ALBERT

Description: The structure of factor IX gene was analyzed in a hemophilia B patient with inhibitor. Genomic DNA, digested with a variety of restriction endonucleases, was hybridized with the cDNA and various genomic factor IX probes. A large subtotal deletion of the gene was observed. The borders of the deletion span from a approximately 125 nucleotide region within the last exon to an unknown domain at least 7.5 kb upstream from the first exon: it thus involves approximately 33 kb of the factor IX locus. The abnormal gene was inherited by the daughter of the propositus, who showed both the normal and the deleted allele.

Description: Two molecular defects involving the spectrin heterodimer (SpD) contact site of the alpha chain (the alpha I domain) were previously identified using limited tryptic digestion followed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both are characterized by atypical peptide maps which reveal a marked decrease of the 80,000-dalton alpha I domain and a formation of new major peptides of either 74,000 (Sp alpha I/74) or 46,000 (Sp alpha I/46) daltons. We now report a third variant of the spectrin alpha chain, designated Sp alpha I/65, in three unrelated black families. In all three probands, the percentage of SpD in the low ionic strength (O degrees C) membrane extracts was increased to 19% to 32%. One- and two- dimensional electrophoretic separations of limited tryptic digests of spectrin from all three probands revealed a decrease of the alpha I domain of spectrin and the concomitant appearance of peptides at 65,000 daltons and isoelectric points ranging from 5.2 to 5.3. The abnormal 65,000-dalton peptides could be stained with an antiserum which had been raised against the alpha I domain, indicating that it was derived from the alpha I domain.

Description: Monoclonal antibodies (MoAbs) to myeloid differentiation antigens have a potential use in purging bone marrow of leukemia cells in autologous bone marrow transplantation (ABMT) therapy for acute myelogenous leukemia (AML). Because the efficiency of purging by MoAb and complement (C) is important to the success of ABMT, we have designed an assay to determine optimal conditions for leukemia cell lysis. In order to mimic the conditions of remission bone marrow, normal buffy coat cells were mixed with cells from the HL-60 promyelocytic leukemia cell line at a concentration that approximated the normal-leukemia cell ratio found in remission marrow. The cell mixture was treated at variable times and temperatures in the presence of C and PM-81, an IgM MoAb that reacts with both normal granulocytes and monocytes as well as with HL-60 cells. PM-81 binds to the majority of cells from 90% of patients with AML yet does not react significantly with normal stem cell populations. Because of the potential use of PM-81 in ABMT, it seemed especially important to show that the antibody was capable of mediating cytotoxicity of HL-60 cells in the presence of an excess of antigen-positive cells. A clonogenic assay that permitted the growth of HL-60 cell colonies but not normal progenitor cells in methylcellulose cultures was used to measure the efficiency of HL-60 cell lysis. We found that under certain conditions, PM-81 was capable of removing the small percentage of HL-60 clonogenic cells admixed with normal buffy coat cells. This information was useful for determining the optimal conditions for purging bone marrow of leukemia cells for ABMT.

Description: The expression of Rhesus antigens on hematopoietic progenitor cells was studied using monoclonal antibodies. Because these antibodies are not capable of lysing mature red blood cells in a complement-dependent cytotoxicity assay, fluorescence-activated cell sorting was performed. Using the monoclonal anti-Rh 29 antibody B10, 68% +/- 6% of the mature erythroid progenitor cells (CFU-E) were sorted into the positive fraction, while only 2% +/- 1% of the relatively immature erythroid progenitor cells (BFU-E), and 3% +/- 1% of the granulocyte-macrophage progenitor cells (CFU-GM) were cultured from this same fraction. Thus up to a 15-fold enrichment of CFU-E could be obtained. In two experiments more than 4% of the cells in the positive fraction consisted of CFU-E; in one experiment even more than 7% did. Using fractionated cell sorting, the Rhesus antigens appeared to have a lower density on CFU-E than HLA-DR determinants. Antibodies against the Rhesus antigens can be applied to enrich erythroid-committed stem cells and to separate mature from immature erythroid progenitor cells.

Description: Surface membrane glycoproteins (SMGs) of cells from the parental wild- type HL-60 cell line and from three sublines variably cross-resistant to the granulocyte differentiation-inducing effects of retinoic acid (RA), dimethylsulfoxide (DMSO), and certain purine bases (6-thioguanine [6TG] or hypoxanthine) were studied by one-dimensional and two- dimensional gel electrophoresis. After both oligosaccharide (periodate/borotritide) and peptide (1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycouril) ectolabeling procedures, striking common changes were noted in the gel electrophoretic patterns of the SMGs from the RA- and 6TG-resistant sublines compared to those from the wild-type HL-60 line or the DMSO-resistant subline. Most prominently, this included the presence in the RA- and 6TG-resistant cells of an apparent high molecular weight acidic glycoprotein(s) (mol wt, 200 to 285 kilodaltons [kD]; isoelectric point range [pl], 4.5 to 6.0) not observed in the wild-type or DMSO-resistant cells and, conversely, the presence of a lower molecular weight glycoprotein(s) (mol wt, 120 to 165 kD; pl, 4.2 to 5.9) in the wild-type and DMSO-resistant cells, which was absent or much reduced in the RA- and 6TG-resistant cells. These acidic SMGs did not change as a function of the induction of granulocyte differentiation. However, some other more basic SMGs varied as a function of granulocyte differentiation in both the wild-type and differentiation inducer-resistant sublines, including the loss of the transferrin receptor and the gain of a mol wt 55- to 60-kD neutrophil- associated protein. In the context of previously reported information, these results indicate (1) that the overall pattern of SMG changes in the RA- and 6TG-resistant cells closely resembles that associated with multidrug (pleiotropic) resistance to cytotoxic agents in a variety of mammalian cells; (2) that the RA/6TG resistance-associated SMG changes are not granulocyte differentiation stage-specific; and (3) that either the RA/6TG resistance-associated SMG changes are not related to the resistance mechanism or they are involved in the resistance/cross- resistance mechanism(s) for RA/purine bases but not for DMSO.

Description: The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+- dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.

Description: The density and size of human erythrocytes has been roughly correlated with cell age, with the denser and smaller cells being older. Observations of this type have led to a hypothesis that the membranes of circulating erythrocytes are dynamic with respect to composition and that material is lost from the membrane during cell maturation and circulation. In this study, flow cytofluorimetry was used to investigate the distribution of the human erythrocyte anion transport protein (protein 3) in heterogeneous samples of circulating red cells. We verified that protein 3 can be specifically and quantitatively labeled in intact human erythrocytes with eosin-5-maleimide, a luminescent probe. Individual cells were accordingly analyzed for size by forward light scattering and for protein 3 content by quantitation of eosin fluorescence. Initial results indicated that the smallest erythrocytes had a protein 3 content equal to that of the largest circulating erythrocytes. This result was independently verified by light scatter-activated cell sorting; direct measurement of cell diameters by microscopy verified that the cell sizes of erythrocytes showing the 10% greatest and 10% smallest light-scattering signal were indeed distinct. Independent analysis of the size-sorted erythrocytes for protein 3 content was accomplished by gel electrophoresis of stroma from 150,000 large and small erythrocytes. Quantitative scanning densitometry of silver-stained gels of prepared stroma showed that protein 3 content of each set of fractionated cells was equal and did not vary as a function of cell size. Taken in combination with the reported correlation between increasing red blood cell age and decreasing cell size, these results indicate that any loss of membranous material during the cell aging process is not random.

Description: A long-term liquid culture system of hemopoietic tissue derived from adult hamster spleen has been described. These primary liquid cultures can maintain stem cell proliferation and differentiation for more than three months without secondary repopulation. A characteristic of the liquid cultures is the formation of clusters of hemopoietic cells around adherent stromal cells. Some islands were composed exclusively of megakaryocytes and adherent cells. Isolation of these clusters of differentiating megakaryocytes and their adherent cellular substrate permitted the analysis of the morphological and ultrastructural features of the interaction between cells of megakaryocytic lineage with the adherent stroma.

Description: We have used immunogold staining to locate thrombospondin (TSP) on thrombin-activated human platelets, and have compared its distribution with that of fibrinogen (or fibrin) on thrombin- and ADP-stimulated platelets. To do this, isolated platelets were incubated with monospecific antibodies to TSP or fibrinogen (fib) and the bound IgG located with a second antibody adsorbed to gold particles. Thrombin- induced secretion in Tyrode-Ca2+ was followed by both anti-TSP and anti- fib binding, with large clusters of gold particles observed on the platelet surface. Little or no labeling was observed on unstimulated platelets with either antibody. When secretion was effected in Tyrode- EDTA, anti-TSP IgG still bound to the activated platelets, but the number of particle clusters was significantly reduced. Little binding of anti-fib IgG now occurred. Platelets activated with ADP in the presence of added fib, and subsequently incubated with anti-fib IgG, showed small particle clusters over the whole platelet surface. Thrombin-stimulated platelets from two patients with thrombasthenia bound anti-TSP IgG similarly to normal platelets activated in Tyrode- EDTA. No anti-fib binding occurred. Our results suggest that fib and TSP bind to specific domains on the stimulated platelet membrane. Such sites may be responsible for the mediation of platelet surface contact interactions.

Description: Protein-heparin complexes, prepared by a water-soluble carbodiimide coupling technique, were used to produce anti-heparin antibodies in rabbits. Antiserums that recognized carbodiimide-treated heparin, but not untreated heparin, were obtained. Carbodiimide-treated heparan sulfate exhibited 10% to 20% cross-reactivity compared with a similarly treated heparin, whereas there was no cross-reactivity with five other carbodiimide-treated mucopolysaccharides. 3H-1-ethyl-3-(3- trimethylammoniumpropyl) carbodiimide iodide was used to demonstrate that carbodiimide forms a stable adduct with heparin and other mucopolysaccharides. Using an antibody fraction that eluted from 1- ethyl-3-(3-trimethylammoniumpropyl) carbodiimide iodide-treated heparin- Sepharose with 2 mol/L KI, it was demonstrated that, for the antibody population studied, the addition of one carbodiimide per heparin molecule resulted in complete epitope expression without loss of anticoagulant activity. The addition of up to eight additional carbodiimide molecules to heparin did not increase the extent of epitope formation, although anticoagulant activity was lost. Except for heparan sulfate, the addition of radiolabeled carbodiimide to other mucopolysaccharides did not result in epitope formation. These data demonstrate that antibodies to an epitope derived from heparin can be formed, that the epitope is fully expressed while anticoagulant activity is present, and that the antibody is specifically directed against an altered portion of the polysaccharide.

Description: We tested conditioned media from 12 patients with T lymphocyte neoplasms and four T cell lines for their ability to stimulate the in vitro growth of erythroid-burst-forming units (BFU-E) from bone marrow mononuclear cells in a methylcellulose culture system. Nine patients suffered from acute lymphocytic leukemia, two from chronic lymphocytic leukemia, and one from non-Hodgkin's lymphoma. The T lymphocytes were characterized by a series of monoclonal antibodies and their stage of development was correlated with their ability to produce burst- promoting activity (BPA). Conditioned media from cells classified as prothymocytes (three cases), common thymocytes (one case), mature thymocytes (three cases), and mature lymphocytes of the helper subtype (two cases) increased BFU-E proliferation four- to 19-fold over control values using normal bone marrow as target cells. Conditioned media from OKT8+ malignant T lymphocytes (three cases) did not enhance BFU-E proliferation. Conditioned media from cells classified as immature T cells stimulated CFU-GM proliferation in only one of seven cases even though they secreted BPA. Conditioned media from three of the four cell lines stimulated by phytohemagglutinin, enhanced BFU-E growth. Our results indicate that malignant cells that have characteristics of immature T cells are able to produce BPA. Studies using techniques to isolate homogeneous populations of normal T cell subsets are required to determine whether normal immature T lymphocytes have the same capability.

Description: Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well- characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross- reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin- binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.

Description: The inhibition of delta-aminolevulinic acid (ALA) synthase activity by heme is commonly thought to regulate the overall rate of heme synthesis in erythroid cells. However, since heme inhibits erythroid cell uptake of iron from transferrin, we have tested the hypothesis that in reticulocytes heme regulates its own synthesis by controlling the cellular acquisition of iron from transferrin rather than by controlling the synthesis of ALA. We found that hemin added to reticulocytes in vitro inhibits not only the total cell incorporation of 59Fe from transferrin but also the incorporation of [2–14C]-glycine and transferrin-bound 59Fe into heme. However, hemin did not inhibit [2 –14C]-glycine incorporation into protoporphyrin. Furthermore, cycloheximide, which increases the level of non-hemoglobin heme in reticulocytes, also inhibited [2–14C]-glycine into heme but not into protoporphyrin. With high concentrations of ferric pyridoxal benzoylhydrazone (Fe-PBH), which, independent of transferrin and transferrin receptors, can be used as a source of iron for heme synthesis in reticulocytes, significantly more iron is incorporated into heme than from saturating concentrations of Fe-transferrin. This suggests that some step (or steps) in the pathway of iron from extracellular transferrin to protoporphyrin limits the overall rate of heme synthesis in reticulocytes. In addition, hemin in concentrations that inhibit the utilization of transferrin-bound iron for heme synthesis has no effect on the incorporation of iron from Fe-PBH into heme. Our results indicate that in reticulocytes heme inhibits and controls the utilization of iron from transferrin but has no effect on the enzymes of porphyrin biosynthesis and ferrochelatase. This mode of regulation of heme synthesis may be a specific characteristic of the hemoglobin biosynthetic pathway.

Description: Leukemic blasts from 774 children with newly diagnosed acute lymphocytic leukemia (ALL) have been phenotyped by microcytotoxicity testing with a panel of monoclonal antibodies and heteroantisera as part of a Pediatric Oncology Group classification study of acute leukemia. One hundred twenty-two cases, or 16% were designated as T cell leukemia based on the reactivity of blast cells with previously well-characterized antisera (PT) against a T lymphocyte-associated antigen. Using this antisera-based definition as a standard, we looked for a monoclonal antibody combination that would be a suitable substitute. An algorithm calling for reactivity with either monoclonal antibody 3A1 or Leu-1 was a 92% sensitive and 97% specific predictor of PT reactivity. Only 27 of 755 cases of leukemia were incorrectly classified using this algorithm. Subsequently, Ficoll-Hypaque-separated bone marrow cells from 118 additional patients with ALL (21 of whom had T cell ALL) were stained by immunofluorescence using a combination of directly fluoresceinated 3A1 and Leu-1. Reactivity of 20% or more of the cells with this antibody combination was a 100% sensitive and 94% specific indicator of T cell ALL defined by PT positivity; with a higher cutoff value for positive values, or the use of supplemental tests, even this small number of false-positives could be eliminated. We conclude that this monoclonal antibody combination is a satisfactory replacement for our heteroantisera definition of T cell ALL.

Description: The effect of a zinc metalloprotease from Serratia marcescens on platelet surface glycoproteins (GP) Ib and V was analyzed. Increasing protease treatments caused progressive loss of GP Ib with appearance of the major fragment, glycocalicin, in the supernatant solution. No GP V was detected in the supernatant solution, and protease-pretreated platelets had the same capacity as control platelets to release fragment 1 of GP V in response to thrombin. The Serratia protease- pretreated platelets did show the lag before thrombin-induced dense granule secretion, characteristic of platelets modified by pretreatment with other nonstimulating proteases. Treatment with Serratia protease gives the only demonstrated selective loss of GP Ib without apparent effect on GP V. It suggests that GP V (1) does not depend on GP Ib for its association with platelets and (2) is not the substrate for protease modification of platelet function.

Description: The blood platelet is the only human cell known to have a circumferential band of microtubules. However, the mechanisms involved in assembly of the multi-looped coil, its interaction with the cell membrane to support discoid shape, and constriction into tight rings around centrally concentrated organelles in activated platelets are unknown. Separation of the microtubule rings from intact platelets would permit new approaches to solution of these questions. The present study has used simultaneous detergent extraction and fixation to isolate intact microtubule coils in significant numbers from suspended platelets for the first time. Isolated coils closely resembled the circumferential band observed in thin sections of plastic embedded platelets and in platelets prepared by the negative-stain whole-mount method. Enough microtubule coils could be recovered from suspensions of concentrated platelets to permit counting and quantitation on microscope grids. Results of this study will permit new approaches to clarification of the structural physiology of platelet microtubule coils.

Description: Twenty-two patients with hairy cell leukemia were treated with biosynthetic (recombinant) alpha-2-interferon in an open-label, single- arm efficacy study. Patients received 2 X 10(6) U/m2 recombinant alpha- 2-interferon three times weekly. Therapy was well tolerated subjectively with minimal short-term hematologic toxicity. Two patients had bacterial infections during the period of study, and one patient experienced a short-lived readily reversible rejection of a corneal transplant. Statistical comparison of the mean hematologic indices at study entry and after three to six months of therapy with recombinant alpha-2-interferon indicates a significant improvement in hemoglobin, granulocyte, and platelet counts. Bone marrow biopsies in six of 14 patients after six months of therapy showed a greater than 50% decrease in the infiltration of leukemia cells. We conclude that recombinant alpha-2-interferon is highly effective therapy for hairy cell leukemia.

Description: Mitogen-stimulated murine spleen cells produce humoral substances capable of supporting murine hematopoiesis and pluripotent stem cell proliferation in vitro. Thus, we evaluated conditioned media generated by human spleen cells (SCM) in the presence or absence of mitogens for factors stimulatory for human pluripotent (CFU-GEMM), erythroid (BFU- E), and myeloid (CFU-GM) precursors. Two and one half percent to 10% SCM stimulated proliferation of all three types of precursor cells from nonadherent buoyant human marrow target cells. Mitogen-stimulated SCM augmented CFU-GM (175% to 225%), whereas CFU-GEMM and BFU-E growth was essentially unchanged. Cell separation procedures used to determine which cells provided these microenvironmental stimuli indicated that nonadherent mononuclear spleen cells provided the bulk of the CSF-GM, whereas adherent cells (95% nonspecific esterase + monocyte- macrophages) and nonadherent cells provided similar proportions of CSF- mix and erythroid burst-promoting activity (BPA). The nonadherent cells generating high levels of CSF-mix, BPA, and CSF-GM were predominantly Leu-1-negative, ie, non-T, cells. In the presence or absence of mitogens, SCM was a more potent source (1.3- to 3.8-fold) than peripheral leukocyte CM of the growth factors for the three progenitor cell types. Specific in situ cytochemical stains for analyzing morphology of myeloid colonies demonstrated that SCM stimulated the proliferation of the same types and proportions of colonies as human placental CM, suggesting that these CMs may contain similar CSF-GMs. These data show the contribution of spleen cell subsets to the generation of hematopoietic growth factors and the responsiveness of these cells to various mitogenic stimuli.

Description: Pregnancy in female carriers of abnormal hemoglobins with great avidity for oxygen provides a unique opportunity to assess the importance of the usual difference in oxygen affinity between fetal and maternal blood. Outcome of pregnancy was recorded for carriers of hemoglobins Bethesda, Osler, and Yakima, whose p50s (9.5, 9.1, and 12 mm Hg at pH 7.4) were far lower than that of a normal fetus (23 mm Hg at pH 7.3). Neither spontaneous abortions nor intrauterine growth retardation could be attributed to the presence of high oxygen affinity in the mothers. In vitro simulations suggested that neither maternal or fetal polycythemia alone was sufficient to adjust for perturbation of the normal situation, and increased uterine and/or fetal blood flow probably provided additional compensation.

Description: Neutrophil function was studied in a patient with polymorphonuclear leukocyte (PMN) glycoprotein-180 deficiency and in her parents. PMNs of the patient had abnormal chemotaxis, phagocytosis, adherence, surface charge, and membrane-associated events of activation. Selective defects to C3b, immunoglobulin G (IgG), phorbol myristate acetate (PMA) and N- formyl-methionyl-leucyl-phenylalanine (FMLP) are described, although C3b receptor density was normal. The parents were found to have abnormal adherence to nylon-wool fibers, abnormal transmembrane potential depolarization with PMA, and reduced amounts of glycoprotein- 180 in their PMNs. These studies provide further evidence that the oxidative burst has several different pathways for activation. They demonstrate that the absence of a single PMN surface glycoprotein is associated with a broad spectrum of PMN functional abnormalities. Finally, the observations made in the parents support an autosomal recessive mode of inheritance.

Description: Ecto-5′nucleotidase (5′NT) activity of peripheral blood (PB) lymphocytes was determined in 31 patients with serum monoclonal gammopathies (MG). Twenty-one patients had a diagnosis of multiple myeloma (MM), and ten patients had monoclonal gammopathy of undetermined significance (MGUS). The proliferative activity of the bone marrow plasma cells (LI%) was investigated in 28 of these MG patients by means of tritiated thymidine uptake evaluated by simultaneous autoradiography and cytoplasmic immunofluorescence. 5′NT activity was significantly lower in MG patients as compared with normal controls. MM patients had lower 5′NT activity than MGUS patients, but the difference was not significant. By contrast, MM had significantly higher LI% than MGUS patients. There was a linear regression of 5′NT on LI% which was statistically significant: the higher the LI%, the lower the 5′NT. Because the LI% is an accurate prognostic and monitoring factor in MG, this correlation indicates that 5′NT may be of assistance in predicting the clinical progress of MG patients. In seven MGs, the PB T and B lymphocytes were studied separately. The T cell subpopulation was 5′NT deficient compared to the normal controls, shown as a significant linear regression of T cell 5′NT on the LI%. This suggests that in MG there may be an alteration of nonneoplastic T lymphocytes correlated with tumor growth. The OKT8+ lymphocytes were mainly responsible for the 5′NT deficiency of unseparated T lymphocytes.

Description: Peripheral blood leukemia cells from patients with acute monoblastic leukemia (AMoL) were tested for killer cell activity against target cells that detected natural killer cell-mediated or monocyte-mediated spontaneous cytotoxicity. The fibrosarcoma cell line Wehi 164, pretreated with actinomycin D to induce susceptibility to lysis, specifically detects the activity of unstimulated human monocytes. In four of six cases of AMoL, high killer cell activity could be measured against this target. In three of these four cases, the killer cell activity could be assigned exclusively to the leukemic clone, based on the high leukocyte counts and the resultant dilution of normal cells, as evidenced by marker and by functional analysis. While leukemic cells with killer cell activity against Wehi 164 contained 34% to 45% cells that were positive for binding of the 63D3 monoclonal antibody, the two leukemic samples without killer cell activity contained only 1% and 12% 63D3-positive cells. Cell sorting of 63D3-positive and -negative cells from two leukemias with killer cell activity demonstrated that the killer cell activity was restricted to the 63D3-positive fraction of AMoL cells. These data demonstrate that monoblastic leukemia cells can be potent killer cells and that killing activity is linked to the 63D3- defined cell surface molecule.

Description: The Belgrade laboratory rat (b/b rat) has hereditary, hypochromic, microcytic anemia with a variety of red cell abnormalities. Although this anemic syndrome has been recently ascribed to the defective delivery of iron to the developing red cell, the basic hematopoietic defect is still unknown. In this article we present evidence that the b/b rat has an additional hematologic defect. We have found that the megakaryocyte number in the marrow of the b/b rat is decreased to one half that of the normal rat, but the maturation rate of recognizable megakaryocytes is accelerated and the size is increased. The platelet count is moderately reduced. These findings indicate that megakaryocytopoiesis in the anemic b/b rat is abnormal and suggest that the genetic defect may involve the progenitors of the megakaryocyte cell lineage. Alternatively, the megakaryocytic abnormalities may be secondary to the severe anemia.

Description: DNA from mononuclear blood and tumor cells from 33 patients undergoing bone marrow transplantation for leukemia was examined for the presence of Epstein-Barr virus (EBV) genomes by blot hybridization. Four groups of patients were studied soon after engraftment, during long-term remission, after relapse of the original leukemia, and after development of secondary B cell neoplasms. Only the cells of patients with secondary neoplasms demonstrated EBV genomes, where all five adequately studied samples were positive. Samples from all other patient categories were negative for EBV genomes. We conclude that EBV genomes do not frequently persist in normal engrafted lymphocytes or in mononuclear cells of patients suffering recurrent leukemia. These results are consistent with EBV playing a role in the genesis of secondary B cell neoplasms following bone marrow transplantation.

Description: Fibrinogen from plasma was compared with fibrinogen from platelets using two-dimensional electrophoresis. The source of platelet fibrinogen was isolated alpha-granules, thrombin- and collagen-released platelet material. The B beta- and gamma-chains from the different sources showed similar two-dimensional patterns, while gamma'-chains were not observed in platelet fibrinogen preparations. Furthermore, the A alpha-chain could hardly be identified in platelet preparations. When individual fibrinogen was studied in persons heterozygous for genetic B beta- and gamma-chain variants, the two-dimensional variant pattern could be demonstrated in plasma fibrinogen as well as in platelet fibrinogen. This observation strongly indicates that the structural genes for plasma and platelet fibrinogen B beta- and gamma-chains are identical.

Description: We report a detailed longitudinal study of the first patient to be treated (in 1973) for paroxysmal nocturnal hemoglobinuria (PNH) with syngeneic bone marrow transplantation (BMT). The patient subsequently relapsed with PNH in 1983, and still has PNH to date. Analysis of thePIG-A gene in a recent blood sample showed in exon 6 an insertion-duplication causing a frameshift. Polymerase chain reaction (PCR) amplification of the PIG-A exon 6 from bone marrow (BM) slides obtained before BMT showed that the duplication was not present; instead, we found several single base pair substitutions in exons 2 and 6. Thus, relapse of PNH in this patient was not due to persistence of the original clones; rather, it was associated with the emergence of a new clone. These findings support the notion that the BM environment may create selective conditions favoring the expansion of PNH clones. © 1998 by The American Society of Hematology.

Description: Patients who receive desmopressin acetate (dDAVP) after cardiopulmonary bypass bleed less during operation and in the first 24 hours after operation than do patients who receive a placebo. To study the mechanism of improved hemostasis in bypass patients, we examined the relationship between von Willebrand factor (vWF) and blood loss in 70 cardiopulmonary bypass patients, one-half of whom received desmopressin intraoperatively. vWF concentration and multimeric composition were analyzed before and after bypass, after drug treatment, and 24 hours after operation. Before operation, patients with valvular disease had lower percentages of vWF high-mol-wt multimers (HMWMs) than did healthy subjects or patients with coronary artery disease, but subsequent blood loss, vWF activity, and bleeding times were not related to this finding. Irrespective of drug treatment, patients who had low preoperative vWF and who had a net loss of the protein during bypass bled more after bypass than did similar patients who had a net increase of vWF during bypass. HMWMs rose to above normal levels after bypass regardless of desmopressin infusion. Differences in the concentration of vWF between desmopressin and placebo patients after receipt of the drug, although small, were better correlated with reduced blood loss than were differences in HMWM distribution. We conclude that the beneficial effect of desmopressin on hemostasis following cardiopulmonary bypass cannot be attributed to a drug-induced change in HMWM distribution but may be related to an increase in overall vWF concentration.

Description: Morphological and functional modifications occurring in Langendorff rabbit heart preparations perfused with purified human leukocytes (PMNL), as an organ model of sulfidopeptide-leukotrienes (sLT) transcellular biosynthesis, were studied. Coronary perfusion pressure (CPP), monitored as an index of coronary vasospasm, increased by 295% after challenge with the Ca(2+)-ionophore A-23187 (0.5 micromol/L) for 30′, accompanied by a significant formation of sLT. Increase in CPP was prevented by PMNL pretreatment with the 5-lipoxygenase inhibitor MK-886 (1 micromol/L) or by heart pretreatment with LTD4-receptor antagonist SKF 104353, indicating a pivotal role of PMNL-derived 5-lipoxygenase (5- LO) products in the observed functional modifications. Similar effects were obtained using granulocyte macrophage-colony stimulating factor- primed PMNL challenged with the tripeptide n-formyl-methionyl-leucyl- phenylalanine. Scanning electron microscopy (SEM) of coronary arteries showed craters on the vessel luminal surface, PMNL adhering to endothelial cells (EC), increased number of microvilli on EC, presence of nonviable, desquamating, fusiform EC. SEM and transmission electron microscopy of myocardial microvessels, showed presence of perivascular and intermuscle edema, presence of activated PMNL and decreased number of patent microvessels. These morphological alterations were significantly blunted by MK-886 or SKF 104353. These data provide evidence of close interaction between PMNL and myocardial EC, resulting in enhanced sLT formation via transcellular biosynthesis, originating from transfer of PMNL-derived LTA4 to EC. These potent proinflammatory autacoids are responsible for coronary vasospasm and the morphological alternations observed.

Description: Lactoferrin (Lf) is a negative regulator of myelopoiesis which operates by suppressing the release from mononuclear phagocytes of GM colony- stimulating factor (GM-CSF) or monokines which can then induce the release of GM-CSA from accessory cells. In this study, endotoxin- depleted, purified iron-saturated human Lf was assessed for its effect on the production of interleukin-1 by cultured monocytes and their subsequent effect on colony-stimulating factor release from cultured fibroblasts. Monocytes were grown with or without Lf and Lf that had previously been incubated with monoclonal anti-Lf. The monocyte- conditioned medium was then either assayed for the presence of interleukin-1 (IL-1) with an enzyme-linked immunosorbent assay or for its ability to stimulate fibroblasts to release growth factors for CFU- GM, BFU-E, or CFU-Mix colonies. In the presence of Lf (10(-7) or 10(-8) mol/L), GM colony-stimulating activity (GM-CSA) was suppressed by 31% to 73%, whereas stimulating activities for BFU-E and CFU-mix colony formation were suppressed by 93% to 100%. Antibody to Lf completely abrogated the suppressive effects observed with Lf, whereas antibody to IL-1 ablated the induction by monocyte-conditioned medium of CSA release by fibroblasts. Lf at 10(-7) and 10(-8) mol/L also reduced IL-1 synthesis by cultured monocytes from 60% to 77%. The inhibitory effects of Lf were only observed when Lf was added before adherence of the monocytes for culture. If Lf was added at the time of adherence or after adherence, no suppression was observed. We conclude that the inhibition of GM-CSA production/release by Lf is mediated through inhibition of the synthesis/release of IL-1 by mononuclear phagocytes. This inhibition of IL-1 prevents accessory cells from producing and/or releasing GM-CSA.

Description: Gene transfer into human hematopoietic stem cells with expression targeted to the maturing myelomonocytic progeny has applications for gene therapy of genetic diseases affecting granulocytes and macrophages. We hypothesized that promoters of myeloid-specific genes that are upregulated with myelomonocytic differentiation would also upregulate expression of an exogenous gene in a retroviral vector. Moloney murine leukemia virus (MoMuLV)-based retroviral vectors using promoters from hematopoietic genes (CD11b, CD18, and CD34) were compared with vectors with viral promoters (MoMuLV long terminal repeat [LTR], cytomegalovirus [CMV], and simian virus 40 [SV40]). Human glucocerebrosidase (GC) cDNA was the reporter gene. HL60 cells were transduced with these vectors and vector-derived GC activity was compared in undifferentiated HL-60 cells and the same cells differentiated into granulocytes using dimethyl sulfoxide or monocyte/macrophages using phorbol myristate acetate. In undifferentiated HL-60 cells, vector-derived GC activity was the highest when it was controlled by the MoMuLV LTR. In HL-60 cells differentiated into granulocytes, vector-derived GC activity transcribed from the CD11b, MoMuLV LTR, and CMV promoters was equivalent to 1.7, 1.5, and 1.5 times the normal endogenous GC activity, respectively, and 0.8, 2.0, and 3.6 times the normal GC activity, respectively, in those differentiated into macrophages. With granulocytic differentiation, the CD11b promoter showed maximal induction in GC activity (8-fold); with macrophage differentiation, the CD11b promoter showed a fourfold induction in GC expression. The CD11b promoter also generated significant levels of GC activity in the myelomonocytic progeny of transduced CD34+ cells. Expression from the CD11b promoter, unlike that from the CMV or the MoMuLV LTR promoters, was relatively myelomonocyte-specific, with minimal expression observed in Jurkat T cells or HeLa carcinoma cells. The induction of expression from the CD11b promoter with differentiation in HL-60 cells correlates with the developmental regulation of the CD11b gene. Retroviral vectors using the CD11b promoter have potential utility for gene therapy of disorders affecting the myelomonocytic lineage.

Description: Dermatan sulfate (DS), a catalyst of the thrombin-heparin cofactor II interaction, has antithrombotic activity and is devoid of significant hemorrhagic risk in several animal models. We investigated the pharmacodynamic and pharmacokinetic properties of DS in humans. DS was injected in single bolus intravenous injections of four increasing doses (0.5, 1, 1.5, 2 mg/kg) to six healthy volunteers. The resulting anticoagulant activities were assessed by the activated partial thromboplastin time (APTT) and the thrombin clotting time (TCT). There were dose-dependent prolongations of the APTT and TCT, and the anticoagulant activities disappeared in less than three hours. The pharmacokinetic parameters were calculated from the plasma concentrations of DS measured with a new chromogenic assay. The volume of distribution was approximately 1.8 times greater than the theoretical plasma volume and was independent of dose. In contrast, the clearance decreased with dose and the terminal half-life ranged from 0.45 +/- 0.08 hours at 0.5 mg/kg to 0.72 +/- 0.11 hours (mean +/- SD) at 2 mg/kg. The bioavailabilities of subcutaneous (SC) and intramuscular (IM) administration relative to those of intravenous administration were determined in 12 other volunteers. The respective bioavailabilities were 24.7% +/- 12.9% and 12.4% +/- 9.2% for SC and IM administration. There was no detectable change in the APTT and the TCT when the volunteers were injected with 1.5 mg/kg SC or IM. In addition, the pharmacokinetic parameters derived from plasma concentrations of DS showed considerable interindividual variations by the two later routes of administration. Peak concentrations were noted 2.7 +/- 1.3 hours after SC injection and 4.3 +/- 4.9 hours after IM injection. The average peak concentrations were 0.7 +/- 0.3 and 0.4 +/- 0.2 mg/L after SC and IM injections, respectively. The half-lives of DS were 7.9 +/- 6.5 hours (SC) and 6.3 +/- 7.4 hours (IM). No adverse reaction to DS was recorded during this study.

Description: The normal myeloid hematopoietic regulatory proteins include four growth-inducing proteins called colony-stimulating factors (CSF), including interleukin-3 (IL-3), or macrophage and granulocyte inducers, type 1 (MGI-1), and another type of protein (MGI-2) with no myeloid cell growth-inducing activity that induces differentiation of normal myeloid precursor cells and certain clones of myeloid leukemic cells. An IgG2a monoclonal antibody was prepared and it neutralized two forms of MGI-2 (MGI-2A and MGI-2B) produced by mouse Krebs ascites tumor cells. The monoclonal antibody was used for affinity purification of MGI-2. This antibody also neutralized MGI-2 produced by normal mouse macrophages, normal myeloblasts incubated with IL-3, and MGI-2 produced by the lungs and found in the serum of mice injected with lipopolysaccharide (LPS). The anti-MGI-2 antibody did not inhibit the activity of any one of the four myeloid growth-inducing proteins (CSF or IL-3 = MGI-1), IL-1, tumor necrosis factor, or lymphotoxin. This antibody also inhibited induction of differentiation of myeloid leukemic cells by LPS, which is mediated by the endogenous production of MGI-2, but did not inhibit induction of differentiation in these leukemic cells by dexamethasone or cytosine arabinoside, which is not mediated by MGI-2. Anti-MGI-2 antibody thus inhibited differentiation when MGI-2 was added externally to cells or when it was mediated by endogenously produced MGI-2.

Description: The authors treated a total of 82 patients with Ph′-positive chronic myelogenous leukemia (CML) with recombinant interferon alpha-2b (IFN alpha-2b). Sixty-five patients in chronic phase (CP), 28 of whom were untreated and 37 pretreated, and nine patients in accelerated phase (AP), were started on IFN three times a week. Patients in CP were randomized to receive 2 or 5 X 10(6) IU/m2, while patients in AP were all given the dose of 5 X 10(6) IU/m2, in addition to concomitant chemotherapy. Patients in CP who were unresponsive to the lower dose were crossed to the higher dose. Of 63 evaluable patients in CP, 43 (68%) responded, 29 (46%) achieved complete hematologic response (CHR), and 14 (22%) achieved partial hematologic response (PHR). The response rate appeared to be significantly influenced by the IFN dose in pretreated patients. Of the nine patients in AP, two attained PHR and one CHR. More recently, eight previously untreated CP cases were submitted to daily IFN administration at doses from 2 to 5 X 10(6) IU/m2. This daily schedule was also applied to patients who had obtained, with the intermittent treatment, a PHR persisting unmodified for six months (nine patients) or an unstable CHR (five patients). Seven of the eight previously untreated patients, and five of the nine PHR patients crossed to daily IFN reached CHR. In the total series of previously untreated patients, the response rate proved to be significantly influenced by the initial risk status. Cytogenetic improvement was seen in 37 of 53 responders (70%) treated for more than 3 months, the median of Ph′-positive cells declining from 100% to 65% (range 0% to 95%). Complete suppression of Ph′ chromosome was observed in one case. The cytogenetic response was persistent for over 6 months in 21 patients, but the lowest value of Ph′ positivity was usually unstable. At a median follow-up of 56 weeks, 23 of 36 (64%) CHR patients remain in continued disease control with IFN. A blastic transformation (BT) occurred in seven of 21 unresponsive patients and in one of the 36 CHR patients. The authors' data confirm that IFN alpha- 2b, especially at daily doses, is effective in inducing clinical and cytogenetic response in a good proportion of patients with CML in the benign phase. Longer follow-ups will define the exact influence of this agent on the natural course of the disease.

Description: Conditions presently have been established for the high-level expression and simplified purification of recombinant human erythropoietin produced in Spodoptera frugiperda cells. Expression, as mediated by infection with a recombinant baculovirus, was accomplished in suspension culture using reduced levels of serum and media supplements experimentally determined to provide optimum levels of factor production (500,000 U/L). Purification of this recombinant human erythropoietin to virtual homogeneity (greater than or equal to 99%) was accomplished via a simple three-step procedure involving isocratic elution from DEAE-Sephacel, reverse-phase high performance liquid chromatography (HPLC) on a C4 medium, and the single-step elution of purified hormone from concanavalin A agarose. Overall, an 890-fold purification was accomplished with a recovery of 80% as assayed in vitro. Biologically, this purified erythropoietin is highly active, possessing a specific activity in vitro of 200,000 U/mg protein. Chemically, this erythropoietin (molecular weight [mol wt] 26,200) appears exceptionally uniform in its oligosaccharide constitution (30%) as contrasted with heterogeneously glycosylated erythropoietins derived from mammalian cells (mol wt 30,000 to 38,000; 40% to 50% complex-type oligosaccharide). Thus, human erythropoietin as presently produced in an insect cell line comprises not only an abundant source of highly active, readily purified hormone for studies of its mechanism of action and cell surface receptor, but also represents a uniquely homogeneous form that should prove advantageous for direct structural analyses.

Description: Ten patients, with bone marrow failure or malignant disorders, became refractory to platelet transfusions using random, as well as partial or fully HLA-matched, single-donor platelets. To determine its effect on platelet refractoriness, intravenous gamma globulin (IV IgG) was administered at 400 or 800 mg/kg/d for five days, and postinfusion platelet responses were monitored. Platelet transfusion responses following intravenous gamma globulin (IV IgG) were graded as follows: Excellent, 48-hour posttransfusion count greater than 50,000/microL; good, 48-hour count greater than 20,000 but less than 50,000/microL; Fair, increased increment, 48-hour count less than 20,000; and failed, no increased increment. Six of ten patients (60%) had improved responses to selected single-donor platelets (two were excellent, three were good, and one was fair). The time to achieve a platelet transfusion count greater than 25,000/microL ranged from one to nine days of IgG therapy. One individual had sustained benefit (greater than 1 year); the remaining responses persisted for 6 to 8 weeks. These results suggest that IV IgG may be useful in the management of platelet refractoriness, especially in patients receiving single-donor platelets.

Description: Graft-versus-host disease (GVHD) is currently encountered after bone marrow transplantation and transfusion. GVHD associated with transfusion (TA-GVHD) in apparently immunocompetent recipients has been recently reported with increasing frequency. A consistent finding in many of these cases is that the recipient received blood from a donor homozygous for one of the recipient's HLA haplotypes. However, the observed frequency of TA-GVHD is much lower than the estimated probability of this donor/recipient combination. The potential role of recipient immune responses in controlling TA-GVHD was investigated using an analogous murine model in which GVHD is induced by the injection of parental lymphoid cells into unirradiated F1 hybrid recipients. The effect of various immune manipulations of the recipient of GVHD induction was assessed by determining the number of donor lymphoid cells required to induce GVHD responses. Whereas depletion of recipient CD4+ cells increased the number of donor cells needed to induce GVHD, depletion of recipient CD8+ and natural killer cells resulted in fewer donor cells being needed to induce a GVHD response. These studies suggest a central role for functioning recipient CD8 and natural killer cells in the down-regulation of TA-GVHD development in recipients.

Description: Bone marrow cells from ten normal donors were exposed to ultraviolet (UV)C or UVB light for total exposures of 0.1 to 100 mJ/cm2, and assayed for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and phytohemagglutinin (PHA)- stimulated proliferative responses. After exposure to UVC CFU-GM, BFU-E and PHA responses showed a UV dose-dependent sharp decrease to levels less than 1% of controls with 0.5, 2.0, and 10 mJ/cm2, respectively. With UVB, PHA responses were most sensitive, declining to less than 1% at 5 mJ/cm2. BFU-E decreased to less than 1% of control with 15 mJ/cm2 UVB. CFU-GM, at UVB doses of 0.1 to 2.0 mJ/cm2, increased to 125% to 130% of control and decreased to less than 1% only at exposures greater than 20 mJ/cm2. Thus, these studies show that UVB, but not UVC light, can be used to inactivate bone marrow T lymphocytes selectively while sparing hematopoietic precursor cells. The data suggest that UVB irradiation can be used for T-lymphocyte purging for allogeneic marrow transplantation.

Description: Bone marrow transplantation (BMT) is relatively effective for the treatment of lysosomal storage diseases. To better understand the contribution of specific hematopoietic lineages to the efficacy of BMT, we transplanted β-glucuronidase–positive mononuclear phagocytes derived from either the peritoneum or from bone marrow in vitro into syngeneic recipients with mucopolysaccharidosis type VII (MPS VII). Cell surface marking studies indicate that the bone marrow-derived cells are less mature than the peritoneal macrophages. However, both cell types retain the ability to home to tissues rich in cells of the reticuloendothelial system after intravenous injection into MPS VII mice. The half-life of both types of donor macrophages is approximately 7 days, and some cells persist for at least 30 days. In several tissues, therapeutic levels of β-glucuronidase are present, and histopathologic analysis demonstrates that lysosomal storage is dramatically reduced in the liver and spleen. Macrophages intravenously injected into newborn MPS VII mice localize to the same tissues as adult mice but are also observed in the meninges and parenchyma of the brain. These data suggest that macrophages play a significant role in the therapeutic efficacy of BMT for lysosomal storage diseases and may have implications for treatments such as gene therapy.

Description: In anaplastic large-cell lymphoma (ALCL), the (2;5) chromosomal translocation creates a fusion gene encoding the 80-kD NPM-ALK hybrid protein. This report describes three new monoclonal antibodies, two of which recognize, by Western blotting, the N-terminal portion of NPM present in the NPM-ALK fusion protein and also in two other NPM fusion proteins (NPM-RAR and NPM-MLF1). The third antibody recognizes the C-terminal portion (deleted in NPM-ALK) and reacts only with wild-type NPM. The three antibodies immunostain wild-type NPM (in paraffin-embedded normal tissue samples) in cell nuclei and in the cytoplasm of mitotic cells. Cerebral neurones, exceptionally, show diffuse cytoplasmic labeling. In contrast to normal tissues, the two antibodies against the N-terminal portion of NPM labeled the cytoplasm of neoplastic cells, in four ALK-positive ALCL, reflecting their reactivity with NPM-ALK fusion protein, whereas the antibody to the C-terminal NPM epitope labeled only cell nuclei. Immunocytochemical labeling with these antibodies can therefore confirm that an ALK-positive lymphoma expresses NPM-ALK (rather than a variant ALK-fusion protein) and may also provide evidence for chromosomal anomalies involving the NPM gene other than the classical (2;5) translocation.

Description: Ten dogs were given 9.2 Gy of total body irradiation and autologous bone marrow infusion followed by ten daily transfusions of leukocytes for a total of 11.5 to 36.2 (median, 18.8) x 10(8)/kg obtained via leukapheresis from histoincompatible unrelated donors. Four dogs were given unirradiated leukocytes, and all developed graft-versus-host disease (GVHD). In contrast, only two of three dogs given leukocytes irradiated with 20 mJ/cm2 of ultraviolet (UV) light (200 to 300 nm), and none of three dogs given leukocytes irradiated with 1,000 mJ/cm2 developed GVHD. These data indicate that UV irradiation abrogates the alloreactive potential of transfused leukocytes, and suggest that UV irradiation can be used to prevent the development of transfusion- induced GVHD.

Description: The mechanism of drug-dependent immunologic thrombocytopenic purpura (DITP) was investigated by studying the sera of four patients with classic DITP (two with quinidine-, one with acetaminophen-, and one with phenazopyridine-dependent antiplatelet antibody) using a solid- phase radioimmunoassay with 125I-staphylococcal protein A. Two forms of antiplatelet antibody could be demonstrated: one that required drug to bind to platelets and one that bound to platelets in the absence of drug. Drug-dependent antiplatelet antibody required the simultaneous addition of drug and the Fc domain of the drug-dependent IgG molecule for binding to platelets. It did not require serum complement or factor VIII-related antigen for binding to platelets. Drug-dependent binding of antibody to platelets was saturation-dependent. Non-drug-dependent antiplatelet antibody of two patients (one with quinidine-induced thrombocytopenia and the other with acetaminophen-induced thrombocytopenia) reacted with autologous platelets as well as with homologous platelets, indicating that they were autoantibodies. Both autoantibodies had disappeared when their sera were tested 23 and 138 days, respectively, after withdrawal of their initial positive sera. Non-drug-dependent antiplatelet antibody binding could be demonstrated with the F(ab')2 fragment of the purified IgG of the serum of the second patient with quinidine DITP, who did not have detectable alloantibodies against HLA. None of the four patients with non-drug- dependent antiplatelet antibody had a past or present history of autoimmune thrombocytopenic purpura.

Description: We describe a dominantly inherited β-thalassemia intermedia phenotype observed in a five-generation Portuguese family. Carriers are characterized by moderate anemia, hypochromia, microcytosis, elevated hemoglobin (Hb)A2 and HbF levels, splenomegaly, hepatomegaly, and inclusion bodies in pheripheral red blood cells after splenectomy. The molecular basis of this condition is a small deletion within the 5′ consensus splicing sequence of the second intron of the β-globin gene, IVS-II-4,5 (-AG). Reticulocyte RNA studies performed by reverse transcription-polymerase chain reaction (RT-PCR) and primer extension analysis showed three abnormally processed transcripts, which, upon sequencing, were shown to correspond to (1) skipping of exon 2, and (2) activation of two cryptic splice sites (between codons 59/60, and at IVS-II-47). In vitro translation studies of these patients' reticulocyte RNA have shown that at least one of these aberrant mRNA species is translated into an abnormally elongated peptide whose cytotoxic properties could, in part, be causing the atypical dominant mode of inheritance observed in this family. We suggest that this elongated β chain is unable to combine with an α-globin chain to form a functional Hb molecule. Its degradation would, then, exhaust the proteolytic defense mechanism of the erythroid precursors, leading to inefficient proteolysis of the free α chains in excess.

Description: Thirty-two patients with acute leukemia, chronic granulocytic leukemia, or multiple myeloma received a T lymphocyte-depleted HLA-identical marrow. After being treated with pan-T monoclonal antibodies (MoAbs) and one round of baby rabbit complement, the mean percentage of T cell depletion was 94% +/- 4%. The number of residual viable T cell infused to the patient was 0.99 +/- 0.65 X 10(6) per kg body weight. The patients were conditioned with fractionated total body irradiation (TBI) (12 Gy) preceding high doses of cyclophosphamide (120 mg/kg). Methotrexate was used as an additional immunosuppressant in the first ten patients. For the following 22 patients no posttransplant immunoprophylaxis was administered. Eight patients died within three months due to complications related to transplantation. Engraftment was achieved in all evaluable patients, and no patient has a late graft failure. The proof of total chimerism was established in 24 patients. Twenty-four of 27 evaluable patients (88%) did not have an acute graft- v-host disease (GVHD) greater than grade 0 to 1. Two patients had a grade 2 (skin only), and one patient had a grade 4 acute GVHD (the latter had only 80% of T cell depletion). A medullary relapse occurred in 11 patients (nine of them had previously been defined as “high risk leukemia”). Our data suggest that it may not be necessary to deplete nearly all T cells to prevent acute GVHD in recipients of HLA-identical marrow.

Description: Hematopoietic chimerism was analyzed in serial bone marrow samples taken from 28 children following T-cell depleted unrelated donor bone marrow transplants (UD BMT) for acute lymphoblastic leukemia (ALL). Chimeric status was determined by polymerase chain reaction (PCR) of simple tandem repeat (STR) sequences (maximal sensitivity, 0.1%). At least two serial samples were examined in 23 patients. Of these, two had evidence of complete donor engraftment at all times and eight showed stable low level mixed chimerism (MC) (

Description: We identified a dog with large granular lymphocytic leukemia and cutaneous lymphoma that exhibited constitutive expression of interleukin-2 (IL-2) receptors by the leukemic peripheral blood lymphocytes. The leukemic cells phenotypically resembled natural killer (NK) cells, and their surface IL-2 receptors were functional, as determined by the capacity to bind human recombinant IL-2 with high- affinity resulting in the transduction of proliferation signals and in the development of lymphokine-activated killer cell activity. These cells produced IL-2 spontaneously, and they may have maintained their proliferative state through an IL-2-dependent autocrine growth pathway. Our results indicate that neoplastic lymphocytes of syndromes that involve circulating leukemic cells with dermotropism can originate from NK-like cells. Additionally, the data also suggest that proliferative conditions such as these may be the result of the aberrant production of IL-2. Further, this case illustrates the potential for the use of hematopoietic malignancies in the dog as a suitable animal model for immune targeting of IL-2 receptors as a novel treatment approach for similar malignancies of human beings.

Description: Two T-ALL patients carrying a t(11;14)(p13;q11) translocation were analyzed. Southern blotting experiments demonstrated that both patients had rearranged their J delta genes and that the translocation involved the delta locus in both cases. In one patient, cloning, restriction mapping, and sequencing showed that the translocation occurred on a D delta 1-D delta 2-J delta 2 rearranged gene. In addition, the rearrangement on chromosome 11 occurred in both patients within a segment of less than or equal to 2 kb showing the presence in this region of a point of recurrent recombination.

Description: To evaluate the prevalence of von Willebrand's disease (vWd) we carried out an epidemiological investigation among school children of the Veneto region in northern Italy. A total of 1,218 of 1,281 possible children participated in the study. They were 11 to 14 years of age, and all attended secondary schools in two distinct small areas, 70 km apart, between which there is no social contact. A blood sample was taken from each subject for determination of the blood group and von Willebrand factor (vWf) level (measured as ristocetin cofactor and expressed in IU/dL after calibration of the internal pool against an international standard), and the parents were given a questionnaire concerning hemorrhagic symptoms in the members of the family in the last three generations. Separate normal ranges were calculated for blood group O and non-O subjects (1,166 children and 289 adults) with a nonparametric method because the distribution curves of the reference values did not fit the gaussian distribution. Diagnoses of vWd were considered only for children who had low vWf levels and were members of a family with a convincing bleeding history (case of “probable vWd”). A final diagnosis was assigned if, in addition to these criteria, at least one other family member on the side with hemorrhagic history had a low vWf level. Of the 1,218 children examined, ten were classified as having vWd (0.82%). Taking into account the 90% confidence interval for the lower limit of the normal range, this figure could range from 7 (0.57%) to 14 (1.15%). All these subjects were mildly to moderately affected and presented features of heterozygous classic vWd (type I). Affected subjects were distributed evenly in the two areas examined. Our results suggest that the prevalence of vWd might be much higher than previously reported and that a different screening approach might be of use for patients with mild bleeding diathesis.

Description: Recent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34+ and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1+ endothelial cells on the OP9 stromal layer, while TEK− cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.

Description: To elucidate the mechanism by which activation of the contact system of blood coagulation leads to expression of fibrinolytic activity, we have determined the molecular characteristics of the plasminogen activators present in dextran sulfate-treated euglobulin fractions by electrophoretic-zymographic analysis and specific immunoadsorption. In addition to free and protease inhibitor-bound tissue-type plasminogen activator (t-PA), dextran sulfate precipitates of euglobulins contained the complex formed between plasma kallikrein and C1-inhibitor, an indicator of prekallikrein activation. These precipitates also contained substantial fibrinolytic activity related to urinary-type plasminogen activator (u-PA). Autoradiographic analysis was then used to evaluate the cleavage of 125I-single-chain u-PA (prourokinase) in dextran sulfate euglobulins as well as after exposure to kallikrein or beta-factor XIIa. This analysis supported the conclusion that plasma kallikrein-mediated cleavage and activation of single-chain u-PA is the mechanism operative for the development of lytic activity in euglobulin precipitates following activation of the contact system.

Description: The effects of recombinant, macrophage-derived, murine tumor necrosis factor-alpha (TNF-alpha) on hematopoiesis in vivo has been examined in normal mice and in Friend virus (FV)-induced erythroleukemic mice. Intravenous (IV) administration of a single dose of recombinant murine TNF-alpha (10(5) U per mouse) significantly suppressed normal and leukemic late-stage erythropoiesis as measured by numbers of mature erythroid colony forming cells (CFU-E) in the bone marrow and spleen and by peripheral blood reticulocyte counts. In normal animals, the immature erythroid (BFU-E), macrophage (CFU-M), and granulocyte- macrophage (CFU-GM) compartments were significantly stimulated by TNF- alpha in both the bone marrow and the spleen. In the bone marrow of leukemic mice, the BFU-E, CFU-GM, and CFU-M progenitor cell compartments were also stimulated by treatment with the monokine. In the spleens of leukemic mice (the primary site of FV leukemia cell accumulation), relative numbers of BFU-E and CFU-GM were increased by TNF-alpha, while those of CFU-M were suppressed. TNF-alpha caused a rapid decrease in the markedly elevated spleen weights of progressively leukemic mice, and in multiple doses it caused complete clinical disease regression in a significant percentage of leukemic animals. The combination of TNF-alpha with interferon-gamma (IFN-gamma) increased the incidence of leukemia regression, compared with TNF-alpha alone. These results show that TNF-alpha exerts a suppressive influence on late-stage erythropoiesis in vivo and suggest that this effect might be exploited in the treatment of acute erythroleukemia, erythroid hyperplasias, and related diseases.

Description: We have characterized the effects of plasmin on glycoprotein Ib (GpIb), a platelet membrane receptor for von Willebrand factor (vWF), and on glycocalicin, a fragment of the alpha chain of GpIb that contains the vWF-binding region. The addition of 4.5 X 10(-7) mol/L plasmin to washed platelets caused a time-dependent decrease in ristocetin- induced, vWF-dependent platelet agglutination. epsilon-Aminocaproic acid (EACA) inhibited plasmin release of glycocalicin-related antigen from washed platelets and preserved vWF-dependent platelet agglutination, thus indicating that the lysine-binding sites on plasmin facilitated its degradation of GpIb. To demonstrate a direct interaction between plasmin and the vWF-binding region of GpIb we incubated purified glycocalicin with plasmin. Plasmin degraded the glycocalicin into two small carbohydrate-poor peptides and into a larger carbohydrate-rich fragment. EACA was able to inhibit plasmin- mediated degra dation of glycocalicin in a concentration-dependent fashion. These studies indicated that plasmin degradation of GpIb was due to a direct interaction between plasmin and GpIb and that this effect was mediated by the lysine-binding region of the plasmin molecule.

Description: The role in platelet function of the cell-binding region of fibronectin was explored by the use of synthetic peptides. The prototypical peptide gly-arg-gly-asp-ser was capable of inhibiting thrombin-induced platelet aggregation without altering the degree of platelet activation as judged by the secretion of 14C-serotonin. The peptide also effectively inhibited, in a concentration-dependent manner, the binding of radiolabeled fibronectin to platelets and the adhesion of platelets to fibronectin substrates. The smallest peptide from the cell-binding region of fibronectin which retained full activity was arg-gly-asp-ser. Transposition of amino acids or conservative substitutions of amino acids within this short sequence resulted in inactive peptides. Peptides containing the arg-gly-asp-ser sequence were also capable of inhibiting the adhesion of platelets to fibrinogen and von Willebrand factor substrates. Examination of the entire panel of synthetic peptides for ability to inhibit adhesion to fibrinogen or von Willebrand factor substrates revealed the same structure-function relationships that had been determined in the studies with fibronectin.

Description: Platelet membrane glycoprotein Ib alpha (GPIb alpha) bears the human platelet alloantigen (HPA)-2 and molecular weight (MW) polymorphisms on sodium dodecyl sulfate-polyacrylamide gels. HPA-2 arises from a threonine/methionine dimorphism at residue 145 of the GPIb alpha sequence, whereas different numbers of tandem repeats of a 39-bp sequence encoding 13-amino acids corresponding to a region between serine399 and threonine411 of the GPIb alpha account for the latter. To identify the genetic basis of the MW polymorphism among Japanese, we counted the tandem repeats in 103 individuals. In addition to the reported three variants with one, two, or three tandem repeats, we identified a new variant with four perfect tandem repeats of the 39-bp sequence that corresponded to the largest phenotype. Phenotypic analysis of the MW polymorphism on 12 individuals including all four phenotypes completely accorded in the genotype. We also determined the genotype of HPA-2 and found that methionine145 was in complete linkage disequilibrium, with the larger variants containing three or four tandem repeats. These results imply a model of evolutionary steps in the gene encoding GPIb alpha.

Description: Endothelial cells express surface molecules that are involved in cell- matrix interaction, including the vitronectin receptor and the fibronectin receptor, both members of a family of cell adhesion receptors (integrins). Here we provide evidence that endothelial cells express a membrane molecule, indistinguishable from the platelet VLA-2 complex, which is a collagen receptor and a member of the integrin family. To identify this endothelial molecule, we have used a monoclonal antibody, CLB-10G11, which recognizes the VLA-2 complex from platelets. The molecule recognized by CLB-10G11 from endothelial cells was characterized as follows. (1) The monoclonal antibody precipitated two proteins from surface-labeled endothelial cells that corresponded to the platelet VLA-2 subunits (glycoprotein Ia and IIa) as judged by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional nonreduced/reduced SDS- PAGE. (2) Preclearing of endothelial cells with monoclonal antibody A- 1A5, an antibody that is directed against the common VLA beta subunit, removed all the CLB-10G11-binding material. (3) Crossed immunoelectrophoresis revealed that CLB-10G11 recognizes a single precipitation arc from either platelets or endothelial cells. Analysis of these two cell types in one gel again revealed one precipitation arc. The antigen of either cell type, recognized by CLB-10G11 could be precipitated by either polyclonal antiplatelet or polyclonal antiendothelial cell antiserum. Hence, it appears that endothelial cells express at least three different surface molecules (the vitronectin receptor, the fibronectin receptor and a collagen receptor), which may play an important role in controlling the anchorage of endothelial cells to the extracellular matrix.

Description: Protein markers are often used to corroborate the morphological subtyping of hematopoietic malignancy. Most commonly, surface markers are used for the phenotyping of hematopoietic cells; however, internal proteins have also been used as markers. Glycophorin, hemoglobin A, hemoglobin F, and transferrin have all been used as markers for the erythroid phenotype. We have recently shown that carbonic anhydrase is constitutively and aberrantly expressed in two erythroleukemic cell lines. We here show that it is also present in high levels in primary erythroleukemic blasts and that it is a useful marker for the M6 phenotype when classifying acute nonlymphocytic leukemia.

Description: The balance of tissue-type plasminogen activator (t-PA) production and degradation determines its concentration in blood and tissues. Disturbance of this balance may result in either increased or decreased proteolysis. In the present study, we identified the receptor systems involved in the degradation of t-PA by human monocytes/macrophages in culture. Monocytes were cultured and became macrophages within 2 days. At 4 degrees C, 125I-t-PA bound to macrophages with high (apparent dissociation constant [kd], 1 to 5 nmol/L) and low affinity (kd = 350 nmol/L). At 37 degrees C, the cells internalized and degraded t-PA via the high affinity binding sites, which were partially inhibited by mannan. The low affinity binding sites were 6-aminohexanoic acid- inhibitable and not involved in t-PA degradation. Degradation of t-PA was upregulated during differentiation of monocytes to macrophages. Dexamethasone further upregulated the mannan-inhibitable t-PA degradation. Lipopolysaccharide downregulated both mannan-inhibitable and non-mannan-inhibitable t-PA degradation. Non-mannan-inhibitable degradation was completely blocked by recombinant 39-kD receptor- associated protein (RAP, inhibitor of lipoprotein receptor-related protein [LRP]), whereas mannan-inhibitable degradation was blocked by the addition of a monoclonal antibody against the mannose receptor. No differences between the degradation of t-PA and functionally inactivated t-PA were observed. We conclude that human monocyte-derived macrophages are able to bind, internalize, and degrade t-PA. Degradation of t-PA does not require complex formation with plasminogen activator inhibitors. The macrophages use two independently regulated receptors, namely, the mannose receptor and LRP, for the uptake and degradation of t-PA.

Description: We found that a monoclonal antibody to CD9 antigen, PMA2, induces fibrinogen binding to platelets and examined the mechanism for this. That PMA2 recognized the CD9 antigen was confirmed by its immunoblot- reactivity with a 24,000-dalton protein, reactivity with platelets and common acute lymphoblastic leukemia (ALL) cells, and competitive binding with the ALB6 antibody known as the CD9 antibody. At saturation, PMA2 bound to approximately 46,000 sites per platelet. The binding of 125I-fibrinogen to platelets occurred in a PMA2 concentration-dependent manner and was blocked by EDTA or an anti- glycoprotein (GP)IIb-IIIa monoclonal antibody. PMA2-stimulated platelets caused ATP secretion and thromboxane B2 synthesis under non- stirred conditions. The role of secreted ADP and thromboxane in fibrinogen-binding and subsequent platelet aggregation was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. CP/CPK or aspirin alone reduced fibrinogen binding to 20% to 30%; however, this binding was sufficient to support full platelet aggregation. Combined treatment with CP/CPK and aspirin abolished fibrinogen binding and aggregation. These results demonstrate that the binding of IgG molecules to the CD9 antigen exposes fibrinogen receptors through both secreted ADP and thromboxane and that either one of both can expose the receptors to an extent sufficient to aggregate platelets.

Description: This study demonstrates that when platelets are stimulated by thrombin in the presence of low concentrations of purified human fibrinogen (10 to 20 micrograms/mL, final concentration) binding of released platelet von Willebrand factor (plt-vWF) to the platelet membrane is enhanced. This effect appears to be mediated by fibrin monomer produced by the action of thrombin on the fibrinogen in the incubation suspension. When fibrin polymerization is inhibited, the binding of released plt-vWF to the platelets is markedly increased. This enhanced binding is dependent on platelet glycoprotein Ib (GPIb) as shown by a decreased response with Bernard-Soulier platelets and inhibition by both monoclonal and polyclonal antibodies against glycocalicin. The binding of fibrin to thrombin-activated platelets preincubated with monoclonal antibody against GPIIb/IIIa is increased when the predominant form of fibrin is fibrin monomer. The fibrin binding is also decreased in the presence of antibody against glycocalicin. Our data demonstrate that fibrin monomer facilitates plt-vWF binding to the glycocalicin portion of platelet GPIb on thrombin-stimulated platelets and that binding of fibrin monomer to glycocalicin is necessary for this response to occur.

Description: We have previously reported a patient with cytochrome b-positive X-linked chronic granulomatous disease. Although the O2-production of neutrophils from the patient was completely defective, we presented data suggesting that the patient's cytochrome b was present at a normal level and possibly had normal spectroscopic features. Thus, to look for a mutation in the cytochrome b heavy chain (gp91-phox) gene, DNA analysis of gp91-phox cDNA derived from this patient was performed. As a result, we found that five nucleotides (1521 through 1525) within exon 12 were deleted, and a new sequence of eight nucleotides was inserted. This mutation converted Gln507-Lys508-Thr509 into His-Ile-Trp-Ala. Mismatched polymerase chain reaction showed that the mother has both wild and mutated alleles, confirming that this case was transmitted in an X-linked fashion. This mutation is different from those previously reported by others. The translocation of p47-phox and p67-phox to the membrane fraction occurred, indicating the complete formation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. We conclude that this case suggests that the structure encoded on exon 12 of gp91-phox is important for electron transfer.

Description: High-dose chemotherapy with or without radiotherapy followed by autologous transplantation of hematopoietic progenitor cells is an effective treatment for patients with high-risk or relapsed non- Hodgkin's lymphoma. Chemotherapy and/or hematopoietic growth factors have been used to mobilize progenitor cells in the peripheral blood for transplantation. However, the mobilized blood cell products have been found to be frequently contaminated with tumor cells, and techniques have not been developed to purge tumor cells from these products. In addition, the minimum number of hematopoietic progenitor cells required for engraftment has not yet been fully elucidated. We treated 21 patients with a single infusion of cyclophosphamide (4 g/m2) followed by daily administration of granulocyte colony-stimulating factor (G-CSF). After recovery of the white blood cell count, a single 3-hour apheresis collection was performed. The apheresis product was then applied to a discontinuous Percoll gradient. The low-density fractions resulting from this separation procedure were enriched for CD34+ progenitor cells (total cell yield, 19.5%; CD34+ cell recovery, 81.2%). These enriched cellular products were treated with a panel of anti-B cell or anti-T cell monoclonal antibodies and complement in an effort to remove residual tumor cells. After treatment of the patient with myeloablative therapies, the enriched and purged cells were reinfused. Hematologic recovery was rapid, with median neutrophil engraftment in 10 days [absolute neutrophil count (ANC), greater than 0.5 x 10(9)/L] and 11 days (ANC, greater than 1.0 x 10(9)/L). Median platelet transfusion independence required 13 days. The rapidity of multilineage engraftment correlated with the number of CD34+ cells per kilogram that were infused. Patients who received more than 2 x 10(6) CD34+ cells per kilogram had rapid hematologic engraftment, whereas those patients transplanted with less than 2 x 10(6) CD34+ cells per kilogram had slower platelet recovery. Modeling studies using a lymphoma cell line with a t(14; 18) chromosomal translocation demonstrated the successful removal of tumor cells assayed using the polymerase chain reaction (PCR) after the processing and purging. Four of the 21 patients had PCR-detectable lymphoma cells in the bone marrow and peripheral blood; however, the enriched and purged blood products reinfused in all four did not contain detectable tumor cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Description: IIbb3 integrin is a heterodimeric receptor facilitating platelet aggregation. Both genes are on chromosome 17q21.32. Intergenic distance between them has been reported to be 125 to 260 kilobasepairs (kb) by pulsed-field gel electrophoresis (PFGE) genomic analysis, suggesting that they may be regulated coordinately during megakaryopoiesis. In contrast, other studies suggest these genes are greater than 2.0 megabasepairs (mb) apart. Because of the potential biological implications of having these two megakaryocytic-specific genes contiguous, we attempted to resolve this discrepancy. Taking advantage of large kindreds with mutations in either IIb or β3, we have developed a genetic linkage map between the thyroid receptor hormone-1 gene (THRA1) and β3 as follows: cen-THRA1-BRCA1-D17S579/IIb-β3-qter, with a distance of 1.3 centiMorgans (cM) between IIb and β3 and the two genes being oriented in the same direction. PFGE genomic and YAC clone analysis showed that the β3 gene is distal and ≥365 kb upstream of IIb. Additional restriction mapping shows IIb is linked to the erythrocyte band 3 (EPB3) gene, and β3 to the homeobox HOX2b gene. Analysis of IIb+-BAC and P1 clones confirm that the EPB3 gene is ∼110 kb downstream of the IIb gene. Sequencing the region surrounding the human IIb locus showed the Granulin gene ∼18 kb downstream to IIb, and the KIAA0553 gene ∼5.7 kb upstream. This organization is conserved in the murine sequence. These studies show that IIb and β3 are not closely linked, with IIb flanked by nonmegakaryocytic genes, and imply that they are unlikely to share common regulatory domains during megakaryopoiesis.

Description: This study shows that human postthymic T cells express CD10 when undergoing apoptosis, irrespective of the signal responsible for initiating the apoptotic process. Cells from continuous T-cell lines did not normally express CD10, but became CD10+ when induced into apoptosis by human immunodeficiency virus (HIV) infection and exposure to CD95 monoclonal antibody, etoposide, or staurosporin. Inhibitors of caspases blocked apoptosis and CD10 expression. Both CD4+ and CD8+ T cells purified from normal peripheral blood expressed CD10 on apoptotic induction. CD10 was newly synthesized by the apoptosing cells because its expression was inhibited by exposure to cycloheximide and CD10 mRNA became detectable by reverse transcription-polymerase chain reaction in T cells cultured under conditions favoring apoptosis. To show CD10 on T cells apoptosing in vivo, lymph node and peripheral blood T cells from HIV+ subjects were used. These suspensions were composed of a substantial, although variable, proportion of apoptosing T cells that consistently expressed CD10. In contrast, CD10+ as well as spontaneously apoptosing T cells were virtually absent in peripheral blood from normal individuals. Collectively, these observations indicate that CD10 may represent a reliable marker for identifying and isolating apoptosing T cells in vitro and ex vivo and possibly suggest novel functions for surface CD10 in the apoptotic process of lymphoid cells.

Description: The release of secretory phospholipase A2 (sPLA2) into the mammalian circulation may contribute to the development of hemorrhagic and inflammatory diseases. sPLA2 has previously been shown to alter the behavior of platelets, leukocytes, and endothelial cells, although the molecular basis for these cellular effects has not been established. Our studies indicate that the inhibition of platelet aggregation by snake, bee venom, and pancreatic sPLA2 is dependent on a plasma cofactor. This cofactor resides within the lipoprotein fraction of plasma, with 54%, 31%, and 11% of the activity present in the high- density lipoprotein (HDL), low-density lipoprotein (LDL), and very low density lipoprotein (VLDL) fractions, respectively. Delipidation of HDL and LDL was associated with the complete loss of platelet-inhibitory activity. Incubation of purified sPLA2 with the HDL fraction of plasma resulted in the time-dependent generation of lysophosphatidylcholine (lysoPC). The formation of lysoPC correlated with the inhibition of platelet aggregation. Purified lysoPC (10 to 100 micrograms/mL) inhibited platelet aggregation and dense granule release induced by thrombin (0.05 U/mL), collagen (1 micrograms/mL), ionophore A23187 (2 mumol/L), ADP (12.5 mumol/L), and adrenaline (3.2 mumol/L). The inhibition of platelet aggregation by lysoPC was dose-dependent and correlated with decreased fibrinogen binding to glycoprotein IIb-IIIa. Our studies indicate that the enzymatic generation of lysoPC from plasma lipoproteins is essential for the sPLA2-mediated inhibition of platelet activation in the presence of albumin. These results raise the possibility that the toxic effects of circulating sPLA2 may be due in part to the generation of the bioactive lysophospholipid, lysoPC.

Description: Polymorphonuclear leukocytes (PMNs) adhere to endothelial cells at sites of acute inflammation. To examine this phenomenon in vitro, we have developed a new assay to measure adherence of PMNs to cultured endothelial cells. Human PMNs were labeled with 111indium-oxine and incubated in microtiter wells with monolayers of either human umbilical vein or bovine aortic endothelial cells. Following incubation, the wells were sealed, inverted, and centrifuged at varying speeds. Results are expressed as the percentage of PMNs added initially that remained attached to the monolayers after being subjected to dislodgment forces (ie, relative centrifugal forces) ranging from 1 to 1,200 g. Adherence of PMNs to endothelial monolayers was temperature dependent, dependent on the concentration of extracellular Mg2+ (but not Ca2+), and enhanced significantly by the chemotactic peptides, N-formyl-methionyl-leucyl- phenylalanine (fMLP) and human C5a. It was found that fMLP and C5a not only increased the number of PMNs that adhered to endothelial cells, but also increased the strength of adherence.

Description: We examined various murine hematopoietic cell populations for their capacity to interact with radiolabeled histamine. Only bone marrow cells (BMC) retained substantial amounts of radioactivity, in contrast to thymus, spleen, and peritoneal cells. The characteristics of this interaction are consistent with histamine uptake rather than receptor binding. Indeed, this process is temperature and sodium dependent and reduced by various metabolic inhibitors. Furthermore, the effect of antagonists or agonists of the H1, H2, and H3 receptor subtypes is not in accordance with the involvement of either of these receptors in histamine binding. The target cells of histamine copurify with hematopoietic progenitors in the low-density BM population. They are most enriched in the subset sorted from the blast cell window on the basis of high rhodamine retention. This fraction contains on the average 80% to 90% immature cells and is highly enriched for several clonogenic progenitor subsets. Sixty percent of the Rh-bright cells are labeled by 3H-histamine, as assessed by autoradiography, suggesting that a variety of immature cells participates in this phenomenon. Furthermore, in all sorting procedures used here, the cells capable of histamine uptake coenrich with those producing histamine in response to interleukin-3, indicating at least a partial identity between these cells.

Description: Two families with polycythemia inherited as an autosomal dominant trait are described. Serial hemoglobin determinations in multiple family members and RBC volume measurements in selected affected subjects documented their polycythemia. Measurements of arterial p02s, p50s, and blood oxygen affinity were normal in all affected individuals from each family who were tested. Erythropoietin (EPO) levels were low in affected individuals from family 1 and normal in affected members of family 2. Stimulation of in vitro CFU-E colony growth by low levels of EPO was significantly increased in subjects from family 1, but normal in those affected from family 2. We conclude that although the inheritance pattern for the polycythemia in both of these families appeared to be the same, the biologic defect leading to the disorder in each of these unique families was different. The precise mechanism of the increased EPO sensitivity noted in affected subjects from family 1 awaits elucidation.

Description: The outcome of patients with non-Hodgkin’s lymphoma has been improved by current approaches to treatment. Nevertheless, many patients either do not have a complete remission or ultimately relapse. To identify such patients, it is important to be able to predict the outcome. We previously found that the differentiation inhibitory factor/nm23 was correlated with the prognosis of acute myeloid leukemia. To examine the prognostic effect of nm23 on non-Hodgkin’s lymphoma, we established an enzyme-linked immunosorbent assay procedure to determine nm23-H1 protein levels in plasma and assessed the association of this protein level with the response to chemotherapy, overall survival, and progression-free survival in patients with aggressive non-Hodgkin’s lymphoma. The plasma concentration of nm23-H1 was significantly higher in patients with malignant lymphoma than in normal controls, especially in aggressive non-Hodgkin’s lymphoma. The complete remission rate in patients with higher nm23-H1 levels was significantly worse than that in patients with lower nm23-H1 levels. Overall survival and progression-free survival were also lower in patients with higher nm23-H1 levels than in those with lower levels. The 3-year survival rates in patients with low and high nm23-H1levels were 79.5% and 6.7% (P = .0001). A multivariate analysis of prognostic factors showed that the plasma nm23-H1level was independently associated with the survival and progression-free survival. An elevated plasma nm23-H1concentration predicts a poor outcome of advanced non-Hodgkin’s lymphoma. Therefore, nm23-H1 in plasma may be useful for identifying a distinct group of patients at very high risk.

Description: This report describes the production and characterization of 13 rodent monoclonal antibodies to the human erythrocyte anion transport protein AE1 (syn. band 3). Eleven antibodies (4 murine and 7 rat) recognize epitopes dependent on the integrity of the third extracellular loop of the protein. Two antibodies (1 murine and 1 rat) recognize epitopes on the N-terminal cytoplasmic domain. Quantitative binding studies using radioiodinated IgG and Fab fragments of antibodies to extracellular epitopes on AE1 ranged from 77,000 to 313,000 (IgG) and from 241,000 to 772,000 (Fab) molecules bound at saturation. The results indicate that the epitopes recognized by different antibodies vary in their accessibility and suggest that there is heterogeneity in the organization of individual AE1 molecules in the red blood cell membrane. Quantitative binding studies on South East Asian ovalocytes using several antibodies to AE1 and an anti-Wrb show a marked reduction in the number of antibody molecules bound at saturation. These results are consistent with the existence of highly cooperative interactions between transmembrane domains of AE1 in normal erythrocytes and the disruption of these interactions in the variant AE1 found in South East Asian ovalocytes.

Description: The susceptibility of highly purified human CD34+ cells to monocytotropic (Ba-L) and lymphotropic (A018-post) strains of human immunodeficiency virus-1 (HIV-1) was examined. Liquid cultures initiated with fresh immunomagnetically purified CD34+ cells using the K6.1 CD34 monoclonal antibody (MoAb) (K6.1/CD34+) were positive for HIV expression 2 weeks after exposure to HIV-1 Ba-L. These cells were initially greater than 90% CD34+ and had undetectable monocyte contamination by flow-cytometric staining and side-scatter analyses, respectively, and undetectable T-cell contamination by CD3 polymerase chain reaction (PCR) analysis. However, secondary CD34+ liquid cultures reselected from the primary liquid cultures 24 hours after HIV exposure by panning with the ICH3 CD34 MoAb (ICH3/CD34+) and maintained for an additional 14 days were negative for HIV expression. The ICH3-unbound cells were positive for both spliced and unspliced HIV RNA when exposed to HIV-1 Ba-L, and were DNA PCR positive when exposed to either monocytotropic or lymphotropic HIV-1. To further test that CD34+ cells were not infectible by HIV-1, we exposed K6.1/CD34+ cells continuously to HIV-1 in a culture system capable of maintaining and expanding primitive CD34+ cells. HIV-exposed K6.1/CD34+ cells proliferated and expanded as efficiently as uninfected cultures. However, when reselected magnetically using the K6.1 CD34 MoAb after expansion for 7 days, bound K6.1/CD34+ cells were again negative for HIV-1 expression, whereas unbound cells were positive for HIV-1 expression. These findings suggest that a sequential CD34+ cell-selection process, in which the two selections are separated by a brief culture period, can yield a population of CD34+ cells that are not infected with HIV-1. This process may be useful in the design of stem or progenitor cell- based transplantation therapies for HIV infection.

Description: A 43-year-old male with a phenotypically homogeneous, expanded subset of T cells presented in 1981 with anemia and neutropenia. The surface antigen phenotype of 99% of the peripheral blood lymphocytes was T3+, T8+, T4-, and they were morphologically large granular lymphocytes (LGL). The same cells comprised 37% of the marrow nucleated cells. Eight months after he presented, the peripheral blood T8+, LGL diminished spontaneously, and the anemia and neutropenia completely resolved. The patient remains hematologically normal as of October 1984. To determine if the T8+, LGL represented a clonal expansion, DNA from peripheral blood lymphocytes collected and cryopreserved when the patient was neutropenic and anemic, and when he was hematologically normal, was analyzed for clonal T-cell antigen receptor gene rearrangements. Using Southern blot analysis, a clonal DNA rearrangement was demonstrated, and this clone diminished but was still demonstrable in peripheral blood lymphocytes collected in 1984. The above observations implicate the expanded T8+, LGL in the pathogenesis of the neutropenia and anemia, yet the exact mechanism remains to be elucidated.

Description: Circumferential bands of microtubules (MT) support the discoid shape of resting platelets and participate with the contractile apparatus in shape change and internal contraction following activation. Elucidation of interactions between the circumferential coils and proteins of the stable and contractile cytoskeleton is essential for understanding MT function in platelet physiology. A previous investigation demonstrated that the circumferential rings can be isolated intact from resting platelets following simultaneous exposure to glutaraldehyde and Triton X-100. However, the use of fixation prevented the characterization of protein interactions. The present study has circumvented this problem by developing a procedure for isolating intact microtubule coils from detergent-treated platelets without the use of fixative agents. Incubation of the platelets for intervals of 30 to 60 minutes with the microtubule-stabilizing agent taxol preserved the circumferential bundle after extraction with Triton X-100 even after washing five times. The procedure has made it possible to carry out protein studies on isolated microtubule rings and associated proteins.

Description: The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by immunodeficiency and severe thrombocytopenia in affected males, but no demonstrable clinical abnormalities in carrier females. Through analysis of the methylation patterns of X-linked genes that display restriction fragment length polymorphisms (RFLPs), we studied the pattern of X-chromosome inactivation in various cell populations from female relatives of patients with WAS. The peripheral blood T cells, granulocytes, and B cells of eight obligate WAS carriers were found to display specific patterns of X-chromosome inactivation clearly different from these of normal controls. Thus, carriers of WAS could be accurately identified using this analysis.

Description: Murine bone marrow cells were separated on discontinuous Percoll gradients and assayed for their ability to give rise to megakaryocyte colonies. Ninety-one percent of the megakaryocyte progenitors (CFU-M) sedimented at densities between 1.070 and 1.080 g/mL. Six percent of CFU-M were found at densities between 1.060 and 1.070 g/mL, 2% between 1.050 and 1.060 g/mL, whereas less than 1% had a density either lower than 1.050 g/mL or higher than 1.080 g/mL. The number of doublings and endomitoses achieved by progenitors of density classes higher than 1.050 g/mL were similar. However, colonies derived from CFU-M of densities less than 1.050 g/mL (LD-CFU-M) had a higher probability of polyploidization and a lower probability of cell division in vitro. The inverse correlation found between the number of cells per colony and their DNA content was invariate regardless of the density class of the progenitors. The heterogeneity of the ploidy of cells within colonies increased continuously with increasing cell numbers per colony. The study if a short-period exposure of LD-CFU-M to acute thrombocytopenia could modify the ploidy of their progeny, mice were given rabbit antimouse platelet serum while control animals received normal rabbit serum. Twenty-four hours after injection, marrow was cultured. After a five-day culture period, no change in the number of colonies, doublings, ploidy, and heterogeneity of ploidy were observed between control and thrombocytopenic animals. The data suggests that LD-CFU-M are a distinct category of CFU-M, perhaps more mature than the common CFU-M.

Description: Two human diseases of platelet storage pool deficiency (SPD), Hermansky- Pudlak syndrome and Chediak-Higashi syndrome, are recessively inherited disorders characterized by hypopigmentation, prolonged bleeding, and normal platelet counts accompanied by a reduction in dense granule number. We have recently described seven independent recessive mutations in the mouse regulated by separate genes which are likely animal models for human SPD. Reciprocal bone marrow transplants were carried out between normal C57BL/6J mice and two of these mutants, beige and pallid, in order to test whether the platelet defects are due to a defect in platelet progenitor cells or to humoral factors. Normal and congenic mutant mice were transplanted with marrow after 950 rad whole body radiation. The long bleeding times and low serotonin concentrations of the two mutants were converted to normal values after transplantation with normal marrow. Likewise, normal mice displayed symptoms of SPD when transplanted with mutant marrow. These studies demonstrate that with each of the two mutations, platelet SPD results from a defect in bone marrow precursor cells. Also, the studies suggest that in severe cases, platelet SPD may be successfully treated by bone marrow transplantation.

Description: Megakaryocytes are relatively rare components of human bone marrow, making the study of their maturation difficult. Phorbol esters can act as differentiating agents in a number of cell systems including murine megakaryocytes. We report the effects of phorbol esters on the previously described long-term human megakaryocytic leukemia cell culture, EST-IU. While two nontransforming phorbols fail to affect these cells, the transforming phorbol 12-O-tetradecanoylphorbol-13- acetate (TPA) induces a phenotype with characteristics of more mature megakaryocytes in a dose-related manner. This phenotype includes an increased adherence to untreated plastic or glass, polyploidization, an increase in cell size, and increased expression of both platelet glycoproteins and factor VIII-related antigen. Two-color flow cytometric analysis allowed simultaneous determinations of DNA content and the expression of surface membrane antigens or alpha-granule constituents, providing evidence that nuclear, membrane, and cytoplasmic maturation occur in parallel in this cellular system. TPA- induced maturation of EST-IU cells provides an important new cellular model for the further study of human megakaryocyte development.

Description: Peripheral blood mononuclear cells from five patients with essential thrombocythemia (ET) were cultured in vitro to evaluate restricted megakaryocytic (CFU-Meg), myeloid (CFU-GM), and erythroid (BFU-E) progenitor cell development. Varying concentrations of aplastic canine serum served as the source of megakaryocyte colony-stimulating activity, and cultured megakaryocyte ploidy distributions were determined by Feulgen staining and microfluorometry. Megakaryocyte colony growth was strikingly abnormal in all five patients evaluated. Four of the 5 had a marked expansion in the concentration of circulating CFU-Meg and 3 of 4 manifested abnormalities in cultured megakaryocyte colony size (2 unusually large and 1 small). Unstimulated megakaryocyte colony growth was substantially increased in three patients. However, the fraction of megakaryocyte progenitors in cell cycle was near or below normal in all instances. Endomitotic megakaryocyte development was disordered in each of the four ET patients in whom it was evaluable. In normal subjects, mean megakaryocyte ploidy values vary biphasically with aplastic canine serum concentration and peak at 13.2 N following 12 to 15 days of culture. In contrast, day 12 mean ploidy values in cultures from the ET patients remained low at all aplastic canine serum concentrations and reached a maximum averaging only 8.4 N. Three patients were evaluated serially at extended culture durations of up to 21 days. The cultured megakaryocyte ploidy was unchanged during this interval for two of the patients. For the third patient, ploidy increased steadily, reaching abnormally high ploidy values by day 21. Progenitor cell expansion was limited to the megakaryocyte line in three patients. However, two patients had substantial increases in CFU-GM, one of whom also had a marked increase in BFU-E. There was no significant unstimulated colony growth by either CFU-GM or BFU-E. These data indicate that ET is usually characterized by an expansion in the concentration of circulating CFU-Meg in vivo which manifest both disordered replication and endoreduplication in vitro.

Description: A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with [35S]-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S.

Description: The effect of differentiation induction by a tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) on a clonal human megakaryoblastic cell line, MEG-O1s, was investigated, and a prominent response was demonstrated. The cells became weakly adherent, developed conspicuous cytoplasmic blebs, and displayed mature megakaryocytic characteristics by light microscopy such as the development of azurophilic cytoplasmic granules and a mosaic pattern of oxyphilic patches, multiplication of nuclei, and enhancement of the PAS reaction and alpha-naphthyl acetate esterase staining. Ultrastructural studies demonstrated the development of prominent cytoplasmic blebs, budding of blebs, and multiplication of nuclei. Numerous granules with central nucleoids that are similar to alpha granules had developed as well as granules with high electron density and clearly demarcated zones. Surface marker analysis revealed a moderate increase in IgG Fc receptor levels and a profound decrease in C3 receptor sites. By an immunofluorescent technique using monoclonal and polyclonal antibodies, a dramatic change in the expression of several megakaryocyte-platelet- specific proteins was demonstrated. All the proteins that had been expressed before induction such as platelet glycoprotein (GP) IIb/IIIa, fibrinogen, von Willebrand factor, factor XIIIA, beta-thromboglobulin (beta-TG), and HLA class 1 antigen were profoundly enhanced after induction by TPA. Induction by TPA led to the expression of fibronectin and factor V, which were not detected on nontreated cells. An ultrastructural immunoperoxidase study demonstrated platelet GPIb and GPIIb/IIIa in both plasma membranes and protein synthesis areas such as perinuclear cisternae and endoplasmic reticulum after TPA induction. beta-TG was also observed in some cytoplasmic granules of TPA-treated cells. TPA remarkably increased the secretion of beta-TG into the culture medium of MEG-01s. Ploidy was also increased from 2C to 4C to 4C to 8C. Similar maturation of MEG-01s was induced by other phorbol diester analogues such as phorbol-12,13-dibutyrate, but not by phorbol itself. These results indicate that phorbol diester, TPA, can bring about differentiation and maturation of a human megakaryoblastic cell line (MEG-01s) and that MEG-01s cells will provide a useful model for studying megakaryocytic differentiation and numerous megakaryocyte- platelet-specific proteins.

Description: Glanzmann thrombasthenia is an autosomal recessive disorder of the platelet glycoproteins (GP) IIb and IIIa. These glycoproteins normally serve as receptors for other adhesive glycoproteins, including fibrinogen, von Willebrand factor, and fibronectin. Most patients affected by Glanzmann thrombasthenia have low levels of GPIIb and GPIIIa; however, the separate mechanisms responsible for the deficiency in each remain to be determined. cDNA clones coding for the GPIIb and GPIIIa have been recently isolated, and their corresponding genomic sequences have been colocalized to the long arm of chromosome 17. Since a deletional event involving one or both of these structural genes could explain the disease phenotype, we have studied the DNA of two previously well-characterized cohorts of Glanzmann thrombasthenia patients from Israel. We performed Southern analysis with near full- length cDNA probes on genomic DNA obtained from 20 individuals. Four restriction enzyme digests were completed on each DNA sample. The similarity of banding patterns among probands, family members, and controls indicated that there were no major insertions or deletions in either the GPIIb or GPIIIa genes. Thus, the genetic defect in these patients with Glanzmann thrombasthenia is most likely due to either a small change in the nucleotide sequence of the coding region or a defect in the regulatory region of one or both genes.

Description: The pathway followed by secretory products stored in platelet alpha granules during the release reaction remains controversial. Tannic acid has been used in the present study as an electron-dense stain to follow the secretory process in thrombin-stimulated platelets. Preliminary experiments demonstrated that tannic acid precipitates fibrinogen, and binds osmium tetroxide to fibrinogen and fibrin strands. Examination of platelets fixed at short intervals after exposure to thrombin and incubated in solutions containing tannic acid revealed electron-dense deposits of osmium not apparent in resting platelets. Granules and lumina of channels making up the open canalicular system (OCS) were unstained in discoid cells. However, exposure to thrombin at concentrations of 1 to 5 U/mL for thirty seconds or more resulted in intense staining of alpha granules by osmium. Some granules communicated directly with dilated channels of the OCS, and several were frequently connected to the same canaliculus. The electron-dense substance in swollen granules and channels appeared to be in the process of extrusion through narrow or dilated openings of the OCS onto the platelet surface. Granule-to-granule fusion and formation of sealed vacuoles of fused granule products unstained by tannic acid-osmium were not observed. The findings support the concept that secretion by stimulated human platelets results from development of direct communications between granules and channels of the OCS and subsequent extrusion of products through channel pores to the surrounding medium.

Description: Cytogenetic, immunologic, and electron microscopic studies were performed on the blast cells of 28 pediatric patients with Down's syndrome, 13 with acute leukemia (DS-AL) and 15 with transient myeloproliferative disorders (DS-TMD). Clonal chromosome abnormalities were found in the cells of all patients with DS-AL but not those with DS-TMD. The younger ages and higher hemoglobin concentrations, platelet counts, and WBC counts of DS-TMD patients provided a clinical contrast with the frankly leukemic cases. Myelodysplastic syndrome, characterized by a small percentage of leukemic blast cells, was observed in 11 of the 13 patients with DS-AL compared with none in the DS-TMD group. Electron microscopy disclosed a positive platelet peroxidase reaction in each of the 11 DS-TMD patients and in nine of the 13 DS-AL patients. Immunologic studies revealed antiplatelet- megakaryocyte antigens on the blast cells of the majority of patients in both study groups. Our findings suggest that the blast cells in cases of DS-AL and DS-TMD arise from cells of the megakaryocytic lineage or from a myeloid progenitor with the capacity for megakaryocytic differentiation. The high risk of the development of AL in patients with DS who are less than 3 years old may be related to increased megakaryocyte proliferation in this age group.

Description: The ability of small acetylcholinesterase-positive (SACHE) cells to incorporate tritiated thymidine (3H-TdR) was studied in the bone marrow of mice made acutely thrombocytopenic by injection of guinea pig antimouse platelet serum (APS) and in control mice injected with normal guinea pig serum (NS). 3H-TdR was administered in vivo, and bone marrow was collected at various time points thereafter. The initial labeling index (LI) for both groups was about 30%. After four hours, the LI increased and reached peak values at 48 hours, but was lower in thrombocytopenic animals. The lower peak LI in APS-treated animals may be the result of both a faster rate of influx of unlabeled cells into the acetylcholinesterase (AChE) positive cell compartment and efflux of more heavily labeled, and probably polyploid, SACHE cells into the compartment of recognizable megakaryocytes. In both groups the increasing LI occurred concomitantly with a decreasing mean grain count. This may indicate cellular division by some fraction of SACHE cells. Heterogeneity in nuclear morphology was also demonstrated, and the frequency of individual morphologies was altered in APS-treated animals. In addition, cells that incorporated 3H-TdR were larger than those that did not. This group of small cells appears to represent a pivotal point in megakaryocytopoiesis in which the switch from mitosis to endomitosis is occurring.