Recombinant human interleukin-3 stimulation of hematopoiesis in humans: loss of responsiveness with differentiation in the neutrophilic myeloid series

Recombinant human (rh) interleukin-3 (IL-3) stimulated the proliferation and differentiation of erythroid, granulocyte, macrophage, eosinophil (Eo), and mixed colonies as well as megakaryocytes from human bone marrow cells. rh IL-3 was a weaker stimulus than rh granulocyte-macrophage colony-stimulating factor (GM- CSF) for day 14 myeloid cell colonies. At day 7 of incubation, rh IL-3 stimulated a few G, M, and Eo clusters but no colonies. This loss of responsiveness of myeloid cells to rh IL-3 was accentuated with further differentiation of the cells. rh IL-3 stimulated very few or no clones after five-day incubation with enriched promyelocytes and myelocytes, whereas rh GM-CSF was an efficient stimulus. Responsiveness to rh IL-3 was completely lost in postmitotic mature neutrophils. Incubation of these cells with rh IL-3 did not result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells or superoxide anion production after stimulation with formyl-methyl-leucyl-phenylalanine (FMLP), although they could be stimulated by rh GM-CSF. In addition, preincubation of neutrophils with different concentrations of rh IL-3 failed to increase or decrease their response to rh GM-CSF. In contrast to neutrophils, mature Eos could be stimulated by rh IL-3 to kill antibody-coated tumor cells. These results show that cells of the neutrophilic myeloid series lose their responsiveness to h IL-3 as they differentiate and suggest that although h IL-3 may be an important therapeutic agent to use for hematopoietic regeneration in vivo, the lack of stimulation of mature neutrophil function makes it an unlikely sole candidate as adjunct therapy for treatment of infectious diseases.     

Human erythrocyte antigens: II. The In(Lu) gene regulates expression of an antigen on an 80-kilodalton protein of human erythrocytes

We have previously shown that a murine monoclonal antibody (A3D8) identifies a human erythrocyte protein antigen whose expression is regulated by the Lutheran inhibitor [In(Lu)] gene. In the present study, we demonstrated by immunoprecipitation and Western blot techniques that the antigen defined by A3D8 was on an 80-kD erythrocyte membrane protein. A second 170-kD protein was coprecipitated with the 80-kD protein but failed to show antigen activity by Western blot analysis. The 170-kD protein, when analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis in two dimensions, was composed of 50- and 30-kD disulfide-linked subunits. In(Lu) Lu[a-b-) erythrocytes differed from Lu(a+b+) or Lu(a-b+) erythrocytes in that In(Lu) deoxycholate erythrocyte membrane extracts contained trace amounts of immunoprecipitable 80-kD protein compared with detergent-solubilized erythrocyte membrane extracts prepared from Lu(a+b+) or Lu(a-b+) subjects.     

Fludarabine: a new agent with major activity against chronic lymphocytic leukemia

Fludarabine was used to treat 68 patients with previously treated chronic lymphocytic leukemia (CLL). Nine (13%) patients achieved a complete remission and 30 (44%) a partial remission. The response rates for Rai stages 0 to 2, 3, and 4 were 64%, 58%, and 50% respectively. Seventeen (43%) of the 40 Rai stage 1 to 3 patients and four (19%) of the Rai stage 4 patients returned to Rai stage 0. Survival was strongly correlated with the final Rai stage achieved. The survival of the 11 partial responders with residual disease consisting only of residual bone-marrow nodules was similar to the complete responders (36+ months) and superior to the other partial response patients (16 months). The response to fludarabine was rapid, with 36 (92%) of the 39 responders having achieved at least a partial response following the first three courses. Complete responses occurred in the blood, liver, spleen, and lymph nodes in 48% to 69% of the patients. Eradication of all disease in the bone marrow occurred in only 13% of the cases. Neutropenia and thrombocytopenia occurred in 56% and 25% of evaluable courses. Major infections occurred in 9% of evaluable courses and fevers of unknown origin or minor infections in 12% of courses respectively. Myelosuppression and infection were more common in patients with initial Rai stages 3 and 4 and in nonresponding patients. Other toxicity was mild. No CNS toxicity was noted.     

Analysis of N-RAS exon-1 mutations in myelodysplastic syndromes by polymerase chain reaction and direct sequencing

Mutations in codons 12 or 13 of the first exon of the N-RAS gene have been reported in myelodysplastic syndromes (MDS) in frequencies that vary between 9% and 40% depending on the techniques used in analysis. Gene amplification and direct sequencing provides the only unambiguous method of detecting those mutations that induce amino acid alterations. Using this technique, we analyzed 21 MDS patients for mutations in exon- 1 of N-RAS. Codon 12 mutations substituting aspartic acid (GAT) for glycine (GGT) were found in four cases, and a codon 13 mutation substituting alanine (GCT) for glycine (GGT) was detected in one patient. We conclude that N-RAS exon-1 mutations in one patient. We conclude that N-RAS exon-1 mutations producing amino acid changes occur in about 20% to 25% of MDS cases.     

The Inab phenotype: characterization of the membrane protein and complement regulatory defect

Recent demonstration that Cromer-related human blood group antigens reside on decay-accelerating factor (DAF) has led to identification of an apparent null phenotype (Inab) for erythrocyte DAF. This study examined expression of other phosphatidylinositol (PI)-anchored proteins by Inab erythrocytes and showed that the PI-linked membrane proteins acetylcholinesterase (AchE) and lymphocyte function-associated antigen-3 (LFA-3) are normally expressed by these cells. Furthermore, studies of the complement sensitivity of Inab RBCs demonstrated them to be abnormally complement sensitive, with an apparent defect in downregulation of C3 convertase activity. Thus, the Inab phenotype appears to represent an instance of hereditary erythrocyte DAF deficiency whose mechanism differs from that of paroxysmal nocturnal hemoglobinuria (PNH) and which is unassociated with clinically evident hemolytic disease.     

Inhibition of monocyte esterase activity by organophosphate insecticides

Organophosphate insecticides, such as Vapona, Naled, and Rabon, are highly potent inhibitors of an enzyme found in human monocytes. The enzyme, a specific monocyte esterase, could be inhibited by Vapona in blood samples via airborne contamination at levels easily achieved from commercial slow-release insecticide strips. Fifty percent inhibition (I50)--as measured on the Hemalog D (Technicon Corp.)--occurred at solution concentrations of 0.22, 1.5, and 2.6 X 10(-6) g/liter for Vapona, Rabon, and Naled, respectively. Parathion (a thiophosphate) and Baygon (a carbamate) were less potent, with I50 values of 3.7 X 10(-5) and 1.5 X 10(-4) g/liter, respectively. Dursban (another thiophosphate) and Carbaryl (a carbamate) showed only marginal inhibition. Eserine, malathion, nicotine and pyrethrum had no inhibitory effect up to 0.5 g/liter. The occurrence of this effect in vivo has not yet been shown, nor is it clear what the implications of such an effect would be. The inhibition of this enzyme by airborne contaminants, however, may interfere with the proper functioning of the Hemalog D.     

Accumulation of methotrexate and methotrexate polyglutamates in lymphoblasts at diagnosis of childhood acute lymphoblastic leukemia: a pilot prognostic factor analysis

Lymphoblasts in bone marrow samples, obtained from 43 children with acute lymphoblastic leukemia at diagnosis, were incubated with 1.0 mumols/L [3H] methotrexate for 24 hours in vitro. Nonexchangeable methotrexate and methotrexate polyglutamates were separated and quantitated. Event-free survival at 5 years was 38% +/- 9% for all 43 patients (27 failures), and 44% +/- 10% for the 35 with non-T, non-B- cell acute lymphoblastic leukemia (20 failures). Of these 35 children, those whose lymphoblasts accumulated more than 100 pmol methotrexate and 500 pmol methotrexate polyglutamates per billion cells experienced better 5-year event-free survival than those whose lymphoblasts did not (65% +/- 12% v 22% +/- 9%, P = .010). This difference characterized "good-risk" patients who were female (P = .014), less than age 7 at diagnosis (P = .005), or had low initial white blood cell counts (less than 20 X 10(9)/L, P = .018). Findings were similar for the 43 children with acute lymphoblastic leukemia and for the "good-risk" children in this total group. Thus, the ability of lymphoblasts to accumulate methotrexate and form methotrexate polyglutamates may be important to the curative properties of current therapy of acute lymphoblastic leukemia in children, particularly for "good-risk" patients. In such patients, inherent rather than acquired drug resistance may be the initial event leading to treatment failure.     

Hairy cell leukemia and myelomatosis: chance association or clinical manifestations of the same B-cell disease spectrum

We describe three patients who had typical features of hairy cell leukemia (HCL) and multiple myeloma (MM) at the same time. In two, both diagnoses were made within a short period of time, and in the third, HCL had been present for 2 yr before the appearance of a paraprotein, bone lesions, and plasma-cell infiltrates established the diagnosis of MM. Although this association has not been previously reported, cases of HCL with osteolytic lesions or a paraprotein band have been described. The cases described may represent clinical manifestations of closely related disorders arising from divergent differentiation from a common B-cell precursor rather than a chance association.     

Somatic hypermutation in low-grade mucosa-associated lymphoid tissue- type B-cell lymphoma

The origin of low-grade mucosa-associated lymphoid tissue (MALT)-type B- cell lymphoma is still unclear. Using a novel two-step procedure, we have sequenced the Ig VH genes expressed by cells from four patients with gastric low-grade MALT-type lymphoma. The nucleotide sequences of the complementarity determining region 3 (CDR3) of the genomic DNA were first amplified using consensus oligonucleotide primers, then sequenced. Based on the CDR3 sequence amplified from each MALT lymphoma, individual tumor-specific primers were synthesized and used directly in the polymerase chain reaction (PCR) to analyze the sequences of their Ig heavy-chain variable region. When compared with the germ-line sequence, many nucleotide substitutions, mainly in the CDRs, were found in the variable gene sequences of the four MALT lymphomas. The mutations showed a high replacement-to-silent ratio and were distributed in a way which suggested that the tumor cells had been positively selected through their antigen receptor. Our findings indicate that the MALT-type lymphoma B cells are hypermutated postgerminal center lymphocytes that have undergone antigen selection.