after 2, 8, 24, or 48 hours. After selecting a dose that resulted in serum concentrations greater than 1,000 mU/mL (a concentration that resulted in down-modulation of neutrophil production from neonatal rat progenitors in vitro) other newborn rats were treated for 3 days with that dose (1,000 U epo/kg) or a vehicle control. Administration of epo resulted in increased hematocrits (P less than .001), reticulocyte counts (P less than .001), normoblasts/femur (P less than .05), and normoblasts/spleen (P less than .001). Recipients of epo also had more erythroid colony-forming units (CFU-E) (P less than .001) and higher CFU-E tritiated thymidine suicide rates (P less than .01) than did controls. However, femurs and spleens of epo recipients contained fewer postmitotic neutrophils (femur, P less than .01; spleen, P less than .01), proliferative neutrophils (femur, P less than .01; spleen, P less than .02), granulocyte-macrophage colony-forming units (CFU-GM) (P less than .005), and lower CFU-GM tritiated thymidine suicide rates (P less than .01). Seven and nine days after twice-daily administration of 2,000 U epo/kg, blood neutrophil concentrations had diminished (P less than .05). Thus, administration of high doses of recombinant epo to newborn rats resulted in diminished neutrophil production accompanying accelerated erythropoiesis.

Description: The activation signals leading to proliferation of normal and leukemic CD3+ large granular lymphocytes (LGL) were studied in vitro. Anti-CD3 monoclonal antibody (MoAb) alone (P less than .01) and recombinant interleukin-2 (IL-2) alone (P less than .01) caused significant stimulation of peripheral blood mononuclear cells (PBMC) from four CD3+ LGL leukemia patients, as measured in a 3H-thymidine incorporation assay. Recombinant interleukin-4 (IL-4) alone had no effect (P = .11). The combination signals of anti-CD3 MoAb and either IL-2 or IL-4 produced a proliferative response greater than anti-CD3 MoAb alone (P less than .01) or lymphokine alone (P less than .01). Leukemic LGL, purified by two-color sorting, were subsequently activated by anti-CD3 MoAb and IL-2 and assessed for DNA content by viable Hoechst No. 33342 (HO) staining. Results of these studies demonstrated that leukemic LGL were stimulated directly by anti-CD3 MoAb and IL-2, with the percentage of cells in cell cycle (S + G2/M) ranging from 16% to 72%. Normal CD3+ LGL were also stimulated to enter the cell cycle by anti-CD3 and IL-2. These results show that leukemic LGL proliferate in vitro after activation through the T-cell receptor and/or lymphokine.

Description: Growth factor-independent clones of myeloid leukemic cells can regain a growth factor-dependent state during differentiation. Loss of viability in these differentiating leukemic cells in the absence of growth factor was associated with DNA fragmentation and morphologic changes typical of programmed cell death (apoptosis). The differentiating leukemic cells could be rescued from apoptosis by a hematopoietic growth factor such as interleukin-3 (IL-3) and by the tumor-promoting phorbol ester 12-O-tetra-decanoyl-phorbol-13-acetate (TPA), but not by the nonpromoting phorbol ester 4-alpha-TPA. IL-3 and TPA rescued differentiating myeloid leukemic cells by different pathways and also rescued normal myeloid precursor cells from apoptosis. The rescue of differentiating leukemic and normal myeloid cells by IL-3 or TPA was blocked by amiloride inhibitors of the Na+/H+ antiporter. We suggest that TPA may act as a tumor promoter by inhibiting programmed cell death.

Description: Although insulin-like growth factor (IGF-1) stimulated 3H-thymidine incorporation upon addition to the interleukin-3 (IL-3)-dependent cell line FDC-P1, IGF-1 did not relieve IL-3 dependency for growth. To further examine the effects of IGF-1 on hematopoietic cells, FDC-P1 cells were infected with a retroviral construct (LISN) containing the human IGF-1 receptor (hIGF-1R) and neo genes. IL-3-independent cells were readily isolated after LISN infection when either IGF-1 or supraphysiologic concentrations of insulin were included in the culture medium. These cells were transformed to IL-3 independence by a ligand- dependent mechanism because their growth was dependent on the presence of either IGF-1 or insulin and growth factors capable of supporting autocrine growth were not detected. Furthermore, a monoclonal antibody (MoAb) directed against the human IGF-1R (alpha IR-3) inhibited IGF-1 but not IL-3-induced proliferation and these cells contained 20- to 200- fold more IGF-1 receptors than uninfected FDC-P1 cells. In contrast, when LISN-infected cells were plated in medium without exogenously supplied IGF-1 or insulin, factor-independent cells were rarely isolated. Growth of these cells was also inhibited by the alpha IR-3 MoAb and they expressed 100- to 400-fold more IGF-1 receptors than uninfected FDC-P1 cells. The endogenous IGF-1 and/or insulin present in the calf serum may have enabled their growth because these cells, unlike the parental cells, would proliferate in serum-free defined media and their growth was again inhibited by the alpha IR-3 MoAb. These results demonstrate that IGF-1 can replace IL-3 for growth when FDC-P1 cells overexpress the IGF-1R. Given the fairly ubiquitous expression of the IGF-1 receptor, these and additional experiments might help to determine whether increased expression of endogenous receptors by cells can lead to leukemogenesis and tumorigenesis. Moreover, hIGF-1R-infected cells will be useful in investigating the mechanisms of IGF1-mediated signal transduction because they are now known to proliferate in response to IGF-1.

Description: To investigate the natural history of stage A chronic lymphocytic leukemia (CLL) we reviewed 84 such patients. Among 74 cases evaluable for disease progression, 22 (29.6%) progressed to more advanced clinical stages (9 to stage B, 13 to stage C); the actuarial estimation of such an event at 4 years was 30% (95% CI: 26.3% to 33.6%). Despite a linear trend toward an increasing risk (r = .92), the hazard function analysis showed a constant pattern of progression, suggesting a lack of correlation of such an event with time (r = .04). Furthermore, disease progression when analyzed as a time-dependent variable had a clear-cut impact on survival (P less than .001). With the aim of identifying a subgroup of patients with low probability of disease progression and death, we applied to our set of patients four different proposals for subclassifying stage A. All methods were similar in terms of sample size, 5-year survival rate, and disease progression risk, suggesting that the choice between different proposals is somewhat arbitrary. Whatever the criteria are for defining “smoldering” CLL, such patients (accounting in the present study for 20.5% of overall series and 46.7% of stage A patients) should not be treated until progression occurs.

Description: Antiplatelet antibodies can activate platelets causing platelet aggregation and the release reaction. However, the pathway of activation by these antibodies is unknown and several potential mechanisms are possible. In this report, we describe studies investigating potential pathways of platelet activation by IgG antibodies. We tested 16 different IgG monoclonal antibodies (MoAbs) against a variety of platelet surface components and found that six antibodies were capable of causing platelet aggregation and release. These included MoAbs against glycoprotein (GP) IIb/IIIa, CD9, GPIV, and two other not well-characterized platelet components. There was no relationship between the number of platelet binding sites and the ability of an MoAb to activate the platelets. By adding intact and F(ab')2 preparations of the MoAb to control or Fc receptor-blocked platelets, we found that in all instances the MoAbs initiated platelet activation via interacting with the platelet Fc receptors. Clustering of the platelet protein components using a secondary antibody did not cause activation. Studies into the pathway of Fc-dependent activation demonstrated that the MoAbs were capable of activating platelets by occupying Fc receptors on adjacent platelets (interplatelet activation), as well as on the same platelet (intraplatelet activation).

Description: The relationship between fibrinogen binding to its receptor and platelet aggregation has been investigated by comparing 125I-fibrinogen binding and aggregation velocities of gel-filtered platelets in the presence of adenosine diphosphate (ADP). Aggregometric responses at various fibrinogen concentrations are found to be bell-shaped and show a maximum at fibrinogen concentrations (Fmax) similar to the 125I- fibrinogen hemisaturating doses. At higher and lower fibrinogen concentrations, platelet aggregation velocities decrease in a parallel manner. Lowering ADP concentration increases Fmax, in agreement with the modulatory effect of ADP on fibrinogen binding to platelets. Variations of fibrinogen in the range of physiopathologic plasma concentrations affect platelet aggregation induced by any ADP dose. These results clarify the relationship between the fibrinogen binding process and aggregation and demonstrate that plasma fibrinogen concentration has a major influence on the velocity of platelet aggregation.

Description: Canine granulocyte-macrophage colony-stimulating factor (caGM-CSF) was cloned and expressed to allow further investigation of GM-CSF in a large animal model. The cDNA is 850 base pairs (bp) long and encodes a peptide of 144 amino acids. The nucleotide and amino acid sequence homology between caGM-CSF and human GM-CSF (hGM-CSF) is 80% and 70%, respectively. A mammalian expression vector pCMV/CAGM was constructed and used to transfect COS cells for expression of caGM-CSF. Supernatant from transfected COS cells enriched with caGM-CSF was shown to have significant stimulating activity in granulocyte-macrophage colony forming unit (CFU-GM) assays of canine marrow. caGM-CSF, expressed from bacteria, was used to treat seven dogs at varying doses twice daily subcutaneously (sc) for 14 to 16 days. Circulating blood neutrophils and monocytes increased significantly. The increase in circulating eosinophils was variable. Thrombocytopenia developed during administration of caGM-CSF but corrected rapidly after cessation of treatment. Evaluation of survival times of 51Cr-labeled autologous platelets suggested increased consumption as the primary reason for thrombocytopenia. A species-specific GM-CSF will be a useful tool for hematologic or immunologic studies in dogs.

Description: Interleukin-1 (IL-1) has profound stimulatory effects on hematopoiesis but the mechanism(s) of action remain unknown. The direct action of IL- 1 on hematopoietic progenitor cells requires the presence of a specific IL-1 receptor (IL-1R). In this report, we tested the effect of in vivo IL-1 treatment on the expression of IL-1R on bone marrow (BM) cells. Injection of mice with IL-1 results in a marked upregulation of IL-1R on light-density BM cells as on a subpopulation enriched for myeloid precursors. Pretreatment of mice with anti-type I IL-1R antibody (35F5), which has been shown to prevent the radioprotective effect of IL-1, also blocked IL-1-induced IL-1R expression on BM cells. This antibody did not directly bind and block IL-1 binding to the type II IL- 1R expressed on hematopoietic cells, suggesting that IL-1R upregulation by IL-1 is indirect. It is therefore possible that IL-1 acts on type I IL-1R-expressing accessory cells such as stromal cells or T cells to induce production of hematopoietic growth factors (HGFs). In support of this, granulocyte colony-stimulating factor administration can induce the increase of IL-1R on BM cells. Thus, the increased expression of IL- 1R on hematopoietic BM cells by IL-1 is indirect, probably mediated in part through endogenous HGF production. These results also suggest that the restorative hematopoietic effect of IL-1 occurs through both indirect and direct mechanisms.

Description: The occurrence of rosetting of Plasmodium falciparum-infected human red blood cells (IRBC) with uninfected red blood cells (RBC) and its potential pathophysiologic consequences were investigated under flow conditions using the perfused rat mesocecum vasculature. Perfusion experiments were performed using two knobby (K+) lines of P falciparum, ie, rosetting positive (K+R+) and rosetting negative (K+R-). The infusion of K+R+ IRBC resulted in higher peripheral resistance (PRU) than K+R- IRBC (P less than .0012). Video microscopy showed that under conditions of flow, in addition to cytoadherence of K+R+ IRBC to the venular endothelium, rosette formation was also restricted to venules, especially in the areas of slow flow. Rosettes were absent in arterioles and were presumably dissociated by higher wall shear rates. The presence of rosettes in the venules must therefore reflect their rapid reformation after disruption. Cytoadherence of K+R+ IRBC was characterized by formation of focal clusters along the venular wall. In addition, large aggregates of RBC were frequently observed at venular junctions, probably as a result of interaction between flowing rosettes, free IRBC, and uninfected RBC. In contrast, the infusion of K+R+ IRBC resulted in diffuse cytoadherence of these cells exclusively to the venular endothelium but not in rosetting or large aggregate formation. The cytoadherence of K+R+ IRBC showed strong inverse correlation with the venular diameter (r = -.856, P less than .00001). Incubation of K+R+ IRBC with heparin and with monoclonal antibodies to glycoprotein IV/CD36 abolished the rosette formation and resulted in decreased PRU and microvascular blockage. These findings demonstrate that rosetting of K+R+ IRBC with uninfected RBC enhances vasocclusion, suggesting an important in vivo role for rosetting in the microvascular sequestration of P falciparum-infected RBC.

Description: The mechanism of platelet thrombus growth on an artificial surface is incompletely understood. While glycoprotein (GP)Ib and GPIIb/IIIa are required for normal attachment and thrombus formation on subendothelium, their roles in platelet deposition to artificial surfaces remain unclear. Using selected platelet inhibitors (aspirin [ASA], low molecular weight dextran, monoclonal antibodies 10E5 [v GPIIb/IIIa], and 6D1 [GPIb]) we examined the mechanism of platelet deposition to polyethylene (PE) surfaces under steady laminar and oscillatory flow conditions. Polyethylene-100 (PE-100) tubes (0.86 mm internal diameter) were perfused under steady laminar flow with citrated human whole blood reconstituted with 111indium-labeled platelets at 312 seconds-1 shear rate in the presence and absence of platelet inhibitors. The effect of oscillatory flow on platelet deposition was examined in a microwell system using 3/16-inch diameter discs of National Heart, Lung, and Blood Institute primary reference PE as the test surface. ASA and dextran did not significantly (P greater than .05) inhibit platelet deposition in laminar flow (not tested in oscillatory). Antibody 10E5 was a potent inhibitor (laminar less than 1%, P less than .0001, oscillatory less than 1.6%, P less than .01) of platelet deposition in both systems, and in this case, true adhesion (first attached layer) was blocked. Antibody 6D1 unexpectedly inhibited 70% of platelet deposition (P less than .01) in steady laminar flow and 56.5% in oscillatory flow (P less than .01). Scanning electron microscopy demonstrated platelets atop platelets in the controls, rare platelets in the 10E5 group, and a patchy monolayer of platelets in the 6D1 group. Transmission electron microscopy of cross-sections confirmed these observations. We conclude that the adhesion of the first platelet layer to an artificial surface requires GPIIb/IIIa. The data also suggest that GPIb is required for the development of the second layer in vertical platelet thrombus growth.

Description: The effects of recombinant human macrophage colony-stimulating factor (rhCSF-1) in long-term marrow cultures (LTMC) established from normal bone marrow cells were examined. When added during the first 3 weeks of culture (every second day, at 15 ng/mL), rhCSF-1 strongly inhibited the growth of all hematopoietic progenitors analyzed (colony-forming unit- MIX [CFU-MIX], CFU-granulocyte macrophage [CFU-GM], CFU-M, CFU-G, burst- forming unit-erythroid). Paralleling the inhibition of progenitors was the complete loss of adipocytes from the stromal layer of rhCSF-1- treated cultures. The inhibitory effect of rhCSF-1 correlated in all instances with the accumulation in the supernatants of these cultures of an activity (different from CSF-1) that inhibited colony formation in semisolid cultures. When addition of rhCSF-1 was delayed 3 weeks, its inhibitory effects were significantly reduced, which correlated with reduced inhibitory activity detected in the supernatants. Analysis of CSF-1 concentration by radioreceptor assay confirmed that added rhCSF-1 increased culture CSF-1 levels and showed that the decreased inhibition observed when rhCSF-1 is added later in culture was not due to decreased CSF-1 levels at that point. In contrast, the ability of rhCSF-1 to inhibit hematopoiesis and accumulate inhibitory activity in LTMC correlated with its rate of utilization, much higher in the first 2 weeks of culture, when the stromal layer was being established, than later. These observations document the inhibitory effect of rhCSF-1 on all aspects of hematopoiesis conducted in cultures that simulate the hematopoietic microenvironment, demonstrate the importance of accessory/stromal cells in mediating the effects of rhCSF-1 in LTMC, and point to an inhibitory activity as the mediating agent.

Description: The human T-cell lymphotropic viruses (HTLV) type I and type II are capable of inducing a variety of cellular genes, including many of the cytokines that regulate cell proliferation. To determine if the spontaneous proliferation of peripheral blood mononuclear cells from patients infected with HTLV-I and HTLV-II was related to coordinate expression of cytokines, we analyzed the levels of interleukin-1 beta (IL-1 beta), IL-2, IL-3, IL-4, IL-6, tumor necrosis factor-alpha (TNF- alpha) and interferon-tau (IFN-tau) in culture supernatants derived from spontaneously proliferating cells. Significantly elevated levels of IL-6 and TNF-alpha were present in culture supernatants from HTLV- I/II-infected individuals when compared with normal controls (P less than .01). Kinetic experiments showed that both IL-6 and TNF-alpha were elevated by day 5. None of the other cytokines (IL-1 beta, IL-2, IL-3, IL-4, and IFN-tau) were detectable in any of the culture. These data suggest that release of IL-6 and TNF-alpha may regulate lymphocyte proliferation in HTLV-I/II-infected individuals.

Description: Amphotropic helper-free retrovirus vectors containing the bacterial neomycin phosphotransferase gene (neo) and the human adenosine deaminase gene (adenosine aminohydrolase, EC 3.5.4.4; ADA) were used to transduce canine marrow cells. In one approach, dogs were treated for 7 days with recombinant human granulocyte colony-stimulating factor to stimulate hematopoietic cell division. Bone marrow cells were collected and transduced by 24 hours of cocultivation on vector-producing cells followed by incubation in a vector-containing long-term marrow culture system for 4 days. Transduced autologous marrow (0.4 to 1.0 x 10(8) cells/kg) was infused into dogs administered otherwise lethal total body irradiation (TBI) of 920 cGy. Two of four dogs engrafted, and their marrows showed intermittently between 1% and 11% G418-resistant colony-forming unit granulocyte-macrophage (CFU-GM) colonies for up to 2 years after transplantation. In a different experimental approach, autologous marrow, obtained at the time of the PB neutrophil nadir 7 days after a single cyclophosphamide injection (40 mg/kg intravenously), was cocultivated for 24 hours on vector-producing cells and infused at doses of 0.06 to 0.18 x 10(8) cells/kg into dogs administered 920 cGy TBI. One of three dogs engrafted, and the marrow showed intermittently 1% to 10% G418-resistant CFU-GM colonies for at least 2 years. Culture results were confirmed by polymerase chain reaction (PCR) showing the presence of the neo gene in marrow cells, peripheral blood (PB) granulocytes, and PB and lymph node lymphocytes. Dilution experiments indicated that up to 10% of marrow, lymph node, and PB cells contained the neo gene, consistent with the culture results. Samples harboring the neo gene also contained the gene for human ADA. However, repeated analyses of PB and marrow cells for human ADA gene expression by starch gel electrophoresis were negative. PB samples of all dogs were free of helper virus, and no long-term side effects from the transduction were observed.

Description: Seventy-seven cytomegalovirus (CMV)-seronegative marrow transplant patients were randomized in a prospective controlled trial comparing the use of leukocyte-depleted platelets plus CMV-seronegative red blood cells with standard unscreened blood products for the prevention of primary CMV infection during the first 100 days after transplant. Eligible patients included CMV-seronegative patients undergoing autologous transplant or seronegative patients undergoing allogeneic transplant for aplastic anemia or non-hematologic malignancy who had seronegative marrow donors. Patients and marrow donors were serologically screened for CMV and randomized before conditioning for transplant and followed for CMV infection with weekly cultures of throat, urine, and blood and with weekly CMV serologies until day 100 after transplant. Leukocyte-depleted platelets were prepared by centrifugation, a procedure that removed greater than 99% of leukocytes. There were no CMV infections observed in 35 evaluable treatment patients compared with seven infections in 30 evaluable control patients (P = .0013). There was no statistically significant difference in the mean number of platelet concentrates in the treatment patients (164 concentrates) compared with the control patients (126 concentrates). Leukocyte-depleted platelets plus CMV-seronegative red blood cells are highly effective in preventing primary CMV infection after marrow transplant.

Description: Previously we showed that mononuclear cells from about half of human T- lymphotropic virus (HTLV)-seropositive persons exhibit spontaneous proliferation in vitro. We sought to determine if proliferation was associated with other immunologic changes characteristic of HTLV infection. The parameters assessed were (1) percentages of lymphocytes expressing CD4 and/or CD25 (interleukin-2 receptor), (2) serum levels of soluble CD25, (3) serostatus for other viruses, (4) anti-HTLV antibody levels, and (5) HTLV type determined by polymerase chain reaction or serologic reactivity with type-specific peptides. The proliferation+ HTLV (PROL+) group, proliferation HTLV (PROL-) group, and control group showed similar percentages of CD4+, CD25+, and CD4+CD25+ lymphocytes; serum levels of soluble CD25 were also similar. Antibodies to cytomegalovirus, hepatitis B core, and hepatitis C were present in similar proportions of PROL+ and PROL+ groups. However, a significant association was found between spontaneous proliferation and anti-HTLV antibody levels; sera from 67% of PROL+ persons, but only 18% of PROL- persons, required dilution to yield absorbance values within the linear range of the anti-HTLV antibody assay. In the PROL+ group, persons whose sera required the most dilution had proliferative responses significantly higher than those whose sera required no dilution. The PROL+ and PROL groups were similar with regard to the relative distribution of HTLV-I and HTLV-II infection. These findings indicate that HTLV-related spontaneous lymphocyte proliferation is related to levels of circulating anti-HTLV antibodies, and characterizes both HTLV-I and HTLV-II infection.

Description: Major histocompatibility complex class I gene expression plays a central role in cellular immunity and tumor surveillance. A substantial proportion of spontaneous tumors are class I-deficient and numerous experiments have suggested that alterations in class I expression may alter oncogenicity and, as a result, have potential therapeutic impact. Interferons (IFNs) are able to upregulate class I expression by mechanisms that remain to be elucidated, but which appear to be IFN- and cell-type specific. We have characterized in detail the in vivo class I transcriptional response to IFN-gamma in two human hematopoietic tumor cell lines, the class I-deficient K562 cell line and the class I-positive Ramos cell line. In each, IFN-gamma induces a rapid increase in class I transcription, which is sustained in Ramos cells, but transient in K562 cells. In each, stimulation by IFN-gamma is dependent on ongoing protein synthesis, suggesting the requirement for production of a “primary response” protein. These data suggest that more than one type of IFN-gamma-induced signal is operative in the transcriptional response to IFN-gamma. Cycloheximide alone is also capable of inducing a rapid increase in class I transcription in both cell types, suggesting that constitutive attenuation of class I transcription may be a common phenomenon, and that IFN-gamma may act, in part, by interfering with such attenuation.

Description: A new case of heterozygous dysfibrinogenemia characterized by the replacement of NH2-terminal amino acid of fibrin beta-chain was found in a 50-year-old man. Despite a prolonged thrombin time, the propositus' fibrinogen had a normal reptilase time with the normal release of fibrinopeptide A. Release of fibrinopeptide B by thrombin was strongly affected, but a very high concentration of thrombin almost completely released fibrinopeptide B with a normal elution pattern on reversed-phase high performance liquid chromatography (HPLC). Lysylendopeptidase-cleavage of purified B beta-chains analyzed on HPLC showed the decrease of one peptide compared with the normal and the appearance of an abnormal peptide peak. These peptides were treated with thrombin and further separated on HPLC. Amino acid sequence analysis of the abnormal peptide demonstrated that B beta glycine-15, NH2-terminus of the fibrin beta-chain, was replaced by cysteine. These findings will be of particular importance because they strongly support the hypothesis that the NH2-terminal portion of the fibrin beta-chain is involved in the polymerization reaction by thrombin. The propositus' daughter and two sisters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Ise. During these studies, we found that a very high concentration of thrombin cleaves not only the A alpha Arg19-Val20 bond but also the COOH- terminal region of alpha-chains, which results in the generation of further degraded alpha-chains with apparent molecular weights of 44,000 or less.

Description: This report describes the development and characterization of a method for the amplification of rearranged V-J segments of the human T-cell receptor gamma chain (TCRG) locus using an adaptation of the polymerase chain reaction (PCR) technique. The technique uses a single pair of ‘consensus’ primers to amplify rearrangements involving the V gamma I subgroup genes, which are common in malignant cells from acute lymphoblastic leukemia (ALL) patients. Using this method we were able to detect rearrangements in the TCRG locus in disease cells from patients with T-cell ALL (12 of 12), common ALL (10 of 14), and Null cell ALL (2 of 2) at presentation. Monoallelic and biallelic rearrangements involving V gamma I subgroup genes were identified by restriction analysis of PCR products from DNA samples from a T-cell leukemic cell line, T-cell clones, and disease cells from patients with ALL of T-and B-cell lineage at presentation. These results confirmed the presence of cell clones within the presentation samples and, in one case, confirmed the persistence of the original malignant cell clone at relapse. This is a rapid and specific method for the detection and characterization of rearrangements of the TCRG locus without recourse to Southern blotting. Therefore, the PCR technique described herein can provide the basis for the study of clonal evolution and minimal residual disease on a high proportion of patients with ALL.

Description: We report the results of a preclinical study comparing four different purging protocols using a promyelocytic human cell line HL-60 and myeloid leukemic progenitor cells (colony-forming unit-leukemic [CFU- L]) from acute myelogenous leukemia (AML) patients assayed in semisolid culture. We studied the antileukemic effect of (1) Single-cycle complement-mediated lysis by two different monoclonal antibodies (MoAbs) (M195 [CD33] and F23 [CD13] 40 micrograms/mL), reactive with distinct antigens found on early myeloid cells and monocytes, used alone and in combinations; (2) 4-Hydroperoxycyclophosphamide (4-HC) (80 mumol/L or 100 mumol/L) alone; or (3) combined with VP-16 (5 micrograms/mL) and (4) a cocktail of 1 through 3 as above (combined immunochemotherapy). More than 4 logs of HL-60 tumor cell elimination were observed after 1 hour of incubation with both MoAbs plus 4-HC + VP- 16 while the single treatment (immunotherapy or chemotherapy) provided 1.5 and 3.5 logs of colony-forming inhibition, respectively. When the same protocols were tested on cryopreserved leukemic cells from eight patients with AML, we observed a mean value of CFU-L inhibition of 92.3% +/- 2.5% SD, 95.5% +/- 1.4% SD, and 99% +/- 0.8% SD after MoAbs and complement lysis, 4-HC, and 4-HC + VP-16 treatment, respectively. The combined treatment of MoAbs and 4-HC + VP-16 produced more than 3- log reduction of CFU-L colony formation. By comparison, the mean recovery of committed normal bone marrow progenitors after incubation with MoAbs and complement was 12% for CFU-granulocyte-macrophage (CFU- GM), 22.9% for burst-forming unit erythroid (BFU-E), and the recovery following 4-HC + VP-16 treatment was 4.4% for CFU-GM and 5.6% BFU-E. In subsequent experiments, highly purified CD34+ blast cells, enriched by positive selection, and stimulated in liquid culture by cytokines (interleukin-1 [IL-1], IL-3, and combination of both) or MO-conditioned medium (MoCM), demonstrated that immunochemotherapy spares hematopoietic colony-forming cells earlier than day 14 CFU-GM, in vitro.

Description: Platelet von Willebrand factor (vWF) has been suggested to play an important role in the hemostatic process. Clinical and experimental data indicate that bleeding time (BT) and platelet-vessel wall interaction cannot be normalized unless the defect of platelet vWF is also corrected. We have examined the effect of normal platelet concentrate transfusion 1 hour after cryoprecipitate infusion in five type III von Willebrand disease (vWD) patients. The cryoprecipitate infusion attained normal circulating levels of ristocetin cofactor, vWF antigen, and factor VIII activity. In two patients, cryoprecipitate infusion did not modify the BT (greater than 30 minutes), whereas in the remaining three patients BT was only partially corrected (from greater than 30 to 12, 18, and 21 minutes). However, the immediate platelet transfusion completely corrected the BT in four cases, and in one case it shortened the BT to 8.30 minutes (n = 3 to 8 minutes). In the perfusion study, cryoprecipitate infusion only resulted in a slight increase in platelet deposition (surface coverage range: 2.4% to 11.3%), whereas the platelet concentrate transfusion elicited a more marked improvement (range: 8.2% to 26.4%; P less than .02 v post- cryoprecipitate). These results suggest an important in vivo role of the platelet vWF in supporting platelet-vessel wall interaction. They also give support to the occasional addition of normal platelet transfusion to the cryoprecipitate infusion for the control of serious bleeding episodes resistant to cryoprecipitate in severe vWD patients.

Description: Transforming growth factor beta (TGF-beta) is a potent and selective growth inhibitor of early hematopoietic progenitors and leukemic cells. The cellular mechanism(s) underlying this antiproliferative effect is, however, currently unknown. In the present study, we demonstrate that TGF-beta inhibits the expression of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), and granulocyte-CSF (G-CSF) receptors on murine factor-dependent and independent hematopoietic progenitor cell lines without a significant change in receptor affinity. A maximum reduction in GM-CSF receptor numbers of 65% to 77% was observed by 96-hour incubation with TGF-beta. The TGF- beta induced trans-down-modulation of GM-CSF receptors was prolonged, noncytotoxic but reversible, and not due to endogenous production of GM- CSF. The TGF-beta induced reduction in CSF receptor numbers preceded TGF-beta's growth inhibitory action. In addition, the ED50 (1 to 10 pmol/L) for TGF-beta's CSF receptor modulatory and antiproliferative effect was similar. The effect of TGF-beta on cell surface CSF receptor expression was specific, because the expression of other cell surface proteins (Ly 5 and Ly 17) was not affected by TGF-beta treatment, and because other growth inhibitors (tumor necrosis factor and interferon) did not affect CSF receptor expression. These data suggest that the downregulation of the growth of hematopoietic progenitor cells by TGF- beta involves reducing the cell surface expression on growth factor receptors.

Description: The efficacy, toxicity, and mechanism of effect of intravenous Anti-D (Winrho) were studied in 43 Rh+ patients with immune thrombocytopenia purpura (ITP) who had not undergone splenectomy and in three already splenectomized patients. The mean platelet increase for the 43 nonsplenectomized patients was 95,000/microL (median 43,000/microL). Children had greater acute platelet responses than did adults. Human immunodeficiency virus status and duration of thrombocytopenia did not affect response. Maintenance treatment was given to patients as needed: the average interval between infusions was 24 days. The three splenectomized patients had no platelet response whatsoever. Toxicity was minimal; infusions were completed in less than 5 minutes. The generally accepted mechanism of effect of Anti-D has been Fc receptor blockade by substitution of antibody-coated red blood cells for antibody-coated platelets. Evidence is presented suggesting that the effect of IV Anti-D is not limited to Fc receptor blockade, including: (1) no correlation of parameters of hemolysis with platelet increase; (2) a 48- to 72-hour delay before platelet increase; (3) a tendency of the change in monocyte Fc receptor I expression to correlate with platelet increase; and (4) increased in vitro production of antibodies to sheep red blood cells following IV Anti-D infusion.

Description: Recently, several malignant cell types have been reported to express colony-stimulating factor-1 (CSF-1) transcripts; however, the clinical significance of CSF-1 in malignancy has not been investigated. Using a CSF-1 radioimmunoassay, we surveyed concentrations of biologically active CSF-1 in the peripheral blood of 316 patients with malignant and premalignant hematologic disorders; 75 had a myelodysplastic syndrome (MDS), 12 acute myelogenous leukemia (AML), 7 chronic myelogenous leukemia, 21 chronic lymphocytic leukemia (CLL), 106 non-Hodgkin's lymphoma (NHL; of low-, intermediate- and high-grade malignancy), 46 Hodgkin's disease (HD), 46 multiple myeloma (MM), and 3 monoclonal gammopathy of undetermined significance. Controls were 64 healthy subjects. The CSF-1 concentration was correlated with the type of disease, status of the disease, treatment status, and hematologic parameters. CSF-1 concentration was significantly elevated in 83.5% of the patients with active disease, and for each active disease group it was significantly greater (P less than .0001) than in the control. Thus, the high circulating CSF-1 concentration was not associated with a particular malignant phenotype or MDS subtype, but did correlate with the disease activity of both NHL and HD, and the tumor burden in MM, AML, and CLL. There was no correlation of the CSF-1 level with total counts of monocytes or neutrophils in patients with MDS or other malignancies. The cellular basis for the elevated circulating CSF-1 was not investigated. However, the results are consistent with the possibility that the premalignant or malignant cells themselves produce CSF-1 or regulate its production by normal cells.

Description: Fibronectin is an adhesive protein that can promote phagocytosis and endothelial cell adhesion. Plasma fibronectin declines following burn in animals and patients, potentially due to its complexing with circulating collagenous debris as well as its rapid binding to sites of tissue injury. Such depletion of fibronectin initiates an opsonic deficiency of the plasma. In view of the sensitivity of fibronectin to proteolytic enzymes, an additional factor that could contribute to the decrease of plasma opsonic activity after burn is the proteolytic fragmentation of fibronectin in the blood. In the current study, we determined if fibronectin fragments appear in the blood of anesthetized rats after a sublethal full-thickness skin burn of 15% to 16% of body surface. Plasma fibronectin concentration was quantified by enzyme- linked immunosorbent assay and the presence of fibronectin fragments in plasma was determined by immunoblot analysis. All blood was collected in an antiprotease mixture to yield final plasma concentrations of 0.15% EDTA, 3mmol/L phenylmethylsulfonyl fluoride, and 3 mmol/L iodoacetate to prevent degradation of fibronectin after sampling. Plasma fibronectin decreased 60% to 70% within 30 minutes post-burn, and this low level lasted for at least 4 hours. Within 30 minutes post- burn, two prominent fragments of fibronectin with a molecular weight of 110 +/- 2.2 kd and 122 +/- 3.3 Kd, respectively, were also detected in the plasma. Peak concentration of these fragments was detected at 60 minutes post-burn, but their level declined by 4 hours. By 4 hours, both bands appeared to resolve into doublets. To rule out the possibility that the fragments of fibronectin detected in the plasma were actually generated by coagulation enzymes activated at the site of peripheral blood sampling, rapid direct inferior vena cava sampling was performed, which also yield the presence of the fragments. Thus, fibronectin fragments exist in the plasma following thermal injury. Because fragments of fibronectin can compete with the intact fibronectin molecule with respect to its ability to stimulate macrophage phagocytosis, such fragments may contribute to altered systemic phagocytic host defense following thermal injury. Furthermore, because fibronectin peptides can compete with matrix fibronectin and impair adhesion of cultured endothelial cells, such circulating fragments may also influence the integrity of the vascular barrier.

Description: After decreasing in the first trimester of pregnancy, the total red blood cell mass increases in the second and third trimesters to peak at term at about 120% to 125% of nonpregnant values, but how this is brought about by changes in the rate of erythropoiesis is not known. We evaluated erythropoiesis by measuring serum transferrin receptor (TfR) levels in 406 women during normal pregnancy (N = 317), at delivery (N = 63), or in the early postpartum (N = 27). Despite the presence of the placenta and the frequent occurrence of iron deficiency, TfR levels remained low in the first two trimesters and increased in the third trimester and at delivery. To explain why erythropoiesic activity was relatively low in early pregnancy, we also measured serum immunoreactive erythropoietin (Epo) in relation to the degree of anemia. There was a very strong correlation between serum TfR and Epo levels in the entire group (r = .59, P less than .0001) as well as in each period of pregnancy. Epo levels remained low for the degree of anemia and did not correlate with hematocrit in the first two trimesters, but recovered afterwards. In the early postpartum, Epo production and erythropoiesis were normal. We conclude that: (1) erythropoiesis is decreased in the first part of pregnancy but increases afterwards; and (2) blunted Epo production in early pregnancy could be responsible for that observation.

Description: In this study, the question of whether glycoprotein Ib (GPIb) mediates both high and moderate affinity pathways of alpha-thrombin-induced platelet activation was examined. Flow cytometric studies, using a panel of monoclonal antibodies (MoAbs), showed that Serratia marcescens protease treatment removed greater than 97% of the glycocalicin portion of GPIb but did not affect the changes in the expression of GPIX or GMP- 140 that were induced by high concentrations of alpha-thrombin (10 nmol/L). However, Serratia treatment almost completely abolished the increase in platelet surface GMP-140 induced by low concentrations of alpha-thrombin (0.5 nmol/L) and diminished the downregulation of platelet surface GPIX by 60.9% +/- 5.6% (mean +/- SEM, n = 3). When present in 20-fold molar excess, an MoAb directed against the alpha- thrombin/von Willebrand factor (vWf) binding domains of GPIb completely blocked the ristocetin-dependent binding of vWf to platelets but inhibited only to about 50% the binding of alpha-thrombin and the activation-dependent binding of vWf. In platelets treated with Serratia marcescens protease to remove GPIb, a concentration of this MoAb 16,000- fold in excess of the maximum possible remaining copies of GPIb failed to inhibit platelet activation by alpha-thrombin. These studies demonstrate that activation of intact platelets by alpha-thrombin proceeds by both GPIb-dependent and GPIb-independent mechanisms.

Description: The c-myc gene plays a pivotal role in mediating the competence state for cell cycle transversion. This biologic role is in contradiction to reports of elevated expression of the gene in multiple myeloma, a tumor with restricted self-renewal capacity. To more clearly define the role of this gene in plasma cells of myeloma patients, c-myc messenger RNA (mRNA) and/or oncoprotein expression were semiquantitatively analyzed on the single cell level in 19 cases of multiple myeloma, among them 1 biclonal case and 1 case with coexistent chronic lymphocytic leukemia (CLL). Performing anti-sense/mRNA in situ hybridization, mature c-myc gene transcripts were detected in 92% (12 of 13) of cases and could definitely be attributed to the plasma cells by our study. The number of Ki 67-positive plasma cells actively passing the cell cycle was less than 1% and independent of c-myc gene expression. However, because the presence of the 152-c-MYC epitope was correlated to extent of marrow plasmacytosis (r = .64; P = .043) and content of plasmablasts (P = .09), the c-myc gene might serve a function different from proliferative activity, but also associated with tumor cell mass. In CLL cells (21 of 22 cases) and their benign counterparts, ie, bone marrow and peripheral blood lymphocytes, the anti-sense/c-myc mRNA hybridization signals remained below the threshold considered as cutpoint between negative and positive. The low amounts of c-myc transcripts were correlated to neither stage of disease (P = .52) nor lymphocyte counts (P = .24). Because the numbers of peripheral blood lymphoma cells were independent of tumor mass and of c-myc gene transcripts expressed, peripheral blood lymphocytosis might more likely reflect homing processes than proliferative activity in CLL.

Description: An elucidation of the interaction between the bone marrow microenvironment and hematopoietic stem cells is critical to the understanding of the molecular basis of stem cell self renewal and differentiation. This interaction is dependent, at least in part, on direct cell to cell contact or cellular adhesion to extracellular matrix proteins. Long-term bone marrow cultures (LTMC) provide an appropriate microenvironment for maintenance of primitive hematopoietic stem cells and a means of analyzing this stem cell-stromal cell interaction in vitro. Although LTMC have been successfully generated from murine and human bone marrow, only limited success has been reported in a primate system. In addition, few permanent stromal cell lines are available from nonmurine bone marrow. Because the primate has become a useful model for large animal bone marrow transplant studies and, more specifically, retroviral-mediated gene transfer analysis, we have generated immortalized bone marrow stromal cell lines from primate bone marrow using gene transfer of the Simian virus large T (SV40 LT) antigen. At least one stromal cell line has demonstrated the capacity to maintain early hematopoietic cells in long-term cultures for up to 4 weeks as measured by in vitro progenitor assays. Studies were undertaken to characterize the products of extracellular matrix biosynthesis and growth factor synthesis of this cell line, designated PU-34. In contrast to most murine bone marrow-derived stromal cell lines capable of supporting hematopoiesis in vitro that have been examined, the extracellular matrix produced by this primate cell line includes collagen types I, laminin. Growth factor production analyzed through RNA blot analysis, bone marrow cell culture data, and factor- dependent cell line proliferation assays includes interleukin-6 (IL-6), IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, leukemia inhibitory factor, and a novel cytokine designated IL- 11. This immortalized primate bone marrow stromal cell line may be useful in maintaining early progenitor cells for experimental manipulation without the loss of reconstituting capacity and as a potential source of novel hematopoietic growth factors.

Description: The Lewis blood group system comprises two main carbohydrate antigens, Le(a) and Le(b). Lewis typing has traditionally been based on serologic determinations using erythrocytes and saliva. Several recent studies have demonstrated that erythrocyte Lewis phenotype may change during pregnancy or disease, and inappropriate Lewis antigens have been found in both normal and neoplastic tissue. To evaluate whether these observations are in conflict with the presently proposed genetic and biosynthetic basis of the Lewis blood group system, we performed a combined enzymatic, immunohistologic, and immunochemical study of Lewis antigen expression in normal and neoplastic tissues, as well as erythrocytes, plasma, and saliva of Le(a-b-)-typed individuals. Of six cancer-bearing patients typed Le(a-b-), three were identified as nongenuine owing to the presence of alpha 1----4fucosyltransferase activity (alpha 1----4FT) and Lewis antigens in saliva and three were identified as genuine (lacking alpha 1----4FT and Lewis antigens in saliva). These genuine Le(a-b-) individuals were shown to express significant alpha 1----4FT in tissues, and Lewis antigens were detected in tissues by immunohistology as well as immunochemistry. We conclude that the Lewis phenotype obtained by serologic determination of erythrocytes and saliva does not apply to all tissues. We discuss biosynthetic and genetic consequences of this finding.

Description: The effects of competitive inhibition of hydroxymethylglutaryl coenzyme A (HMG CoA) reductase by compactin on the in vitro proliferation of peripheral blood myeloid leukemia cells were studied using the cells from 45 patients with acute myeloid leukemia or chronic myelogenous leukemia in blast phase. The cells from 58% of these patients showed a dose-related inhibition of DNA synthesis when incubated with compactin. Unexpectedly, cells from 18% of the patients were resistant to the inhibitory effects of compactin on DNA synthesis and responded to the HMG CoA reductase inhibition with an actual increase in the incorporation of 14C-labeled thymidine into DNA. Another 18% of the patients studied displayed both inhibition and stimulation of DNA synthesis in a biphasic response depending on the particular concentration of compactin used. The maximum enhanced rates of cellular DNA synthesis were observed with lower compactin concentrations (5 x 10(-7) mol/L) than were required for maximum inhibition of DNA synthesis (10(-5) mol/L). Leukemia cells displaying a stimulated response to compactin had a significantly lower baseline DNA synthetic rate than did cells that showed an inhibitory response of DNA synthesis to compactin. There was no correlation between these cells' varying DNA synthetic response to compactin and measures of baseline HMG CoA reductase activity or acetate conversion to cholesterol. Whereas the observation of cellular DNA synthesis stimulation by HMG CoA reductase inhibition has not been observed in other mammalian cells and seems paradoxical, explanations may emerge in light of our growing knowledge concerning the importance of isoprenylation for the function of certain cell regulatory proteins.

Description: Genetic markers of two general types have been used to assess the number of simultaneously productive stem cells in vivo, retrovirus markers and enzyme or hemoglobin variants. Use of the two techniques has led to different conclusions regarding stem-cell population organization, kinetics, and usage. To better understand this discrepancy, we have combined the two methods by retrovirally marking and transplanting stem cell populations of allophenic mice in which all tissues, including the hematopoietic system, are chimeric. Hematopoietic and lymphoid tissues of engrafted recipients were analyzed by Southern blotting to determine the number and extent of participation of individually marked stem cells. Genotypic chimerism of the same tissues was determined by quantitating electrophoretic variants of glucose phosphate isomerase. This procedure permitted the genotypic identification of individual stem-cell clones. The results demonstrate the participation of few pluripotent stem cells in the repopulation and maintenance of engrafted hematopoietic and lymphoid tissues. Furthermore, stem cells used during the period of early engraftment tended to be of one genotype (DBA/2), whereas stem cells used for long-term maintenance tended to be of the other, coexistent genotype (C57BL/6). We propose that this genotypic specificity reflects functional differences in stem-cell subpopulations and their relative prevalence in different mouse strains suggests a genetic component in the organization and usage of stem cells.

Description: 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.

Description: The unstable hemoglobin Montreal with a deletion of three amino acid residues (Asp, Gly, Leu) at positions 73, 74, and 75 of the beta chain and an insertion of four residues (Ala, Arg, Cys, Gln) at the same location was observed in a 7-year-old Canadian boy suffering from a moderate hemolytic anemia. The introduction of an extra amino acid residue and of other changes in the crevice where the heme group is located is the likely cause of the instability of this hemoglobin variant. The above listed changes were detected through analyses of tryptic peptides of the beta-Montreal chain, sequencing of amplified DNA, and hybridization of amplified DNA with appropriate, 32P-labeled, oligonucleotide probes. It is suggested that a mispairing involving the AGTG sequences at codons 66 and 67 and at codons 72 and 73 of the normal beta gene caused a repetition of a 16-bp segment, while a deletion of 10 nucleotides due to recombination or slippage followed by a second short deletion during DNA repair resulted in the modified sequence of the beta-Montreal gene.

Description: Severe type II essential mixed cryoglobulinemia (EMC) bears a poor prognosis. Treatment with corticosteroids and/or cytotoxic drugs infrequently results in long-term remissions, and is associated with significant toxicity. We conducted a prospective study with interferon (IFN) in 21 patients with severe type II EMC unresponsive to immunosuppressive regimens. They were treated with recombinant IFN- alpha 2a (18 patients) or with natural IFN-beta (three patients), alone, at a dosage of 3 megaunits (MU)/d for 3 months, followed by 3 MU every other day as maintenance. We observed 11 complete remissions, five partial remissions, and five minor responses. Of 16 patients observed for more than 1 year, 11 remained in remission for 14 to 40 months; five of them remained in complete remission for 18 to 40 months after withdrawal of treatment. Four patients discontinued treatment because of side effects. In four patients who relapsed while on maintenance therapy with recombinant IFN-alpha 2a, remission could be reinduced by treatment with natural IFN-alpha. The response rate of 77% achieved in this study prompts the use of IFN-alpha as a first-choice drug for type II EMC.

Description: In the present study, culture supernatants from larger granular lymphocytes (LGL) that were activated with Candida albicans antigens were shown to stimulate the ability of neutrophils to inhibit fungal growth. Identification of the activation factors showed that granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, was involved. Human peripheral blood mononuclear cells were fractionated by Percoll density centrifugation and each subpopulation of cells was stimulated with C albicans yeast cells. GM-CSF was produced in those fractions enriched for LGL, but not T lymphocytes or adherent monocytes. Additionally, the phenotype of the GM-CSF-producing cell was found to be CD2+, CD16+, HLA-DR+, and negative for CD4, CD8, and CD15. Kinetic studies demonstrated that GM- CSF appeared in the supernatants within 2 days of culture and continued to be produced up to 7 days. Optimal stimulation of LGL was seen at a ratio of 3 heat-killed C albicans yeast cells per LGL. Thus, LGL play an important role in host defense against this opportunistic pathogen by producing cytokines, including GM-CSF, which in turn activates the fungicidal activity of neutrophils.

Description: We assessed the effect of interleukin-9 (IL-9) on clonogenic maturation and cell-cycle status of hematopoietic progenitors of fetal (umbilical cord blood) and adult (bone marrow) origin. As a single agent IL-9 supported, in a concentration-dependent fashion, maturation of burst- forming units-erythroid (BFU-E) of adult and fetal origin. However, only 1/3 the number of adult BFU-E colonies developed, as did in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), and only 1/6 the number developed as did in response to IL-3. In contrast, the effect of IL-9 on fetal BFU-E colonies was equal to that of GM-CSF and IL-3. Synergistic effects of IL-9 with low concentrations (0.1 ng/mL) of GM-CSF and IL-3 were seen on adult BFU-E colony formation, but no effect was apparent at higher concentrations (1.0 ng/mL). In contrast, using fetal cells, synergistic effects of IL-9 with low and high concentrations of GM-CSF and IL-3 were apparent. Addition of IL-9 to plates containing fetal cells plus GM-CSF and IL-3 not only resulted in more BFU-E colonies, but also in more multicentered (greater than or equal to 10 individual centers) colonies, and more cells per colony. IL-9 had a wider spectrum of action on progenitors of fetal origin than on progenitors of adult origin, supporting the generation of fetal multipotent colony-forming unit (CFU)-Mix and CFU-GM colonies. Incubation with IL-9 did not accelerate cycling of adult or fetal BFU-E, CFU-Mix, or CFU-GM to the extent observed after incubation with IL-6. Thus, IL-9 primarily supported maturation of erythroid progenitors of adult origin, and its addition to plates containing GM-CSF and IL-3 (1.0 ng/mL) did not result in maturation of additional clones. In contrast, IL-9 had a wider spectrum of action on fetal progenitors and, when combined with IL-3 and GM-CSF, resulted in clonogenic maturation of progenitors that did not undergo maturation after stimulation with IL-3 and GM-CSF.

Description: In a series of 100 acute myeloid leukemia (AML) patients defined by cytochemistry and immunophenotyping, 20 expressed T-lymphocyte associated antigens on the surface of their blasts. While 15 expressed two or more T-cell antigens, five were found to express only CD7. All patients belonged to the French-American-British type M4, and four were under the age of 40. Despite intensive chemotherapy, four never obtained a complete remission and the fifth died of relapse after an allogenic bone marrow transplantation. While 12 randomly selected T- cell antigen negative AML patients showed only few rearrangements in Ig- or T-cell receptor (TCR) genes, such genetic alterations were demonstrated in four of five patients for the TCR delta gene and in all patients for the TCR beta gene. Interestingly, DNA fragments of similar size were demonstrated in three of five patients for both the beta and delta genes. These data suggest that the solitary presence of CD7 among T-cell antigens in otherwise clearcut AML cases identifies a group of patients with similarities in antigen receptor gene configuration as well as outcome.

Description: We have isolated and characterized a genomic clone of the human erythropoietin (Epo) receptor from a placental genomic library using a cDNA probe for the murine Epo receptor. The coding region spans about 6.5 kb with seven intervening sequences ranging in size from 81 bp to 2.1 kb. A stretch of 123 purines is found in the 5′ region from -456 to -578 upstream from the first codon and flanking the Alu repetitive sequences located further upstream. The human Epo receptor contains a palindromic sequence 5′ of the translated region that consists of an almost perfect inverted repeat of 12 nucleotides (CAGCTGC(G/C)TCCG) centered about G at -92 from the first codon. An inverted SP1 binding site (CCGCCC) and an inverted GATA-1 binding site (TTATCT) are located at positions -151 and -179, respectively, and CACCC sequences are located at -585 and further upstream. No TATA or CAAT sequences are in this 5′ flanking region. However, this region as far as -275 has a 72% GC content compared with an overall GC content of 56%. A 1-kb BamHI fragment of the human Epo receptor containing 700 bp of sequences 5′ of the coding region was transcribed in an in vitro transcription assay; initiation of transcription appeared to be around 132 +/- 5 just downstream from the inverted SP1 site at -151. T1 analysis of human Epo receptor messenger RNA also maps the site of transcription initiation to this region. Within 180 nucleotides 5′ to the first exon are three regions with 70% or greater homology with the murine Epo receptor. The study of this gene, including its similarities with the murine Epo receptor, should help elucidate aspects of the transcriptional and possible translational control of the Epo receptor in human erythroid cells and thus its role in signal transduction and erythroid differentiation.

Description: Murine bone marrow chimera models were used to assess the efficacy of host total body irradiation (TBI) given at different doses, dose rates, and fractionation schemes in providing for engraftment of syngeneic and allogeneic bone marrow. B6-Hbbd congenic and LP mice, respectively, were used as donors (10(7) bone marrow cells) for syngeneic and allogenic (H-2 compatible) transplantation in standard B6 recipients. Stable marrow chimerism was determined from host and donor stem cell- derived hemoglobin phenotypes (Hbbs and Hbbd) on gel electrophoresis at 3 months posttransplant. Partial engraftment of syngeneic marrow was seen at single doses as low as 2 Gy, with the donor component increasing steadily with increasing TBI dose to a level of 100% at 7 Gy. Immunologic resistance of the host appeared to prevent allogeneic engraftment until 5.5 Gy. A very steep radiation dose response was then observed so that the level of chimerism with 6 Gy and above became comparable with syngeneic engraftment. Low dose rate (5 cGy minute-1) and fractionated TBI required higher total doses for equivalent engraftment (radiation dose-sparing) in both syngeneic and allogenic bone marrow transplantation. This displacement in the dose-response curve on fractionation was seen with interfraction intervals of 3 and 6 hours. A further dose-sparing effect was observed on extending the interval to 18 and 24 hours, but only for allogeneic transplantation, and may therefore be related to recovery of immune-mediated graft resistance. The involvement of multiple target cell populations in determining allogenic engraftment rendered the application of the linear-quadratic model for radiation cell survival problematic in this case. The recovery in dose when low dose rate and 6-hour interfraction intervals were applied in either syngeneic or allogeneic BMT is consistent with appreciable sub-lethal damage repair in the primitive self-renewing stem cell population of the host marrow. These results contrast with the poor repair capacity of the 11-day spleen colony- forming units (CFUs) population after fractionated irradiation and support the notion that ablation of early stem cells in the pre-CFUs compartment is essential for long-term marrow engraftment.

Description: Interleukin-8 (IL-8) stimulated an increase in cytoplasmic-free Ca2+ ([Ca2+]i) and intracellular pH (pHi) in parallel at low concentrations (0.5 to 5 ng/mL), and stimulated O2- release and membrane depolarization in parallel at high concentrations (50 to 5,000 ng/mL). IL-8-induced O2- release was potentiated by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF) in a dose-dependent manner, whereas it was inhibited by cyclic AMP agonists. These characteristics and the time- courses of the responses stimulated by IL-8 were similar to those stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP), except that the cells stimulated by IL-8 showed shorter duration and less magnitude in some responses. In addition, IL-8 was found to be a potent priming agent and to enhance O2- release stimulated by FMLP. The priming effect of IL-8 was very rapid and was maximal within 5 minutes of preincubation. The dose-response curves for priming were identical to those for triggering of an increase in [Ca2+]i and pHi. The potency of the maximal priming effects on FMLP-induced O2- release was TNF greater than GM-CSF greater than IL-8 greater than G-CSF. The combination of IL-8 and the suboptimal concentrations of TNF or GM-CSF resulted in the additive priming effect, whereas the combination of the optimal concentration of IL-8 and the optimal concentration of TNF, GM- CSF, or G-CSF resulted in the effect of more potent priming agent alone. These findings suggest that IL-8 stimulates or primes human neutrophils according to its concentrations and cross-talks with TNF, GM-CSF, G-CSF, or FMLP at the inflammatory sites.

Description: A major feature of human immunodeficiency virus (HIV) infection is disordered B-cell function, which paradoxically includes both pathologic overactivity (elevated serum antibodies, lymphadenopathy, and increased risk for lymphoma) and underactivity (impaired antibody immunity, particularly to bacterial polysaccharide antigens). B-cell immune dysfunction contributes significantly to HIV-related morbidity and also represents an obstacle to eventual definitive treatment by anti-HIV immunization. Our laboratory has recently identified in normal B-cell populations certain VH gene subfamilies with a developmentally regulated pattern of utilization. In particular, B cells bearing rearranged VH3L were rare in the germinal center but uniformly abundant in the blood and lymphoid mantle zone. We used this index gene subfamily as a clonal criterion for the pattern of B-cell development in lymphocytes of HIV-positive individuals. In a series of 19 HIV- positive subjects, a striking deficit of VH3L B cells was observed; in contrast, none of the 16 normal subjects showed this abnormality. Other VH subfamilies (VH1N, VH4/6, and VH5N) were unaffected in the HIV- positive patients. This VH3L clonal deficit and other recent phenotype and histopathologic findings suggest that the general B-cell dysfunction in HIV is due to a discreet maturational arrest at the germinal center stage.

Description: The present study examined the effects of the androgen steroid, dihydrotestosterone (DHT), on murine T-cell production of a number of lymphokines. Direct exposure of murine T cells to DHT in vitro was found to reduce the amount of interleukin-4 (IL-4), IL-5, and gamma- interferon (gamma IFN) produced after activation with anti-CD3 without affecting the production of IL-2. Exposure of T cells to either androstenedione or testosterone (the metabolic precursors of DHT) affected no change in the biosynthesis of either of these lymphokines. We have determined that macrophages possess 5 alpha-reductase, and are thus competent to metabolize testosterone to DHT. This physicochemical information is complemented by a functional analysis of macrophage metabolism of testosterone. By incubating bone marrow macrophages with testosterone, before their use as accessory cells, the IL-4 and IL-5 producing potential of the activated T cells cocultured with them was depressed. That the observed effect was mediated by the conversion of testosterone to DHT was further corroborated by illustrating that the inhibition of IL-4 production was abrogated if 4MA, a specific 5 alpha- reductase inhibitor, was added to macrophage cultures containing testosterone. The biologic role of DHT in lymphokine and immune response regulation in vivo was addressed using several lines of investigation. First, transdermal delivery of DHT to groups of mice altered the capacity of T cells residing in the draining lymph nodes, only, to produce lymphokines. Second, treatment of either aged mice or the T cells isolated from them with a combination of dehydroepiandrosterone and DHT restored the capacity of their T cells to produce IL-2, IL-4, and gamma IFN to levels equivalent to that of younger mice. Finally, we observed a difference between males and females of a given age to produce IL-2, IL-4, and gamma IFN, with both IL-4 and gamma IFN production being elevated in females. Collectively, our findings indicate that DHT, similar to other steroid hormones, may play an important role in lymphokine regulation in vivo.

Description: Measurements of circulating transferrin (Tf) receptor are useful in assessing erythropoiesis; however, steps involved in the generation of soluble Tf receptor from cellular receptor are incompletely understood. To obtain a better understanding of this process, we investigated the loss of Tf receptor during terminal maturation of rat reticulocytes in vitro. Previous studies have identified Tf receptor-containing vesicles in the culture medium of maturing reticulocytes. In the present study, vesicle-free reticulocyte culture medium was found to contain functional and immunoreactive soluble Tf receptor, which increased over time. During a 44-hour incubation, Tf receptor on reticulocytes decreased by approximately 69%, while, of the Tf receptor shed to the medium, 65% was present in vesicles and 35% was in a soluble form. Isolated vesicles reincubated in fresh medium released soluble Tf receptor to the medium. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated soluble receptor protein was mainly 190 Kd and 95 Kd under nonreducing and reducing conditions, respectively, similar in size to the vesicular and cellular receptor. Our studies show that loss of Tf receptor from rat reticulocytes during maturation in vitro involves shedding of cellular Tf receptor in vesicles and release of soluble receptor from these vesicles.

Description: The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein IIb/IIIa) were conjugated with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) using the cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates (rscu-PA/MA-TSPI-1, rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, and rscu-PA/MA-LIBS-1), purified by immunoadsorption and gel filtration, were obtained with recoveries of 34% to 45%, with an average stoichiometry of 1.6 to 1.8 IgG molecules per rscu-PA molecule, and with unaltered specific activities and affinities. Preincubation of human platelet-rich plasma with rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, or unconjugated rscu-PA resulted in partial inhibition of ADP-induced aggregation; 25% inhibition was obtained with 63 micrograms/mL rscu-PA and with 6 micrograms u-PA/mL rscu-PA/MA-PMI-2 or 1.2 micrograms u- PA/mL rscu-PA/MA-PMI-1. In an in vitro system composed of a 125I-fibrin- labeled platelet-rich human plasma clot immersed in normal human plasma, the conjugates had threefold to greater than 15-fold less fibrinolytic potency than unconjugated rscu-PA. The thrombolytic potency of rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 was compared with that of rscu-PA and that of a control conjugate rscu-PA/MA-1C8 in a pulmonary embolism model in the hamster, using clots prepared from platelet-poor or platelet-rich human plasma. Lysis was measured 30 minutes after the end of a 60-minute intravenous infusion of the thrombolytic agents. rscu-PA, rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, as well as rscu-PA/MA-1C8 had comparable thrombolytic potencies (percent lysis per dose administered) towards platelet-poor human plasma clots. In contrast, the thrombolytic potency of rscu-PA/MA-PMI-1 and of rscu- PA/MA-LIBS-1 towards platelet-rich clots was 2.3- to 3-fold higher than that of rscu-PA (P less than .005) and fivefold to sevenfold higher than that of the control conjugate (P less than .01).

Description: The distribution of intercellular adhesion molecule-1 (ICAM-1), a ligand for lymphocyte function antigen-1, on hematopoietic tissue was determined using the anti-ICAM-1 monoclonal antibody CL203.4 with flow cytometry and short-term semi-solid hematopoietic progenitor cultures. After timed incubation in media with fetal bovine serum, 29% of erythroid burst-forming units (BFU-E), 24% of erythroid colony-forming units (CFU-E), and 52% of granulocyte-macrophage colony-forming units (CFU-GM) bone marrow progenitors expressed ICAM-1. This finding, which is consistent with the detection of ICAM-1 on acute non-lymphoblastic leukemic blasts, is at variance with recent reports. ICAM-1 was also detected on bone marrow blasts, proerythroblasts, promyelocytes, and cells of monocyte/macrophage lineage, but was not detected on erythroblasts, normoblasts, neutrophilic myelocytes, metamyelocytes, bands, or on most lymphocytes. These results indicate that maturation of cells of the erythroid and myeloid lineage is associated with loss of ICAM-1. The distribution of ICAM-1 on bone marrow progenitors, early precursor cells, and accessory cells in conjunction with the function of this molecule in cell-cell interactions suggests that ICAM-1 may play a role in the cell-cell and cell-stromal interactions that regulate hematopoiesis.

Description: Granulocyte-macrophage colony-stimulating factor (GM-CSF) exerts its biologic activities through binding to specific high-affinity cell surface receptors. After binding, the ligand/receptor complex is rapidly internalized in most hematopoietic cells. Using a human factor- dependent cell line, MO7, and normal human neutrophils, we found that the internalization is exquisitely temperature-dependent, such that ligand/receptor internalization does not detectably occur at 4 degrees C. Activation of the GM-CSF receptor has previously been shown to stimulate a number of postreceptor signal transduction pathways, including activation of a tyrosine kinase and activation of the serine/threonine kinase, Raf-1. The GM-CSF-stimulated increase in tyrosine kinase activity occurs rapidly at both 4 degrees C and 37 degrees C, and therefore is likely to be independent of receptor internalization. At 37 degrees C, the protein tyrosine phosphorylation was transient in MO7 cells, with maximum phosphorylation observed after 5 to 15 minutes, followed by a rapid decline. At 4 degrees C, the protein tyrosine phosphorylation of the same substrates was greater than at 37 degrees C, and no decline in substrate phosphorylation was observed for at least 90 minutes. In contrast to tyrosine phosphorylation, the activation and hyper-phosphorylation of Raf-1 observed at 37 degrees C in both MO7 cells and neutrophils was markedly diminished at 4 degrees C. These results indicate that at least one postreceptor signal transduction mechanism, activation of a tyrosine kinase, does not require ligand/receptor internalization, and indicate that receptor internalization may be a consequence, rather than the initiator, of signal transduction.

Description: We have previously shown that tumor necrosis factor-alpha (TNF alpha) strongly potentiates interleukin-3 (IL-3)-induced short-term proliferation of human CD34+ hematopoietic progenitor cells (HPC). Using longer term cultures of CD34+ HPC, we demonstrate here that this initial potentiation ceases after 10 to 12 days; whereupon TNF alpha displays inhibitory effects. Thus, TNF alpha was found to inhibit cells of granulocytic affiliation while it potentiates the development of maturing cells of the monocytic lineage both in liquid and semi-solid (day 14 colony-forming unit) cultures. TNF alpha was demonstrated to reversibly block granulocytic differentiation at the level of uncommitted CD13-, CD15- blast cells that accumulate in IL-3 + TNF alpha cultures. Furthermore, growth of committed granulocytes (CD15+) from IL-3 cultures was also inhibited by TNF alpha through an arrest of cell cycle in G0/G1. Finally, the use of neutralizing anti-TNF alpha monoclonal antibody and limiting dilution studies indicate that the inhibitory effects of TNF alpha are direct. Taken together, our data demonstrate that, following a phase of potentiation of proliferation of early HPC, TNF alpha displays direct inhibitory effects due to negative interference with both granulocytic differentiation and proliferation of granulocytic cells.

Description: We have identified the mutation in a phosphoglycerate kinase variant (PGK-Matsue) associated with severe enzyme deficiency, congenital nonspherocytic hemolytic anemia, and mental disorders. The mRNA coding for PGK was reverse transcribed and amplified by the polymerase chain reaction. Nucleotide sequencing of the variant cDNA showed a point mutation, a T/A----C/G transition in exon 3 of the variant gene. No other mutation was found in all coding regions of PGK-Matsue. The nucleotide change created an additional NciI cleavage site in the variant gene; thus, the NciI fragment types detected by Southern blot hybridization differ in the variant DNA and normal DNA. The mutation should cause Leu----Pro substitution at the 88th position from the NH2- terminal Ser of PGK. Because the Leu----Pro substitution is expected to induce serious perturbation and instability in the protein structure, the severe enzyme deficiency is mainly caused by more rapid in vivo denaturation and degradation of the variant enzyme.

Description: Homozygous mutant rats at the newly found white spotting (Ws) locus were anemic and deficient in mast cells and melanocytes. Because the phenotype of Ws/Ws rats resembled the phenotype of mice possessing a double-gene dose of mutant alleles at the W locus and because the c-kit gene was mapped at the W locus of mice, we characterized the c-kit gene of Ws/Ws rats. The authentic sequence of the rat c-kit cDNA was determined by using a cDNA library prepared from the hippocampus of Sprague-Dawley rats. The c-kit cDNA of Ws/Ws and normal (+/+) control rats was obtained by reverse transcriptase modification of the polymerase chain reaction. When compared with the authentic sequence, a deletion of 12 bases was found in the c-kit cDNA of Ws/Ws rats. This change was shown to be a result of the deletion of the genomic DNA. Four amino acids encoded by the deleted 12 bases (ie, Val-Lys-Gly-Asn) were located at two amino acids downstream from the tyrosine autophosphorylation site in the c-kit kinase and were conserved not only in mouse and human c-kit kinases but also in mouse and human c-fms kinases (ie, receptors of colony-stimulating factor-1). Taken together, the Ws/Ws rat is the first characterized mutant of the c-kit gene in an animal species other than the mouse.

Description: In this study five monoclonal antibodies (MoAbs) to T-cell receptor (TCR) proteins (WT31, alpha F1, beta F1, TCR delta-1 and delta TCS-1) were used to identify discrete maturative stages in 40 cases of T-cell acute lymphoblastic leukemia (T-ALL). These MoAbs reacted exclusively with CD3+ T cells and did not label B-lineage and myeloid cells. In 17 of the 40 T-ALL cases studied the leukemic blasts lacked membrane and cytoplasmic TCR chains (group I). In 12 cases cells did not have membrane CD3/TCR but expressed cytoplasmic TCR proteins heterogenously: nine cases had cytoplasmic TCR beta chains (beta F1+, alpha F1-; group II), one case had cytoplasmic TCR alpha chains (alpha F1+, beta F1-; group III), and two cases were labeled by both alpha F1 and beta F1 MoAbs (group IV). The remaining 11 cases were mCD3+: nine were TCR alpha beta+ (group Va) and two exhibited TCR gamma delta (TCR delta-1+, delta TCS-1+; group Vb). The analysis of the TCR beta, -gamma, and - delta gene configurations in 23 of the 40 T-ALLs showed that: (1) the lack of TCR protein expression was due to the lack of TCR gene rearrangements only in one of nine cases; (2) five of five TCR beta+, TCR alpha- cases studied had germline TCR alpha genes (ie, no detectable TCR delta gene deletions); (3) seven of eight cases with TCR delta gene deletions expressed TCR alpha proteins, whereas in 12 of 20 of the T-ALLs with TCR beta gene rearrangements the synthesis of the corresponding protein occurred; only 2 of 16 cases with rearranged TCR delta genes expressed TCR delta chains. The T-ALL categories identified with anti-TCR MoAbs did not have additional characteristic phenotypic patterns and may correspond to the normal stages of T-cell development more precisely than those defined by other differentiation antigens.

Description: We describe several novel analogs of the seco-steroid 1,25(OH)2-vitamin D3[1,25(OH)2D3] and their effects on differentiation and proliferation of HL-60 human myeloid leukemic cells in vitro as well as their effects on calcium metabolism in vivo. The 1 alpha-25(OH)2–16ene-23yne-26,27F6- vitamin D3 is the most potent analog reported to date, having about 80- fold more activity than the reference 1,25(OH)2D3 for inhibition of proliferation and induction of differentiation of HL-60 cells. Also, this analog decreased RNA expression of MYC oncogene in HL-60 by 90% at 5 x 10(-10) mol/L. Intriguingly, intestinal calcium absorption and bone calcium mobilization mediated in vivo by 1 alpha-25(OH)2–16ene-23yne- 26,27F6-D3 was found to be markedly (15-fold) less than that of 1,25(OH)2D3. In addition, 1 alpha-25(OH)2D3 bound to 1,25(OH)2D3 receptors of both HL-60 and intestine more avidly than did 1 alpha- 25(OH)2–16ene-23yne-26,27F6-D3. This novel analog may open up new therapeutic strategies for several hematopoietic, skin, and bone abnormalities and may provide a new tool to understand how vitamin D3 seco-steroids induce cellular differentiation.

Description: We studied the nature of blast cells in 41 patients with acute leukemia following a previous primary myelodysplastic syndrome (MDS) by a combined multiparameter analysis including morphologic, immunophenotypic, and molecular genetic (Igs, T-cell receptor (TCR)- beta, -gamma, and -delta and the major breakpoint cluster region [M- bcr]) investigations. In addition, the clinical and hematologic characteristics according to the immunophenotype of blast cells were analyzed. Our results show that, although the granulocytic and/or monocytic lineages are those most commonly involved in these acute leukemias, other cell components, including the megakaryocytic and lymphoid, may be present (12% and 15% of the cases, respectively). Moreover, both morphologic and phenotypic studies show the frequent coexistence of two or three cell populations. Interestingly, in all cases the lymphoblastic component constantly displayed an early B phenotype (CD19+, CD10-, TdT+). Upon analyzing whether the type of MDS conditioned any differences in the immunophenotype of blast cells, we observed that, although the lymphoid lineage may be involved in all MDS subgroups, some differences emerge within the myeloid leukemic transformations. Thus, the refractory anemias with excess of blasts (RAEB) and RAEB in transformation displayed a significantly higher incidence of myeloblastic and megakaryoblastic transformations, while in the RA, RA with ring sideroblasts and chronic myelomonocytic leukemia, the granulo-monocytic phenotype predominated. In addition, our results show that the clinical and hematologic characteristics of these patients may be partially related to the immunophenotype of the blast cells. Ig heavy chain gene rearrangements were found in two of 19 patients analyzed (11%), one with a hybrid leukemia (lymphoid-myeloid) and the other with a granulo-monocytic phenotype. Two other hybrid transformations analyzed were in germline configuration. Gamma and delta gene rearrangements were found in 21% and 37% of these acute transformation, respectively. The TCR-beta and M-bcr were in germline configuration in all 19 cases studied. In summary, immunophenotype and molecular studies point to a pluripotent stem cell with preferential myeloid commitment as the target cell of leukemias following a primary MDS.

Description: We have examined the effect of graft-versus-host disease (GVHD) on the reconstitution of donor hematopoiesis in a murine bone marrow transplant (BMT) model of GVHD to minor histocompatibility antigens. GVHD had no effect on peripheral blood counts, which normalized by 1 month after BMT, and did not affect numbers of hematopoietic progenitors in the BM, which remained decreased in all transplant recipients. Donor stem cells (colony-forming unit-spleen day 8) and stem cell self-renewal remained low in all mice for 5 months after transplant, but GVHD further damaged the stem cell compartment. Peripheral counts 1 month after transplant were supported by increased numbers of stem cells in cycle and increased splenic hematopoiesis. However, GVHD altered the pattern of extramedullary hematopoiesis, causing dramatically decreased activity in the spleen and increased activity in the liver. We conclude that GVHD further decreases hematopoietic reserve and causes damage to the donor stem cell compartment during hematopoietic reconstitution after transplant, despite unaffected progenitor frequencies and peripheral blood counts.

Description: The common acute lymphoblastic leukemia antigen (CALLA, CD10), which is expressed on early lymphoid progenitors and neutrophils, is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, “enkephalinase”). The CD10 cell surface enzyme is known to hydrolyze a variety of biologically active peptides including met-enkephalin, formyl-met-leu- phe (f-MLP), and substance P. These three CD10/NEP substrates induce the migration and aggregation of neutrophils, suggesting that each of the peptides can function as a mediator of neutrophil inflammatory responses. Recently, inhibition of CD10/NEP was found to reduce the concentration of metenkephalin needed to activate human and invertebrate granulocytes by several orders of magnitude. Herein we show that f-MLP and substance P induce rapid changes in neutrophil morphology, migration, and adhesion molecule expression, including upregulation of Mo1 (CD11b/CD18) and shedding of LAM-1 (also known as LECAM-1, Leu8, or TQ-1, the human homologue of murine gp100MEL14). Importantly, these coordinated changes are potentiated by inhibition of cell surface CD10/NEP enzymatic activity. Neutrophil cell surface CD10/NEP enzymatic activity is also shown to be regulated by the activation state of the cell during the time period in which the enzyme has its most pronounced effects. These results suggest that in neutrophils, CD10/NEP functions to control responsiveness to multiple inflammatory peptides.

Description: Several studies have suggested that the glycoprotein (GP) IIb-IIIa complex, which serves as the platelet fibrinogen receptor, also plays a role in the regulation of Ca2+ influx across the platelet plasma membrane. To examine this possibility further, we have compared Ca2+ transport in platelets and human erythroleukemia (HEL) cells, a megakaryoblastic cell line which synthesizes GP IIb-IIIa complexes that appear to be identical to those found on platelets. As with platelets, the results show the presence in unstimulated HEL cells of a rapidly exchangeable cytosolic Ca2+ pool that is in equilibrium with an intracellular sequestered Ca2+ pool and with extracellular Ca2+. Allowing for differences in cell size, the rate constants for Ca2+ exchange in HEL cells were similar to those in platelets. As in platelets, thrombin caused an increase in cytosolic Ca2+ that was due partly to enhanced Ca2+ influx and partly to the mobilization of internal Ca2+ stores. Incubation of the HEL cells with EDTA at 37 degrees C irreversibly altered the GP IIb-IIIa complex as evidenced by decreased binding of a complex-specific monoclonal antibody. In platelets this was accompanied by a 40% decrease in the rate of Ca2+ influx. However, in HEL cells there was neither a diminution in Ca2+ influx nor a reduction in the magnitude of the increase in cytosolic Ca2+ caused by thrombin. These results show that the parameters of Ca2+ distribution and movement are similar in HEL cells and platelets and that in HEL cells, as in platelets, the GP IIb-IIIa complex can be altered by removing Ca2+. However, unlike platelets, dissociation of the HEL cell IIb-IIIa complex has no discernible effect on plasma membrane Ca2+ transport. This suggests that earlier observations in platelets correlating changes in the rate of Ca2+ influx with changes in the number of intact IIb-IIIa complexes reflect an indirect, rather than a direct, role of these proteins in Ca2+ transport.

Description: Bone marrow samples from patients with pre-B acute lymphoblastic leukemia (pre-B ALL), either at diagnosis or at relapse, were transplanted into scid mice to determine whether these freshly obtained leukemic cells could proliferate in vivo and whether there were any differences in their in vivo growth characteristics. Cells from three patients who relapsed within 13 months of diagnosis proliferated rapidly in the murine bone marrow, spleen, and thymus, invaded peripheral organs, and resulted in morbidity and mortality of the animals within 4 to 16 weeks. Cells from two patients who relapsed 3.5 years after diagnosis grew much slower than the early relapse samples, taking up to 30 weeks to infiltrate the bone marrow of recipient mice. In contrast, leukemic cells were absent or were detected at low numbers in scid mice transplanted with cells obtained at diagnosis from three patients who have not yet relapsed. These results show an increased ability of leukemic cells from patients with aggressive lymphoblastic leukemia of poor prognosis to proliferate in scid mice.

Description: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40–77, 78–94, or 110–127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.

Description: Interleukin-1 (IL-1) profoundly affects a number of functions of vascular cells. Two distinct IL-1 receptors (IL-1R) are expressed on different cell types: the 80 Kd IL-1RI on T cells and fibroblasts, and the 68 Kd IL-1RII on B cells and myelomonocytic cells. The presence and functionality of IL-1R on vascular cells has been investigated by using polyomatransformed mouse endothelial cell (EC) lines (sEnd.1 and tEnd.1). These cells expressed specific and saturable binding sites for IL-1 (1,273 sites per cell with kd 9.5 x 10(-11) mol/L for sEnd.1, and 771 sites per cell with kd 8.5 x 10(-11) mol/L for tEnd.1, with radioiodinated IL-1 alpha as ligand). Binding of IL-1 was also evident at single cell level by autoradiography. By cross-linking studies, the molecular weight of the IL-1 binding protein on EC was approximately 80 Kd. This was confirmed by the presence in EC of mRNA for the 80 Kd IL- 1RI. The IL-1RI on EC was apparently functional, since EC responded to IL-1 with IL-6 mRNA expression and IL-6 bioactivity production. These results were extended to human EC and vascular smooth muscle cells, which were also found to express mRNA for IL-1RI.