Neutralizing and nonneutralizing monoclonal antibodies to the human granulocyte-macrophage colony-stimulating factor receptor alpha-chain

A panel of monoclonal antibodies was raised against the low-affinity human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor alpha-chain expressed as recombinant protein on murine FDC-P1 cells. All the selected antibodies were of the IgG2A isotype and bound to protein A. They each recognized both native and recombinant receptors by indirect surface immunofluorescence and by immunoprecipitation. Several of the antibodies also recognized presumably denatured receptors as detected by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three different epitopes on the extracellular domain of the GM-CSF receptor alpha-chain were defined by these antibodies, and two of the epitopes did not appear to be involved in binding hGM-CSF or in interactions with the beta-chain of the GM-CSF receptor that are required for high-affinity binding of GM-CSF. On the other hand, the epitope recognized by antibody 2B7-17-A appeared to be critically involved in the binding of GM-CSF because this antibody completely abrogated both high- and low- affinity binding of GM-CSF to native and recombinant receptors. Antibody 2B7-17-A had a relatively high affinity for the GM-CSF receptor alpha-chain (kd = 3 nmol/L) and slow dissociation kinetics (kd = 0.002 min-1). These properties made the 2B7-17-A antibody a potent inhibitor of hGM-CSF biologic action in several different bioassays, with a half-maximal inhibitory dose of about 6 nmol/L (1 microgram/mL). This antibody could prove useful in alleviating any pathologic states mediated by excess GM-CSF levels and in defining the domains of the GM- CSF receptor required for ligand binding.     

Significance of Ph1-negative marrow cells in Ph1-positive chronic granulocytic leukemia

Fifty-six of 195 Ph1-positive patients with chronic granulocytic leukemia were found to have Ph1-negative metaphases in marrow aspirates on one or more occasions. In 22 cases, Ph1-negative cells were found prior to initiation of antileukemic therapy. Five patients were in the blastic stage of the disease when Ph1-negative mitoses were seen. The finding of Ph1-negative cells appeared to be related principally to short duration of CGL and to administration of antileukemic therapy (conventional agents and doses, in most cases). Ph1-negative cells were usually not found more than 2 yr after the diagnosis of leukemia, but in a few cases, they were seen as long as 5-10 yr after diagnosis. Only a minority of metaphases analyzed were Ph1-negative, except in the case of 6 patients who transiently had 50% or more Ph1-negative cells after antileukemic therapy. The presence of Ph1-negative cells in marrow was not associated with any survival advantage in this series.     

Plateletpheresis in 90- to 110-pound donors using the CS-3000 blood cell separator

BACKGROUND: Increases in the use of single-donor apheresis components have increased the need for platelet donors. In the United States, persons must weigh 110 pounds or more to qualify as blood donors, and the same weight limitation has been placed on apheresis donors. Because automated plateletpheresis with some instruments differs considerably from whole-blood donation with respect to the volume of blood removed from the donor, the feasibility of using persons weighing between 90 and 110 pounds as platelet donors was evaluated by the use of the CS- 3000 blood cell separator. STUDY DESIGN AND METHODS: The study was performed using female subjects who met all usual donor requirements except for minimum weight. The standard platelet collection procedure of the instrument was used, except that the blood processing rate was manually selected so as to optimize the blood withdrawal and return rate in individuals. Vital signs were recorded before and after donation as were signs or symptoms of any type of donor reaction. RESULTS: Twenty-six of 28 women completed the donation procedure; in two instances, collection was terminated prematurely because of an inability to maintain adequate venous access. An average of 4.5 × 10(11) platelets were collected during a mean donation time of 110 minutes. All donors tolerated the procedure well, and no serious adverse reactions were seen. Because of the administration of priming solution and anticoagulant during apheresis, there was a net positive fluid balance following the procedure, in spite of the removal of approximately 220 mL of platelet concentrate. CONCLUSION: These preliminary studies suggest that 90- to 110-pound persons may serve as plateletpheresis donors. Additional studies are needed to more fully document the safety and efficacy of this approach. The use of lower- weight donors may significantly increase the number of persons available to provide single-donor platelet components.     

The sensitivity to cytosine arabinoside of the blast progenitors of acute myeloblastic leukemia

Two culture methods are available for the study of the blast cells of acute myeloblastic leukemia (AML). One is an assay for clonogenic precursors; it depends on their ability to form blast colonies in culture in the presence of methylcellulose and suitable growth factors. The other assesses the growth of blast cells in suspension culture, where growth is measured by increasing numbers of clonogenic cells. We have compared the two methods as assays for the cytotoxic effects of the chemotherapeutic drug cytosine arabinoside (Ara-C). Marked patient- to-patient variation was found using either method; however, the slopes of the dose-response curves were usually greater when cells were exposed to drug in suspension rather than in methylcellulose. Control experiments showed that the difference could not be explained by drug carry-over from the suspension cultures to the methylcellulose plates when clonogenic cells in the suspensions were assessed. Further, the survival curves for Adriamycin were very similar, regardless of which assay was used. No correlation was found between D10 Ara-C values measured in suspension or in methylcellulose. However, a significant association with outcome was found between D10 Ara-C in suspension and response to treatment with a regimen in which Ara-C was the only chemotherapeutic agent used. No such association was detected when the D10 values obtained with the clonogenic assay were compared with outcome for the same group of 15 patients. Finally, a feasibility experiment was performed in which blast cells were exposed to Ara-C repeatedly during exponential growth over 238 days. A dose-related inhibition of growth was observed; no evidence was seen of emerging drug-resistant cells. Nor did the morphology of the cells change as a result of drug exposure. We conclude that drug sensitivities of AML blast cells in culture are dependent on measurement methods, even when techniques affecting cell proliferation are compared. Measurements of drug sensitivity in culture may best be interpreted when the bases of the assay systems are understood.     

Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors [see comments]

Human bone marrow mononuclear cells (BMMNCs) and enriched CD34 positive (CD34+) cells were transduced with adenovirus vectors encoding Escherichia coli beta-galactosidase gene. Tranductions were carried out by 24-hour coincubation with adenovirus vectors at different multiplicities of infections (moi). Efficacy of gene transfer into BM cells and expression of the gene product (ie, beta-galactosidase) were studied using X-Gal histochemical staining and flow cytometric analysis. X-Gal staining demonstrated that the percentage of positive cells at mois of 5 to 500 was 3.4% to 34.5% for BMMNCs and 6.0% to 20.0% for enriched CD34+ cells. Similar results (1.5% to 35.7% for BMMNCs and 5.4% to 24.2% for enriched CD34+ cells) were obtained with flow cytometric analysis using fluorescein di-beta-D-galactopyranoside (FDG). Multicolor flow cytometry analysis, which included FDG, demonstrated that BM progenitors (CD34+ or CD34+CD38-), T cells (CD2+), B cells (CD19+), natural killer cells (CD56+), granulocytes, and monocytes all expressed the adenovirus transgene. To ascertain the effects of adenovirus vectors on normal BM progenitors, the numbers of colony forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit- erythrocyte (BFU-E), and high-proliferative potential-colony-forming cells (HPP-CFC) after 24-hour coincubation with adenovirus vectors were determined. When BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at mois of 5 and 50, no significant differences in the numbers of CFU-GM, BFU-E, and HPP-CFC were observed compared with the uninfected control cells. However, the numbers of CFU-GM were significantly (P < .01) decreased when BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at a moi of 500, compared with the uninfected control cells. The adenovirus infected cells, purified by cell sorting for FDG expression, were capable of growing in culture and gave rise to various colonies (ie, CFU-GM, BFU-E, and HPP-CFC). These data indicate that recombinant adenovirus vectors can be used to transfer genes to human BM hematopoietic cells with expression of the exogenous gene at a high transduction efficiency.     

Effect of HLA Bw4/Bw6 compatibility on platelet transfusion responses of refractory thrombocytopenic patients

Platelet transfusions from donors matched for cross-reactive antigens have been shown to be effective in providing hemostasis in alloimmunized thrombocytopenic patients. A significant number of these transfusions, however, fail to provide posttransfusion platelet recoveries. We investigated incompatibility in the Bw4/bw6 system as a possible explanation for these failures. The Bw4/Bw6 system is a biallelic antigen system closely associated with HLA-B. HLA-B locus antigens that are cross-reactive frequently differ in their Bw4/Bw6 specificity. Posttransfusion platelet recoveries from 21 alloimmunized thrombocytopenic patients homozygous for Bw4 or Bw6 and transfused with both Bw4/Bw6 compatible and incompatible platelets were analyzed. The mean 1-hr posttransfusion recovery was 84% following Bw4/Bw6-compatible platelets versus 52% with Bw4/Bw6-incompatible platelets (p less than 0.02). Twenty-four hours following transfusion, mean recoveries were 44% and 24%, respectively, (p less than 0.01). A subgroup of 8 patients (38%) was identified who had markedly lower responses following Bw4/Bw6- incompatible transfusions as compared to Bw4/Bw6-compatible transfusions (mean recoveries: 1 hr--compatible 100%, incompatible 27%, p less than 0.001; 24 hr--compatible 45%, incompatible 7%, p less than 0.01). These data suggest that the Bw4/Bw6 antigen system has clinical significance for some patients requiring platelet transfusion therapy and, when appropriate, should be considered in the selection of donors.     

Rapid detection of deletions causing delta beta thalassemia and hereditary persistence of fetal hemoglobin by enzymatic amplification

A considerable number of deletions of variable size and position that involve the beta-globin gene complex on chromosome 11 are associated with the clinical entities of hereditary persistence of fetal hemoglobin (HPFH) and delta beta thalassemia. Specific deletions appear to be associated with consistent phenotypes and some are known to be recurrent. To facilitate the molecular diagnosis of uncharacterized patients with HPFH and delta beta thalassemia, oligonucleotide primers have been designed to enzymatically amplify deletion-specific products for nine known deletions, which include those responsible for HPFH-1, HPFH-2, HPFH-3, Spanish (delta beta)zero thalassemia, hemoglobin (Hb) Lepore, Sicilian (delta beta)zero thalassemia, Chinese G gamma(A gamma delta beta)zero thalassemia, Asian-Indian inversion-deletion G gamma(A gamma delta beta)zero thalassemia, and Turkish inversion-deletion (delta beta)zero thalassemia. Using this approach, we have successfully characterized the molecular basis for delta beta thalassemia in 23 individuals from 16 families of diverse ethnic origins. Thirteen individuals from this group were shown to be heterozygous for the 13.4- kb Sicilian deletion, two were heterozygous for the 100-kb Chinese G gamma(A gamma delta beta)zero deletion, four were heterozygous for the Turkish form of inversion-deletion delta beta thalassemia, and three were heterozygous for the Asian-Indian form of inversion-deletion G gamma(A gamma delta beta)zero thalassemia. One Vietnamese subject was heterozygous for a 12.6-kb deletion, which we have fully characterized at the molecular level. Sequence analysis of the breakpoint regions of the Chinese deletion and the Turkish rearrangement indicates that, in each case, the mutation is likely to have arisen from a single origin. This hypothesis is supported by the evident geographical clustering of the various deletions described here.     

Ultrastructural demonstration of tubular inclusions coinciding with von Willebrand factor in pig megakaryocytes

The appearance of von Willebrand factor (vWF) in bone marrow megakaryocytes was studied by standard electron microscopy (EM) and immuno-EM using an original purification technique. Eighty percent pure megakaryocytes were isolated from porcine rib bone marrow using Percoll gradients followed by counterflow centrifugation. Activation was prevented by prostacyclin and prefixation with low concentrations of glutaraldehyde. In early megakaryoblasts, standard EM revealed the presence of tubular structures in the small vesicles located in the Golgi area, in the small immature alpha-granules and in the rare mature alpha-granules. Immunolabeling for vWF was simultaneously observed in small vesicles and small alpha-granules, mainly in the Golgi zone. In mature megakaryocytes, standard EM showed that tubular structures were numerous, regularly spaced, and aligned in parallel. Immunolabeling for vWF was intense, restricted to the alpha-granules, and distributed in a similar manner to porcine platelets. Gold particles were located eccentrically at one pole of the alpha-granule, labeling only its periphery or outlining one side of an elongated granule. Tubule profiles could be seen underlying the immunolabeling and were usually located at one side of the granule. In conclusion, this study demonstrates the presence of tubular structures in megakaryocyte alpha- granules, their association with vWF, and the appearance of both in the Golgi-associated vesicles.