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: 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: 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: Dogs given 1200 r of total body irradiation were cross-circulated with dogs having normal marrow function. Irradiated controls died in from 4 to 11 days with marrow aplasia. Dogs cross-circulated daily for 6 to 9 days showed histologic evidence of bone marrow repopulation after 1 week. Male dogs cross-circulated with female partners showed typical female drumsticks on mature granulocytes after repopulation had occurred. Cytogenetic studies of an irradiated male dog cross-circulated with a female partner showed all mitotable cells from the bone marrow and peripheral blood to be of female donor type. Allogeneic bone marrow engraftment was associated with an early and severe secondary syndrome which resulted in the death of the animals in the second week. When methotrexate was given, survival was increased to 3 weeks. It was concluded that (1) cross circulation provided leukocytes and platelets adequate for support during the period of radiation-induced marrow aplasia, (2) allogeneic marrow engraftment was produced consistently by cells transferred in the peripheral blood of the normal cross circulation partner, (3) the grafts were associated with an early and severe form of secondary disease, and (4) methotrexate given during the early period of engraftment reduced the severity of the secondary disease.

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: 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: 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: The enzymes of the "salvage" pathway for thymidine in normal and leukemic leukocytes separated on glass-bead-columns were studied. Levels of thymidine kinase activity in blast cells were high, but were low in granulocytes, normal and leukemic lymphocytes, and in RBC. Phosphatase and thymidine phosphorylase activity was high in granulocytes and in normal and leukemic lymphocytes, but became apparent in blast cells only in the absence of ATP. All the cells studied could phosphorylate TMP to some extent, but the amount of TTP formed depended greatly on the presence of an optimum ATP concentration. Without added ATP breakdown to thymine was at its peak, while phosphorylation of TMP was negligible. Excess ATP reduced or inhibited the activity of most of the enzymes, preventing both phosphorylation and breakdown. Concentrations of Mg equimolar with the ATP gave maximum TTP production, while inhibition by high ATP concentrations occurred despite equimolar Mg concentrations. Both TTP and thymine production ceased in the absence of Mg. Changes in ATP levels in cell loci may function in regulating thymidine metabolism.

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: 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: Leukokinetic studies were performed using granulocytes labeled in vitro with radioactive diisopropylfluorophosphate (DFP32). The half-time of the granulocytes in the circulation, blood granulocyte mass and granulocyte turnover rates were determined. In control subjects the mean half-life was 6.44 hours with a range of 5.1 to 7.7 hours. The mean blood granulocyte mass was 38 x 109 cells with a range of 19.9 to 36.4 x 109 cells and the granulocyte turnover rate was 4.08 x 109 cells per hour with a range of 2.51 to 5.50 x 109 cells per hour. There was a direct relationship between the half-life and the blood granulocyte mass in the control subjects. In 6 subjects with infection the blood granulocyte mass was uniformly increased. The mean half-life and mean granulocyte turnover rate were both increased above the normal range. In 11 subjects with carcinoma several different leukokinetic patterns were found. The blood granulocyte mass was raised in 5 patients, but in only one of these was the granulocyte turnover rate increased above the normal range. In 6 subjects the blood granulocyte mass was within the normal range and deviations from the mean control value were accompanied by proportionate changes in the granulocyte turnover rate in all but 1 patient. No relation was found between the half-life and the blood granulocyte mass in subjects with infection and/or carcinoma. The possibility that this was due to the establishment of a new steady state of blood granulocyte mass at altered levels of granulocyte production, or that steady state conditions did not exist has been considered. However the data are interpreted no evidence for suppressed granulopoiesis was found in subjects with advanced malignant disease.

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: 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: Three hundred and three patients with polycythemia vera have been treated with 32P more than five years ago. Seven patients have been excluded because they have been treated by 32P elsewhere before their first visit. Of the 296 other patients, 269 have been followed up regularly for more than 5 years. The median survival after hematologic diagnosis is 14.5 years. It is shorter in older patients and in those patients who had the shortest remissions after the initial 32P treatment. Of 145 patients who had died, death was due in 53 cases to hematologic complications (29 acute leukemia, 4 chronic leukemia, 16 myeloid metaplasia or myelofibrosis, 4 aplastic anemia). The latter complications were seen most commonly in those patients who had received the highest total dose of 32P, those with a high initial white count and those in whom the spleen was palpable at least 2 cm. beneath the costal margin. The influence of the quantity of 32P given is very significant for equivalent clinical situations, the influence of white cell count and spleen size is significant for equal doses of 32P. This suggests that these two types of factors favor the early occurrence of hematologic complications.

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: 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: 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: 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: Pregnancy in mice was accompanied by increased erythropoiesis which was most marked in the spleen and was reflected peripherally by increased percentages of circulating erythrocytes. During the last four or five days of pregnancy, there was an equally significant drop in erythropoiesis to normal or below normal values and this occurred despite a falling hematocrit. It is suggested that the changing oxygen requirement of the fetuses may be the primary determinant of these erythropoietic changes.

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.

Description: The distribution and transport in platelets of human coagulation Factor V was investigated by immunofluorescent and immunoelectron microscopy. In resting intact platelets, little surface staining was observed by immunofluorescence. In permeable resting cells, punctate staining similar to that reported for fibrinogen (Fbg), thrombospondin (TSP), fibronectin (Fn), von Willebrand factor (VWF), B-thromboglobulin (BTG), and platelet Factor 4 (PF4) was observed. Double label immunofluorescent staining for Fbg and Factor V demonstrated colocalization, suggesting their presence in the same intracellular structure. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins which exactly colocalized. Thus, at the light level, Factor V and Fbg are localized in the same structure in resting and thrombin-stimulated cells. On the ultrastructural level, an alpha granule localization for Fbg has previously been established. We have extended our immunofluorescent observations regarding the localization of Factor V in human platelets by use of transmission electron microscopy of antibody-stained ultrathin frozen sections. In resting cells, staining of virtually all alpha granules was observed for Factor V. In contrast, consistent staining was absent from other organelles including plasma membranes, mitochondria, and vacuolar structures which may represent the open canalicular system. These data thus establish at the ultrastructural level an alpha granule localization of human coagulation Factor V.

Description: The intracellular flow of tritiated lysine in human eosinophilic myelocytes was studied by electron microscope autoradiography so that information could be obtained on the formation of eosinophil granules. Bone marrow particles obtained from a patient with a marked increase in the number of bone marrow eosinophils were incubated in vitro for periods up to 150 minutes. The percentage of cytoplasmic grains over the Golgi complex rose from 11 percent at 5 minutes to 28 percent by 30 minutes and fell to 15 percent at 150 minutes. Grains over cytoplasmic granules steadily rose to 37 percent by 150 minutes. These results are statistically significant and demonstrate that: human eosinophilic myelocytes are able to form cytoplasmic granules under the in vitro conditions employed, and that intracellular amino acids or proteins flow through the Golgi complex before incorporation into granules.

Description: Lymph nodes from each of the four histologic types of Hodgkin's disease were examined for the presence of eosinophils and for eosinophil degranulation by immunofluorescent localization of eosinophil granule major basic protein (MBP). Eosinophil degranulation shown by MBP deposition outside of eosinophils was found in six of eight nodes from patients with nodular sclerosing disease and in two of eight nodes from patients with lymphocyte depletion-type disease. Three nodes of the mixed cellularity type, one node of the lymphocyte predominance type, and one lymph node of the lymphocyte depletion type showed one or two small foci of extracellular MBP deposition. Lymph nodes from patients without Hodgkin's disease showed no extracellular deposition of MBP. Large numbers of eosinophils were found in seven of eight nodes of the nodular sclerosing variant, but were less frequently seen among the other types of Hodgkin's disease. The presence of extracellular MBP in lymph nodes of patients with Hodgkin's disease indicates that eosinophil degranulation commonly occurs and suggests that the released eosinophil granule proteins may participate in the inflammatory reaction in this disorder more extensively than is presently appreciated.

Description: Surface phenotypic characterization of megakaryoblasts, identified by platelet peroxidase activity, was investigated in four patients who showed increased proliferation of megakaryoblasts: one patient with typical features of acute leukemia, one presenting with acute myelofibrosis, and two with Down's syndrome in whom blasts disappeared spontaneously (transient abnormal myelopoiesis, TAM). MY10 and/or MY9 antigens were expressed on the surface of some megakaryoblasts, but MY7, and MY4, antigens specific to granulocytic or monocytic cells, were not. Some megakaryoblasts were positive for only anti-HLA-DR antibodies. It was speculated that, during the differentiation of the megakaryocytic lineage, MY9 antigen appears transiently on the surface of megakaryoblasts that have lost HLA-DR antigens and have gained the glycoprotein IIb/IIIa antigen. This study also demonstrated that the proliferating blasts in some patients with TAM were mainly megakaryoblasts and suggested that the target cells in TAM are CFU-GEMM.

Description: Reported findings of elevated total calcium (Ca) contents in erythrocytes (RBCs) from patients with beta-thalassemia intermedia (beta-TI) prompted the question of whether the state and transport of Ca in these RBCs are similar to those in sickle cell anemia (SS) RBCs where the increased Ca is compartmentalized in endocytic inside-out vesicles and extracted by exposure of the cells to the Ca ionophore A23187 and a Ca chelator (ethylene glycol tetraacetic acid) and the levels of cytoplasmic free ionized Ca [( Ca2+]i) are normal. We confirmed a high total Ca content of 51 +/- 13 mumol/L RBCs in splenectomized (SPX) beta-TI and 24 +/- 1 mumol/L RBCs in non-SPX beta- TI. Unlike SS RBCs, however, most of the increased Ca was in the lighter, presumably younger beta-TI RBCs, and about half the Ca was not ionophore mobilizable but apparently firmly bound, possibly to remnants of organelles in nucleated and other young RBCs. In the denser RBCs from non-SPX beta-TI, total and extractable Ca amounts were normal. beta-TI RBCs loaded with the Ca chelator Benz 2 showed an initial influx of 45Ca in the normal range, which indicated normal Ca permeability, and near-steady-state levels of [Ca2+]i that were normal (22 +/- 7 nmol/L RBCs in non-SPX beta-TI) or minimally increased (40 +/- 19 nmol/L RBCs in SPX beta-TI). Serial-section electron microscopy of beta-TI ghosts from the denser cell fractions showed more fully enclosed vesicles in non-SPX ghosts than were seen in normal ghosts and many large vesicles and structured, electron-dense material in SPX ghosts. A delayed extrusion of ionophore-preloaded 45Ca only by the SPX beta-TI RBCs together with normal [Ca2+]i suggested compartmentalization of the loaded Ca in these RBCs, perhaps in endocytic inside-out vesicles, and normal Ca pumps. Since beta-TI RBCs show essentially normal levels of [Ca2+]i and normal Ca influx, their high total Ca content should not be associated with any of the deleterious effects observed in vitro with increased levels of [Ca2+]i.

Description: Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen. We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa. Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies. On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media. Populations of approximately 100,000 cells with greater than or equal to 95% viability and containing 70% to 90% megakaryocytes were obtained reliably for study. After the radioactive pulse, the cell suspension was solubilized with nonionic detergent. To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose. The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1–1D-Sepharose) or on a control monoclonal antibody resin. Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. Autoradiograms of diethylamine eluates from HP1–1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions. Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1–1D-Sepharose. Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material. This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.

Description: A BamHI polymorphism has been identified in the human factor IX gene. This polymorphism, which occurs in approximately 6% of X chromosomes, has been used to determine the carrier status of a female in a family with a history of hemophilia B. This family was uninformative for the previously reported TaqI and Xmnl polymorphisms in the factor IX gene.

Description: In this study we provide a characterization of the fibronectin (FN) binding to endothelial cells (EC), and we identify the FN binding site on these cells. 125I-FN binding to EC in suspension was time dependent and reached a plateau at 4 h. Cold FN inhibited this interaction in a concentration-dependent way, but vitronectin, fibrinogen, and IgG were poorly effective. About 80% of the total FN associated to EC at the equilibrium was specifically bound; of this, 60% was reversibly bound, while 20% appeared to be internalized. The FN binding was saturable and an apparent dissociation constant of about 0.23 x 10(-6) mol/L and a maximal number of binding sites of about 9.8 x 10(5) was estimated from binding isotherms. Autoradiography data showed that EC-associated 125I- FN was all in high mol wt form that did not enter the gel. We then characterize the FN receptor (FNR) in EC. An antiserum to the FNR isolated from human placenta inhibited FN binding to EC by 89%, and using the immunoblotting technique, it recognized two bands in the EC detergent extract of mol wt 125/160 Kd. This antiserum also recognized the EC membrane protein complex eluted from the FN affinity column by an arg-gly-asp (RGD) peptide. When this complex was included into liposomes, it poorly bound to FN. However, the binding was strikingly increased by addition of Mn in the buffer and was specific for FN in respect to other substrata. These data define the FN binding site in EC and indicate that it is functionally and structurally related to that isolated from human placenta.

Description: It has been previously shown that fibrinogen (FG) associates specifically with human umbilical vein and bovine aortic endothelial cells (EC) in culture and induces EC migration. In the present study, we have investigated whether the FG-EC interaction is mediated by an Arg-Gly-Asp (RGD) recognition specificity and whether EC membrane proteins related to platelet GPIIb-IIIa are involved. Highly purified radioiodinated human FG, containing no detectable fibronectin, interacted with cultured human and bovine EC in suspension in a time- dependent and specific manner. The binding was inhibited by EDTA. Two polyclonal antibodies to platelet GPIIb-IIIa, which immunoprecipitated a heterodimer molecule from EC, inhibited FG binding to EC. These same antibodies inhibited FG-induced EC migration in a dose-dependent manner as measured in a Boyden chamber. Preabsorption of the antibodies with purified platelet GPIIb-IIIa markedly reduced both inhibitory activities. A series of synthetic RGD-containing peptides inhibited FG binding to EC and FG-induced EC migration. Gly-Arg-Gly-Asp (GRGD) was the most active peptide tested in inhibiting FG binding and EC migration (ID50 of 30 microM), and conservative substitutions in the RGD sequence markedly reduced inhibitory activity (ID50 greater than 1,000 microM). These results indicate that FG binding and EC migration are events mediated by an RGD recognition specificity and that EC surface proteins immunologically related to the GPIIb-IIIa complex on platelets are involved in this recognition.

Description: Certain types of chromosomal abnormalities have been shown to exert strong independent influence on treatment outcome in acute lymphoblastic leukemia (ALL). To identify the changes most closely associated with prognosis, we analyzed the completely banded blast cell karyotypes of 161 children with this disease. One hundred twenty-five cases had one or more chromosomal abnormalities, with 45 showing translocations. The frequency of translocations was highest (58%) among patients with pseudodiploid karyotypes and lowest (0%) in the hyperdiploid group defined by 51 or more chromosomes. During the maximum 6-year follow-up period, 30 of the 45 patients with a translocation failed therapy, compared with only 27 of the 116 who lacked this feature. Life-table estimates of event-free survival indicate that only 14% of the translocation group will be in complete remission at 3 years. The percentages of failures associated with random and nonrandom translocations were virtually identical (68% v 65%). When entered in a Cox proportional hazards model with seven other types of chromosomal abnormalities, and then with 11 clinical and laboratory variables of known prognostic value in ALL, translocation emerged as the strongest single predictor of treatment outcome (P less than 0.0001). The model indicated that translocation increases the risk of treatment failure six times by comparison with the absence of this feature. These findings offer an explanation for the majority of early treatment failures in childhood ALL, including those previously attributed to ploidy classification.

Description: We investigated the activation of the nonenzymatic protein cofactors factor VIII and factor V in plasma when coagulation was initiated by thromboplastin. With sensitive bioassays, we were able to measure specifically the generation of activated factor VIII and activated factor V in plasma. Our results showed that when plasma was triggered with a relatively high concentration of thromboplastin, factor VIII and factor V were completely activated at the clotting time of plasma. However, when the generation of thrombin, but not that of factor Xa, was delayed by addition of hirudin to the plasma, factor Va was generated only at the time thrombin generation overcame the hirudin inhibition. In addition, generation of factor VIIIa correlated with thrombin generation and not with factor Xa generation. Furthermore, addition of large amounts of factor Xa to hirudinized plasma did not show detectable factor VIII or factor V activation. We concluded that in plasma activated with thromboplastin the enzyme responsible for activation of factor V and factor VIII is thrombin, not factor Xa.

Description: Leukemic cell chromosomal findings in 27 infants were analyzed. Among the 18 cases of acute nonlymphoblastic leukemia (ANLL), all but two were classified as monocytic or myelomonocytic. The remaining nine cases were acute lymphoblastic leukemia (ALL), seven lacking the common ALL antigen and two having cytoplasmic immunoglobulin (pre-B phenotype). Twenty-five cases (93%) had an abnormal karyotype, 21 (84%) being pseudodiploid. Chromosomal translocations were detected in 67% of the ANLL cases and in 78% of the ALL cases. Nonrandom chromosomal abnormalities included the t(9;11)(p21–22;q23) in three cases of monocytic leukemia, inversion of chromosome 16 in three cases of myelomonocytic leukemia with bone marrow eosinophilia, and t(4;11)(q21;q23) in one case of ALL. Chromosomal regions preferentially involved in infant leukemia included 11q23–25 (13 cases), 9p21–22 and 10p11–13. All but one of the 24 cases with chromosomal breakage or rearrangement had breakpoints that corresponded to known fragile sites, half of which were at 11q23–25, a finding that may have pathogenetic importance. The CALLA- or pre-B phenotype and the presence of chromosomal translocations in most infants with ALL provide a biological explanation for their poor prognosis.

Description: Human multilineage colony-stimulating factor (multi-CSF)/interleukin-3 (IL-3) induces colony formation from CFU-GEMM, BFU-E, and CFU-Eo when applied to in vitro cultures of highly enriched hematopoietic progenitor cells. No granulocytic colonies are formed in response to IL- 3. However, with appropriate assays, we demonstrate that IL-3 increases the size of G-CSF-induced granulocytic colonies; these colonies contain greater proportions of immature cells as compared with colonies stimulated by G-CSF alone. Furthermore, IL-3 promotes the survival of CFU-G in vitro, whereas in cultures not supplemented with IL-3, CFU-G extinguish within seven days. We conclude that IL-3, although it does not stimulate granulocytic colony formation by itself, regulates the survival and proliferative rate of granulocytic progenitors.

Description: Several previous studies suggested that murine macrophage colony- stimulating factor (CSF-1) might have impaired access to hematopoietic cells in the marrow. The apparent lack of hematopoietic responses to exogenous CSF and the finding of available or unoccupied CSF receptors despite saturating CSF levels in the serum led to studies of a potential blood-bone marrow barrier for this factor. Groups of mice were injected with pure unlabeled CSF-1 by either intravenous (IV) or intraperitoneal (IP) routes. Marrow and spleen cells were obtained at intervals after injection, held at 0 degree C, and assessed for changes in binding of 125I-CSF. Saturation of all available CSF receptors is achieved in vitro with 100 to 150 U CSF/mL. Despite achieving serum levels of 5,000 to 7,000 U/mL after IV injection of 25,000 units of CSF, less than 50% of the marrow receptors and less than 85% of the splenic receptors were saturated or downregulated. The decline in receptor availability was transient, with return of receptor sites in two to four hours. Increasing the IV dose to 125,000 units increased serum CSF values to approximately 20,000 U/mL and led to a virtual disappearance of available receptors for two to three hours. When administered IP, only approximately 40% of marrow and 80% of splenic receptors were affected for two hours. It was necessary to increase the dose of CSF to 250,000 units IP to saturate or downregulate receptors for three to four hours after injection. These observations indicate a marked blood-bone marrow barrier and lesser blood-spleen barrier for the transfer of serum CSF to responsive hematopoietic cells in vivo.

Description: Hereditary elliptocytosis is a heterogeneous disorder resulting from defects in the erythrocyte membrane skeleton. Although some cases of elliptocytosis result from defects in spectrin, the specific structural abnormality has yet to be identified in the majority of cases. Protein 4.1 plays an essential role in erythrocyte membrane physiology, and deficiencies have been implicated in only a few rare cases of elliptocytosis. By using 4.1 immunoblots and a 4.1 radioimmunoassay we identified distinct variants of protein 4.1 in 15 elliptocytic members of three US white families with the Rh-linked form of elliptocytosis. Elliptocytic members of family G were heterozygotes for a low-molecular weight (mol wt) 4.1 variant (65,000 to 68,000 daltons; normal, 80,000) inherited in linkage with the Rz phenotype. Elliptocytic members of family C expressed a simple partial deficiency of protein 4.1 (63% of the normal level) that was inherited in linkage with the r phenotype. Elliptocytic members of family N were heterozygotes for a high-mol wt 4.1 variant (100,000 daltons) also inherited in linkage with the r phenotype. These studies indicate that mutant forms of protein 4.1 are not uncommon in elliptocytosis among whites and that different kindreds probably express different mutations. The observed linkage of elliptocytosis and Rh blood type most likely results from the close proximities of the 4.1 gene (site of the mutation) and the Rh gene, which is located nearby on the short arm of chromosome 1.

Description: In a previous report (Blood 60:265, 1982), we described a family with an abnormal RBC membrane protein doublet, which we considered a shortened protein 4.1 on the basis of biochemical and genetic data. Using an anti-4.1 monoclonal antibody, we confirm here that the shortened protein derives from protein 4.1. One of the members of the family contemporaneously displayed the 4.1 (-) trait, eg, the heterozygous state of this variety of hereditary elliptocytosis that lacks protein 4.1. The 4.1a/4.1b ratio was low whenever the 4.1 trait was present, regardless of the type of protein 4.1 involved. The RBCs of the compound heterozygote, containing only the shortened species of protein 4.1, made it possible to analyze without interference the contact between shortened protein 4.1 and sialoglycoprotein beta, or glycoconnectin. Shortened protein 4.1 did not alter the amount of glycoconnectin in the ghosts nor did it change its extractability into the Triton shells. Limited proteolysis of shortened polypeptides 4.1a and 4.1b showed that they are sequence related. It is conflicting that the persons carrying the shortened protein 4.1 are devoid of specific clinical and morphological abnormalities, apart from those pertaining to the 4.1- trait, when the latter is present.

Description: The rare McLeod blood group phenotype is characterized by weak Kell antigens, lack of the common Kx antigen, and acanthocytic morphology. Previous studies that did not detect membrane or cytoskeletal protein abnormalities suggested a lipid disturbance. In normal red cells, dimyristoyl phosphatidylserine (DMPS) is transported across the membrane by an enzymatic process and accumulates in the inner leaflet of the membrane bilayer causing discocyte to stomatocyte shape changes. Scanning electron microscopy of McLeod red cells shows a mixture comprised of 15% discocytes, 51% with irregular surfaces, and 34% acanthocytes. On incubation with various concentrations of DMPS at 37 degrees C for periods up to two hours, McLeod red cells transported DMPS across the membrane and caused irregularly shaped and acanthocytic McLeod red cells to attain normal discocyte shape and later to become stomatocytes. Chlorpromazine, which at 0 degrees C preferentially partitions into the inner monolayer of the membrane, had a similar effect on the shape of McLeod red cells. This suggests that in McLeod cells acanthocytosis is due to a lack of lipid in the inner leaflet of the membrane bilayer but that the imbalance is not caused by defective transport of phosphatidylserine across the membrane.

Description: We have previously shown that the In(Lu) gene down-regulates expression of an erythrocyte protein antigen identified by murine monoclonal antibody (MoAb) A3D8. In the present study we have examined In(Lu) Lu(a- b-) erythrocytes for expression of additional epitopes on the erythrocyte 80 kilodalton protein (p80) bearing the A3D8 antigen. Using a total of seven additional MoAbs that recognize three epitopes on erythrocyte p80, we have shown that In(Lu) Lu(a-b-) erythrocytes exhibit down-regulation of expression of all three epitopes. In(Lu) erythrocytes also showed a reduction in their reactivity to rabbit antibodies produced against purified p80 from either erythrocytes or lymphocytes. Furthermore the reactivity of the MoAbs was not altered by treatment of the cells with neuraminidase but was substantially reduced by treatment of cells with trypsin or chymotrypsin. The polyclonal anti- p80 sera were shown to react with a fragment of 50,000 daltons, still associated with erythrocyte ghosts, following treatment of the cells with trypsin or chymotrypsin. Treatment of erythrocytes with the thiol- reactive reagent AET decreased their reactivity with the MoAbs but had a variable effect on their reactivity with polyclonal antibodies. Erythrocyte p80 is a glycoprotein with N-linked oligosaccharides, as demonstrated by its binding to concanavalin A (Con A) and Len culinaris lectins. Following Endoglycosidase F treatment, erythrocyte p80 underwent a reduction in apparent mol wt of 11,000. The presence of a reduced amount of an intact p80 glycoprotein, seen by a decrease in reactivity with MoAbs directed at three distinct epitopes and with two different polyclonal antibodies, suggests that the In(Lu) gene interferes with expression by erythrocytes of the entire p80 glycoprotein.

Description: We have explored the polymorphism of the glycophorin system in the human erythrocyte membrane using the immunoblotting techniques and examining 52 individuals selected without prior bias as to their serologic state and ten documented serologic variants of M, N, S, s blood group system. Polyclonal antisera to alpha glycophorin and to alpha glycophorin CNBr carboxyl terminal fragment C (residues 82–131) and M and N specific monoclonal antibodies (MoAbs) were used. The first two reagents detect specific regions of the alpha glycophorin molecule and all electrophoretically resolved species of glycophorins immunologically related to alpha and delta glycophorins (delta glycophorin, [alpha-delta] hybrids and other glycophorins with an alteration in the carboxyl terminal segment); the M and N MoAbs identified the glycophorin species containing or lacking the M or N determinant in the amino terminal octapeptide structures. We find that immunoblotting confirmed in all cases the serologically determined phenotype; we also find that polymorphic forms of the glycophorin system are relatively infrequent; immunoblotting, independent from serologic testing, was capable of detecting five mutants, two most likely S-s-U-phenotypes; a new glycophorin species was detected in normal red cells with both antiglycophorin and antipeptide C sera, which is not evident with MoAbs; immunoblots of known glycophorin variants (En(a-), U-, Mg, Mi I, II, III, V, and Sta) confirmed but also extended our knowledge of the abnormal glycophorins involved; and the He+ and Wrb(-) cells showed normal patterns.

Description: A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29- kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS- PAGE. Amino acid composition indicated that the Rh(D) protein contained sulfhydryl groups which are essential for biological activity.

Description: The behaviors of an anti-Ia antibody (7.2) and an antibody directed at a lymphocyte adhesion molecule (S.5) radiolabeled with 131I were studied in normal dogs. Antibody 7.2 localized to spleen and, to a lesser extent, to marrow and lymph nodes. Antibody S.5 rapidly localized to marrow and spleen, achieving tissue/blood ratios greater than 6:1 within three hours of injection that were maintained for at least 48 hours. Prior treatment with cyclophosphamide (CY) markedly altered the distribution of S.5 but had much less effect on the distribution of 7.2 and almost no effect on the distribution of a control antibody. When animals were treated with increasing doses of 131I labeled to S.5, lethal myelosuppression occurred when a dose of 6 mCi/kg was reached. At this dose, the otherwise lethal effects of 131I could be reversed with autologous marrow transplant support.

Description: Frequencies of 35 HLA A, B, C, and DR antigens were determined in 1,834 leukemic Caucasoids to evaluate possible associations between HLA and leukemia. In comparison with the frequencies of HLA antigens in published controls, the frequency of Cw3 was significantly higher in patients with acute lymphoblastic leukemia (relative risk = 2.64, P less than 0.0002), acute myelogenous leukemia (relative risk = 1.92, P less than 0.0007), and chronic myelogenous leukemia (relative risk = 2.07, P less than 0.002; P values adjusted for multiple comparisons). The frequency of Cw4 was elevated in patients with acute lymphoblastic leukemia (relative risk = 2.01, P less than 0.0003), acute myelogenous leukemia (relative risk = 2.06, P less than 0.0002), and chronic myelogenous leukemia (relative risk = 2.14, P less than 0.0008). The frequency of Aw19 was significantly decreased in patients with acute myelogenous leukemia (relative risk = 0.68, P less than 0.01) and chronic myelogenous leukemia (relative risk = 0.59, P less than 0.005). None of the other 32 HLA antigens investigated had a statistically significant association with leukemia. The data suggest that Cw3 and Cw4 may be markers for leukemia susceptibility genes, while Aw19 may be a marker for decreased susceptibility to leukemia.

Description: Two patients with aplastic anemia were treated with high-dose cyclophosphamide and marrow transplantation from their normal, genetically identical twin. Both patients rapidly recovered normal marrow function, but marrow failure recurred 13 and 18 months later. Because donor and host pairs were identical twins, these cases of graft failure could not have resulted from the usual cause of graft failure, ie, immunological reactivity of host cells against unshared minor histocompatibility antigens of the donor. These results imply that there are at least two mechanisms responsible for graft failure after marrow transplantation for severe aplastic anemia.

Description: The effects of interferon-alpha (IFN alpha) on in vitro proliferation and M-protein secretion in human myeloma cells were investigated. Human myeloma cells were purified from bone marrow aspirates in 12 multiple myeloma patients. Purified myeloma cells were cultured for 48 hours with IFN alpha at its lower concentrations (0.1 to 100 U/mL). The cells were then pulsed with 3H-TdR for the last 12 hours and 3H-TdR uptake was measured (in vitro proliferation). After 48-hour culture, supernatants were harvested and the amount of M-protein in these fluids were measured by enzyme-linked immunosorbent assay (ELISA) (in vitro M- protein secretion). In vitro M-protein secretions of myeloma cells were significantly suppressed even at 0.1 U/mL of IFN alpha, while 3H-TdR uptakes were not so suppressed until 10 or 100 U/mL of IFN alpha were added. The expressions of secretory immunoglobulin (Ig) mRNA of these myeloma cells were also selectively suppressed by IFN alpha. Furthermore, after IFN alpha had been administered intramuscularly, 3 to 6 x 10(6) U/d for at least 1 month, in vitro M-protein secretions of these myeloma cells were decreased compared with those before IFN alpha administration. Therefore, these results suggest that IFN alpha has more sensitive inhibitory effect on M-protein secretion of human myeloma cells rather than on myeloma cell proliferation.