Monoclonal antibodies to the human CSF-1 receptor (c-fms proto-oncogene product) detect epitopes on normal mononuclear phagocytes and on human myeloid leukemic blast cells

The first monoclonal antibodies (MoAbs) to epitopes in the extracellular domain of the human c-fms proto-oncogene product (receptor for the macrophage colony stimulating factor, CSF-1) were used with flow cytometric techniques to study receptor expression on normal human peripheral blood monocytes, bone marrow cells, and leukemic blasts. On normal cells CSF-1 receptors were restricted in their expression to cells of the mononuclear phagocyte lineage. CSF-1 receptors were detected on leukemic blasts from 15 (30%) of 50 children with acute myeloid leukemia, compared with four (15%) of 26 adults. By contrast, detectable CSF-1 receptors were uniformly absent on blasts from 19 children with acute lymphoblastic leukemia. CSF-1 receptors on normal monocytes and myeloid leukemia cells could be induced to downmodulate by incubation with either human recombinant CSF-1 or phorbol esters, confirming that the receptors had functional ligand- binding sites and responded to transmodulation by inducers of protein kinase C. The numbers of receptors per cell and the percentage of positive cases were highest for leukemic blasts with cytochemical and morphological features of monocytes. However, CSF-1 receptors were also detected on a subset of leukemic blast cells with features of granulocytic differentiation (FAB subtypes M1 through M3). Southern blotting analyses of DNA from 47 cases of acute myeloid leukemia demonstrated no rearrangements within the 32 kb of genomic sequences that contain CSF-1 receptor coding exons or in the 50 kb upstream of the first coding exon. Analysis of the upstream region of the c-fms locus revealed that sequences representing the terminal 112 untranslated nucleotides of c-fms mRNA map 26 kb 5' to the first coding exon, suggesting that at least one c-fms promoter is separated from the receptor coding sequences by a very long intron. Whereas expression of the CSF-1 receptor in myeloid leukemic blasts is not restricted to cells with monocytic characteristics, the apparently aberrant pattern of receptor synthesis in a subset of cases with granulocytic features appears not to be due to chromosomal rearrangements within 50 kb upstream of sequences encoding the receptor.     

Gastric mucosal protection with selective inhibition of thromboxane synthesis.

Because thromboxane synthesis enhances gastric mucosal damage we have investigated whether the thromboxane synthesis inhibitor dazmegrel might be protective to the mucosa. Dazmegrel at a dose of 1 and 5 mg per rat (4.8 and 23.8 mg/kg) significantly reduced the damage caused by acidified taurocholate. In parallel experiments dazmegrel exerted a selective and dose dependent inhibition of ex vivo thromboxane synthesis by gastric fragments over the dose range in which protection was observed. As dazmegrel can be given to man, these experiments suggest that investigation of mucosal protection would be justified.     

2-Chlorodeoxyadenosine: an effective new agent for the treatment of chronic lymphocytic leukemia

2-Chlorodeoxyadenosine, a new lymphocyte-selective, anti-neoplastic drug was administered to 18 patients with advanced chronic lymphocytic leukemia of B-cell origin. All patients were resistant to conventional treatment. A total of 44 courses of 2-chlorodeoxyadenosine were completed with minimal toxicity. An overall response rate of 55% was achieved with four of 18 patients demonstrating partial response and six of 18 patients experiencing clinical improvement. Only minor bone marrow suppression occurred during administration of the drug, indicating a high degree of lymphocyte selectivity. Reduction of lymphocyte infiltration in bone marrow occurred in treated patients including one patient who experienced normalization of the bone marrow. Three of four patients with concurrent autoimmune hemolytic anemia experienced resolution of hemolysis, as indicated by elimination of transfusion requirement, fall in reticulocyte count, elevation of hemoglobin, and ability to taper prednisone without recurrence of hemolysis. Duration of responses ranged from 2 to 15 months without maintenance therapy.     

Frequent deletion of p16INK4a/MTS1 and p15INK4b/MTS2 in pediatric acute lymphoblastic leukemia

The tandemly linked p16INK4aMTS1 and p15INK4b/MTS2 genes on chromosome 9, band p21 encode proteins that function as specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6. This locus undergoes frequent bi-allelic deletion in human cancer cell lines, suggesting that the encoded proteins may function as tumor suppressors. However, more recent analysis of primary tumor samples has shown a much lower frequency of abnormalities affecting this region, raising doubt over the importance of these proteins in human malignancies. Hemizygous deletions and rearrangements of chromosome 9, band p21, are among the most frequent cytogenetic abnormalities detected in pediatric acute lymphoblastic leukemia (ALL), occurring in approximately 10% of cases. To determine if the p16INK4a/p15INK4b locus might be the target of these chromosomal lesions, we analyzed both genes in primary clinical samples from 43 pediatric ALL patients using interphase fluorescence in situ hybridization, Southern blot analysis, and the polymerase chain reaction. Deletions of p16INK4a/p15INK4b were identified in 18 of 20 cases with cytogenetically observed abnormalities of 9p and 5 of 23 with apparently normal chromosomes 9p, with the majority containing bi- allelic deletions (16 homozygous/7 hemizygous). Although most homozygous deletions involved both genes, Southern blot analysis showed an interstitial deletion in a single case that was confined to p16INK4a, suggesting that p15INK4b was not the critical target gene in this case. Sequence analysis of both p16INK4a and p15INK4b in all seven cases with hemizygous deletions failed to show mutations within the coding regions of the retained alleles. In this select group of patients, deletion of p16INK4a/p15INK4b was associated with T-cell phenotype, nonhyperdiploid karyotype (< 50 chromosomes), and poor event- free survival. These findings indicate that deletion of the p16INK4a/p15INK4b locus is one of the most common genetic abnormalities so far detected in pediatric ALL, and that loss of one or more of these cell cycle kinase inhibitors is important in leukemogenesis.     

Antibody-dependent cytolysis of the human leukemia cell line K-562 in the absence of effector cells or complement

K-562 human leukemia cells grown in the presence of specific goat anti- K-562 gamma globulin, F(ab)'2, Fab', or Fab showed a decrease in DNA and protein syntheses and a loss of cell viability within several hours. Eventually all cells died and lysed within 2-5 days. These events occurred in the absence of added effector cells or complement. The effect on the cell was not due to proteolytic or other external lytic activity generated at the cell surface, since 51Cr-labeled bystander cells or antibody-sensitized bystander cells (chicken erythrocytes or K-562 cells) were not lysed when added to cultures of K- 562 cells incubated with immune globulin. The anti-K-562 gamma globulin was not observed to patch or cap on K-562 cells, but the ability of bound immunoglobulin to fix complement decreased markedly (50% in 60 min), so that no complement-mediated cytolysis could be observed at 2 hr. However, the immunoglobulin, at least portions of it, reacted with fluorescein--labeled rabbit anti-goat gamma globulin for about 16 hr. 125I-labeled immune globulin appeared to be internalized and released into the medium as small degradation products. 125I-labeled Fab did not appear to be internalized, since it was released intact and more rapidly from cells than the intact globulin.     

Defect in B cell function in HTLV III/LAV positive hemophilia patients

The capacity of the peripheral blood lymphocytes (PBL) to generate an antibody response in vitro T cell-dependent antigen ovalbumin was studied in 12 severe hemophilia patients who were otherwise in good health. PBL from four of 12 patients were not capable of generating such a response after stimulation in vitro, whereas all controls were normal. This negative plaque-forming cell (PFC) response coincided with the presence of antibodies directed toward human T-lymphotropic virus III/lymphadenopathy-associated virus (HTLV-III/LAV). Only one patient with antibodies against HTLV-III/LAV had a normal PFC response. The negative PFC response was not due to a deficient T helper cell activity, nor to an excessive T suppressor cell function. However, in the peripheral blood of these four patients, the presence of activated B cells that are refractory to antigen-specific T helper cell signals and secrete specific antibodies spontaneously could be demonstrated. Most of the patients showed a hyperimmunoglobulinemia. No correlation between the T4/T8 ratio and the level of the PFC response was demonstrable. From the data obtained in these investigations we raise the hypothesis that infection with HTLV-III/LAV in hemophilia patients will lead to in vivo (pre)activation of B cells that results in unresponsiveness or decreased response to antigen-specific signals.     

The relationship between the hemorrhagic and antithrombotic properties of low molecular weight heparin in rabbits

We have compared the hemorrhagic and antithrombotic effects of a low molecular weight (LMW) heparin fraction and standard heparin in rabbits. Similar LMW heparin fractions have antithrombotic effects when tested in animals, but their hemorrhagic effects relative to standard heparin have not been established. Standard porcine mucosal heparin (mol wt 15,000 daltons) was depolymerized by nitrous acid to a low molecular weight fraction (mol wt 4600 daltons). Using equal USP units, the standard and Dep LMW heparin were compared in vitro, ex vivo, and in vivo. In vitro, when diluted in rabbit plasma, the Dep LMW heparin at equivalent anti-Xa activity showed less prolongation of thrombin clotting times or activated partial thromboplastin times. Ex vivo, platelets from rabbits treated with the Dep LMW heparin showed less inhibition of collagen-induced aggregation. The relative hemorrhagic properties of the two heparins were compared in vivo in rabbits using a sensitive blood loss assay, and the antithrombotic properties were compared in a thrombin-induced venous stasis model. By using an optimal threshold heparin dose in each test system, it was possible to demonstrate that equal USP units of Dep LMW heparin caused less blood loss but showed greater antithrombotic activity than standard heparin.     

Identification of a distinct low-affinity receptor for human interleukin-4 on pre-B cells

Biotinylated interleukin-4 (IL-4) was used to examine IL-4 receptor (IL- 4R) expression on a range of human B-cell lines by flow cytometry. Using high concentrations of biotinylated IL-4, we have identified a novel low-affinity IL-4 receptor expressed at high levels on pre-B lines. Expression of this low-affinity receptor did not correlate with detected mRNA levels for the previously cloned receptor or with reactivity of two anti-human IL-4R monoclonal antibodies (MoAb). Radiolabeled IL-4 cross-linking studies using pre-B lines showed a doublet of 65 to 75 Kd in contrast to the 110- to 130-Kd molecule detected on cells expressing the cloned IL-4R. A soluble IL-4 binding protein (IL-4bp) was purified from the supernatants of three pre-B lines expressing the low-affinity receptor on their surface. IL-4bp could block both IL-4-mediated CD23 induction on tonsil B cells and IL- 4-induced inhibition of proliferation of the pre-B line JM1. Partial N- terminal amino acid sequence was obtained from purified IL-4bp that confirmed this protein to be novel. A 12 amino acid peptide based on the IL-4bp sequence was used to produce a polyclonal antiserum that was reactive with purified IL-4bp, and also bound to the surface of pre-B cells but not to murine CTLL cells transfected with the human IL-4R. Blocking MoAb against the previously characterized high-affinity receptor inhibited IL-4-mediated proliferation of hIL-4R+ CTLL cells but had no effect on IL-4-induced inhibition of JM1 cell proliferation, and only partially inhibited IL-4-mediated CD23 and sIgM induction and proliferation of tonsil B cells. The data presented here provide evidence for a novel cell-surface expressed low-affinity IL-4R that also exists as a biologically active soluble IL-4 binding protein.     

Heterogeneity of T-cell lymphoblastic malignancies

To determine the T-cell lineage of the malignant lymphoblast in lymphoblastic lymphoma, tumor cells from nine patients were phenotyped employing a T-cell subset specific heteroantisera, TH2. The normal human peripheral blood T-cell compartment is composed of 80% TH2- negative and 20% TH2-positive T cells, as defined by reactivity with subset specific heteroantisera. Human suppressor cells are TH2 reactive, whereas helper cells are TH2 unreactive. Tumor cells from the majority of patients with lymphoblastic lymphoma were TH2 reactive in contrast to the lack of reactivity previously described in the majority of patients with T-cell acute lymphoblastic leukemia. Comparative clinical studies, including disease presentation and course, were correlated with the presence of the TH2 antigen on the tumor cell. These results provide evidence to support the notion of heterogeneity in the T-lymphoblastic malignancies and suggest that lymphoblastic lymphoma and T-cell acute lymphoblastic leukemia are probably not a single disease process.