bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of folate analogues and vitamin B12 on provision of thymine nucleotides for DNA synthesis in megaloblastic anemia
Authors: Taheri MR , Wickremasinghe RG , Jackson BF , Hoffbrand AV
Published: October 2019

The role of vitamin B12 in the folate dependent biosynthesis of thymidine nucleotides is controversial. In an attempt to clarify this, three methods have been used to assess the relative efficacy of vitamin B12 (hydroxocobalamin) and various folate analogues in titrated concentrations at correcting ‘de novo’ thymidylate synthesis by megaloblastic human marrow cells: (1) The deoxyuridine (dU) suppression test which analyses the reduction in (3H)-thymidine labeling of DNA by unlabeled dU. Marrow cells were also labeled with (6–3H)-dU with assessment of (2) its incorporation into DNA and (3) the accumulation of (6–3H)-deoxyuridine monophosphate (3H-dUMP). The three methods gave similar results. In both, N6-formyl tetrahydrofolate (formyl-FH4) was the most effective agent at correcting thymidylate synthesis in megaloblastic anemia due to vitamin B12 or folate deficiency. Vitamin B12 corrected the lesion in vitamin B12 deficiency but not in folate deficiency. Tetrahydrofolate (FH4) and folic acid were effective in deficiency of vitamin B12 or folate, although in both deficiencies they were less effective than formyl-FH4. Methyl-FH4 was effective in folate deficiency but not in vitamin B12 deficiency. These results confirm the failure of methyl-FH4 utilisation in vitamin B12 deficiency. They suggest that if vitamin B12 is needed in the formylation of FH4, this is a minor role in provision of the correct coenzyme for thymidylate synthesis compared with its major role of provision of FH4 from methyl- FH4.
Karyotypically distinct subpopulations in acute leukemia with specific growth requirements
Authors: Lowenberg B , Hagemeijer A , Swart K
Published: October 2019

Bone marrow and blood cells of a patient with acute monoblastic leukemia with subclones marked with specific karyotypic abnormalities were investigated. In order to more precisely evaluate the proliferative abilities of these populations, leukemic cell enriched fractions were prepared and incubated in two colony assays. Colony forming cells of the disparate clones had growth advantages in different systems which shows that their proliferation depended on the presence of selective stimulatory factors in culture. In one assay, at diagnosis, colonies from the minor clone were demonstrated exclusively. It is suggested that the assays measured distinct cellular stages of myeloid differentiation and the findings indicate that prior to diagnosis the neoplasm had evolved into subsets with progressive dedifferentiation. Differences of growth in vitro correlated with the different roles of these clones in the clinical history of the disease. Approaches based on differential cloning of tumor stem cells as in this example, may be useful for discriminating biological properties of heterogeneous subpopulations within neoplasms, and may facilitate the cytogenetic recognition of minimal clones among composite malignant cell specimens.
Erythropoiesis in ha/ha and sph/sph mice, mutants which produce spectrin-deficient erythrocytes
Authors: Brookoff D , Maggio-Price L , Bernstein S , Weiss L
Published: October 2019

In order to characterize chronically accelerated erythropoiesis, we studied the ultrastructure of bone marrow and spleen of ha/ha and sph/sph mice, two mutants with profound hemolytic anemia secondary to deficiency of the erythrocyte membrane protein spectrin. The marrows and spleens of both varieties were extremely erythropoietic and were without histological abnormalities directly related to spectrin deficiency. Erythropoiesis was consistently associated with distinctive, dark branched cells which constituted large proportions of the stroma of the mutant spleens and marrow. These dark cells were not present in untreated and acutely bled controls. Plasma clot assays for erythroid progenitors revealed that CFU-E concentrations in the mutant marrows were significantly increased over those in untreated controls while BFU-E concentrations were approximately half. In addition, mutant CFU-E often gave rise to abnormal appearing colonies. Spectrin, though crucial to erythrocyte function is probably not important to the process of erythroid differentiation and maturation. The status of erythroid precursors in the marrows of the spectrin deficient mice is similar to that of mice subjected to an acute bleed. The divergent changes in CFU-E and BFU-E may indicate that these two cells play different roles in accelerated erythropoiesis. The dark cells that we describe are similar to stromal cells observed in models of the early stages of erythropoiesis.
A monoclonal antibody that detects expression of transferrin receptor in human erythroid precursor cells
Authors: Lebman D , Trucco M , Bottero L , Lange B , Pessano S , Rovera G
Published: October 2019

A monoclonal antibody, L5.1, obtained by immunizing a Balb/c mouse with HL60 human promyelocytic leukemia cells, was found to react with both HL60 cells and with the K562(S) cell line. This monoclonal antibody binds and immunoprecipitates a glycoprotein (Mr 87,000) present on the cell surface membrane of K562(S) as a disulfide bonded dimer. In competition experiments L5.1 competes with both transferrin and OKT9 (a known antitransferrin receptor antibody) for binding to target K562(S) erythroleukemia cells. Binding of both L5.1 and transferrin to the surface of K562(S) cells is inhibited by treatment with 12--O- tetradecanoyl-phorbol-13-acetate, and the extent and time course of inhibition is similar in both cases. Cell sorting analysis of normal human marrow cells incubated with L5.1 indicates that L5.1 reacts strongly with all the morphologically recognizable erythroid lineage precursors, from the pronormoblast to the orthochromatic normoblast, and with reticulocytes. Erythrocytes, myeloid elements, monocytes, megakaryocytes and platelets, peripheral blood B and T lymphocytes do not bind significantly with this antibody and only a small fraction of promyelocytes was reactive. Antibody L5.1 did not react with leukemic cells of patients with acute lymphoblastic, myeloblastic and promyelocytic leukemias, but it did react with some established B (1 of 5) and T (2 of 3) cell lines, and a myeloid (1 of 3) cell line, and with PHA-stimulated peripheral blood lymphocytes. The nonhemopoietic cell lines tested did not bind with L5.1 with the exception of a colorectal adenocarcinoma and a melanoma cell line, which were both strongly positive. The relationship of antibody L5.1 to other monoclonal antibodies that bind the transferrin receptor is discussed.
Specific protein and glycoprotein deficiencies in platelets isolated from two patients with the gray platelet syndrome
Authors: Nurden AT , Kunicki TJ , Dupuis D , Soria C , Caen JP
Published: October 2019

The gray platelet syndrome is a rare inherited platelet disorder characterized by the absence of alp ha-granules as observed by electron microscopy. Analysis of the glycoprotein composition of the platelets of 2 such patients by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed decreased or absent staining for carbohydrate of several high molecular weight glycoproteins. The major periodic acid Schiff (PAS) staining membrane glycoproteins were normally detected and were normally labeled with 125I during lactoperoxidase-catalyzed iodination. Analysis of the protein composition of gray platelets by single or two- dimensional SDS-PAGE followed by Coomassie blue staining revealed an apparent absence of GP Ig (thrombospondin), markedly reduced platelet fibrinogen and albumin concentrations, and severely reduced levels of 2 low molecular weight polypeptides exhibiting identical rates of migration on SDS-PAGE as platelet factor 4 and beta-thromboglobulin. SDS-PAGE profiles similar to those of the gray platelets were observed with normal human platelets that had undergone the release reaction induced by thrombin. Analysis of gray platelet proteins by crossed immunoelectrophoresis using a rabbit anti-human platelet antibody preparation and rocket immunoelectrophoresis using monospecific antisera confirmed the above findings and showed additional severe deficiencies of factor VIIIR:Ag and cold-insoluble globulin. In contrast, factor XIII (subunit A), a cytoplasmic protein, was normally detected. Our studies provide further evidence that circulating gray platelets specifically lack, or have markedly decreased concentrations of, the alpha-granule proteins.
Differing responses of globin and glycophorin gene expression to hemin in the human leukemia cell line K562
Authors: Tonkonow BL , Hoffman R , Burger D , Elder JT , Mazur EM , Murnane MJ , Benz EJ Jr
Published: October 2019

The human leukemia cell line, K562, produces embryonic and fetal hemoglobins and glycophorin A, proteins normally associated only with erythroid cells. Hemoglobin accumulation is enhanced by exposure of the cells to 0.05 mM hemin. We have examined K562 cells before and after exposure to hemin to determine whether expression of these erythroid proteins was shared by all cells or confined to specific subpopulations. Globin gene expression was examined by quantitation of globin mRNA sequences, using a 3H-globin cDNA molecular hybridization probe. Constitutive cells produced globin mRNA, the content of which was increased 3–4-fold by hemin. Cell-to-cell distribution of globin mRNA was determined by in situ hybridization of 3H-globin cDNA to constitutive and hemin-treated K562 cells. Virtually all cells in the culture exhibited grain counts above background, indicating globin gene expression by all cells, rather than a confined subpopulation. Virtually all hemin-treated cells had 3–5-fold higher grain counts, indicating uniformly increased globin gene expression. The glycophorin content of K562 cells was estimated by fluorescence-activated cell sorting (FACS) of cells labeled with fluorescein-labeled antiglycophorin antiserum. The vast majority of constitutive cells contained glycophorin, but exhibited to apparent increase in glycophorin accumulation after hemin exposure. Thus, glycophorin and globin genes exhibited differential responses to hemin. These differences could reflect normal differences in the patterns of specialized gene expression in stem cells. Alternatively, different aberrations of gene expression could be occurring in response to the determinants of the neoplastic properties of K562.
Collection of pluripotential hematopoietic stem cells by cytapheresis
Authors: Lasky LC , Ash RC , Kersey JH , Zanjani ED , McCullough J
Published: October 2019

Successful complete hematopoietic reconstitution (CHR) using nonleukemic peripheral stem cells (PSC) after marrow ablation has been reported in animals but not man. Previous studies of cytapheresis products from humans, as a prelude to use for CHR, have documented the presence of committed myeloid (CFU-GM) and erythroid (BFU-E) precursors. We have examined mononuclear cell (MNC) products collected on the Fenwal CS3000 Blood Cell Separator for these plus the more primitive mixed (granulo-, erythro-, mono-, and megakaryocytic) cell colony-forming units (CFU-GEMM) and for various lymphocytic subpopulations (LSP). One to two-hour products contained 36 +/- 7 CFU- GEMM/10(6) MNC (mean +/- SE, n = 8) or 490 +/- 131/ml product. This compared favorably with blood (23 +/- .4/10(6) MNC or 46 +/- 8/ml, n = 14) and bone marrow (146 +/- 58/10(6) MNC, n = 12). Collection efficiency for E-rosette-positive cells approximated that for total lymphocytes and was variable for other LSP. Recovery of CFU-GEMM after freezing in 10% dimethylsulfoxide at a controlled rate and storage in liquid N2 was 54% +/- 8% (n = 8). Cytapheresis collection of large numbers of pluripotent hematopoietic precursors and demonstration of adequate recovery of these after cryopreservation, both previously unreported, are significant steps toward eventual CHR using nonleukemic PSC.
Two classes of murine granulocyte progenitor cells expressed in plasma clot diffusion chamber cultures
Authors: Steinberg HN , Page PL , Robinson SH
Published: October 2019

Two distinct classes of granulocyte progenitor cells present in normal mouse bone marrow are expressed sequentially in the vivo plasma clot diffusion chamber culture system. By several criteria, progenitor cells giving rise to granulocyte colonies on day 4 of culture (CFU-D4) are different from those giving rise to colonies on day 7 (CFU-D7). These differences include: cell cycle activity as measured by in vitro incubation with cytosine arabinoside, residual concentration in the bone marrow after in vivo treatment of donor mice with cytosine arabinoside or methotrexate, resistance to osmotic lysis, size as determined by velocity sedimentation, and the morphology of the granulocyte colonies to which these cells give rise. The CFU-D7 appears to represent an earlier progenitor cell than the CFU-D4 in the differentiation pathway of the granulocyte and is analagous in many respects to the BFU-E in the erythroid pathway.
In vitro regulation of immunoglobulin synthesis after human marrow transplantation. II. Deficient T and non-T lymphocyte function within 3- 4 months of allogeneic, syngeneic, or autologous marrow grafting for hematologic malignancy
Authors: Witherspoon RP , Lum LG , Storb R , Thomas ED
Published: October 2019

Immunoglobulin secretion was studied in 37 patients between 19 and 106 days after allogeneic HLA-identical (30 patients), allogeneic one HLA- haplotype-identical (three patients), syngeneic (three patients), or autologous (one patient) marrow grafting. E rosette-positive (T) and E rosette-negative (non-T) peripheral blood mononuclear cells were cocultured with pokeweed mitogen for 6 days. Polyvalent immunoglobulin secretion was determined by counting plaque forming cells in a reverse hemolytic plaque assay. The number of antibody secreting cells in cocultures of autologous T and non-T lymphocytes was low in 40 of 44 tests conducted on samples from the 37 patients. Mononuclear or non-T cells from 38 of 40 tests failed to produce antibody when cultured with normal helper T cells. T cells from 23 of 37 tests failed to help normal non-T cells secrete antibody. T lymphocytes from 23 of 41 tests suppressed antibody production greater than 80% by normal T and non-T cells. The suppressor cells were radiosensitive in 17 of the 25 tests. The abnormal function of lymphocyte subpopulations in patients during the first 3 mo after syngeneic, allogeneic or autologous marrow grafting was similar regardless of the type of graft or the presence of acute graft versus host disease.
Prognosis of chronic lymphocytic leukemia: a multivariate survival analysis of 150 cases
Authors: Rozman C , Montserrat E , Feliu E , Granena A , Marin P , Nomdedeu B , Vives Corrons JL
Published: October 2019

A multivariate survival analysis by means of Cox's multiple regression model was performed on a series of 150 consecutive patients with chronic lymphocytic leukemia (CLL) from a single institution. In addition to the well established prognostic factors, such as anemia and thrombocytopenia, a marked prognostic value of the degree of absolute peripheral lymphocytosis emerged from this analysis. This was evident in the whole population as well as in low and intermediate risk groups of patients (Rai's stages 0, I, and II and International Workshop on CLL stages A and B), pointing out that different subsets of patients can be isolated within these groups.
Platelet-associated complement C3 in immune thrombocytopenic purpura
Authors: Myers TJ , Kim BK , Steiner M , Baldini MG
Published: October 2019

Platelet-associated C3 (PA-C3) was measured with a quantitative immunofluorescence assay. With this assay, PA-C3 levels were determined for 78 normal volunteers, 30 patients with immune thrombocytopenic purpura (ITP), and 20 patients with nonimmune thrombocytopenias. Platelet-associated IgG (PA-IgG) levels were also measured with our standard quantitative immunofluorescence assay. All patients with nonimmune thrombocytopenias and ITP in remission had normal PA-C3 levels. Twenty-four patients with active ITP were classified into 3 groups: 9 (38%) with increased PA-IgG and normal PA-C3 levels, 10 (42%) with elevated PA-C3 and PA-IgG levels, and 5 (20%) with increased PA-C3 values only. A direct correlation was found between PA-C3 and PA-IgG levels. PA-IgG levels were higher in the group of patients with elevated PA-C3 levels than in those with normal values. Platelet survival studied showed reduced survival times of 1.5--2.5 days for the 5 patients with elevated PA-C3 levels only. Elevated PA-C3 levels returned to normal in 7 ITP patients whose platelet counts increased in response to corticosteroid therapy or to splenectomy. Therefore, PA-C3 and PA-IgG assays can be used to identify patients with ITP, to follow their response to therapy, and to classify them into immunologic subgroups similar to red cell classification by Coombs'testing in immune hemolytic anemia.
Characteristics of bone marrow fibroblast colony-forming cells (CFU-F) and their progeny in patients with myeloproliferative disorders
Authors: Castro-Malaspina H , Gay RE , Jhanwar SC , Hamilton JA , Chiarieri DR , Meyers PA , Gay S , Moore MA
Published: October 2019

Chronic myeloproliferative disorders (MPD) are clonal diseases of the pluripotent hematopoietic stem cell frequently associated with myelofibrosis (MF). There is only indirect evidence indicating that the increased deposition of collagen in bone marrow matrix is a secondary phenomenon. A liquid culture system for cloning and growing bone marrow fibroblasts has permitted us to approach more directly the understanding of the pathogenesis of myelofibrosis by comparing the biophysical, growth, and functional characteristics of fibroblasts from normals, MPD patients without MF, and those with MF. In patients with MF, marrow fibroblast colony (CFU-F) formation could not be studied; fibroblasts were grown from marrow explants. CFU-E from normals and MPD patients exhibited similar cell density distribution and similar cell sedimentation rates. These similarities contrasted sharply with the differences seen when the erythroid and granulocyte-macrophage progenitors were studied by the same methods. There was a marked light density shift and a rapidly sedimenting shift of MPD hematopoietic colony-forming cells. Marrow fibroblasts from MPD patients with and without MF displayed the same in vitro growth characteristics as fibroblasts from normals. Both types of fibroblasts exhibited anchorage and serum dependence, and contact inhibition of growth. Marrow fibroblasts were also characterized for the presence and distribution of fibronectin and collagen types by immunofluorescent staining using monospecific antibodies. Extracellular matrix, membrane-, and cytoplasm- associated fibronectin, type I, type III, and type V collagen showed a similar staining pattern in both normal and myelofibrotic marrow fibroblasts. Plasminogen-dependent fibrinolytic activity elicited from normal and myelofibrotic marrow fibroblasts were equivalent. Chromosomal analysis of hematopoietic cells and marrow fibroblasts from Philadelphia chromosome positive chronic myelocytic leukemia patients with and without MF showed that the Philadelphia chromosome was present only in hematopoietic cells. The results of these studies taken together demonstrate that bone marrow collagen-producing cells from MPD patients with and without MF behave in vitro as do those from normals. These findings support the hypothesis that that the marrow fibrosis observed in patients with MPD results from a reactive process rather than from a primary disorder affecting the marrow collagen-producing cells.
Low serum thymic hormone levels in patients with chronic graft-versus- host disease
Authors: Atkinson K , Incefy GS , Storb R , Sullivan KM , Iwata T , Dardenne M , Ochs HD , Good RA , Thomas ED
Published: October 2019

We tested the hypothesis that chronic graft-versus-host disease (GVHD) is due to inadequate thymic function by examining pretransplant serum levels of facteur thymique serique (FTS). Four of five patients with no detectable FTS activity developed chronic GVHD, while one of four with some FTS activity did. Further patient numbers are needed to confirm or reject this hypothesis. We further postulated that chronic GVHD, whatever its cause, involves thymic epithelium as a target organ. When tested 11 mo or more posttransplant, patients with chronic GVHD had lower absolute FTS levels (p less than 0.02) and lower age-corrected levels (p = 0.05) than patients without chronic GHVD. Low values in chronic GVHD were associated with the disease itself and not its therapy. These findings show that thymic epithelial secretory function is impaired in chronic GVHD, and this may in part be responsible for the immunodeficiency characteristic of these patients.
Cyclosporin-A is selectively cytotoxic to human leukemic T cells in vitro
Authors: Totterman TH , Danersund A , Nilsson K , Killander A
Published: October 2019

We have studied the antileukemic effects of cyclosporin-A (CyA) on the cells of a number of human hematopoietic biopsies and established cell lines in vitro. Cya killed the tumor cells but not the nonmalignant control cells from three of four patients with T-lymphocytic leukemia/lymphoma at concentrations comparable to those recommended in clinical long-term immunosuppressive therapy. The leukemic cells from 7 patients with B-type chronic lymphocytic leukemia were insensitive to CyA. The drug was cytostatic and cytolytic to three of five T- lymphoblastic leukemia cell lines tested. With the possible exception of a plasma cell line, all other control cell lines (B-lymphocytic lymphoma, histiocytic lymphoma, fibroblast, and glia cell lines) were resistant to CyA. These observations indicate that CyA or its derivatives may be useful as a highly selective antitumor agent in different T-cell malignancies.
Hereditary high concentration of glutathione in canine erythrocytes associated with high accumulation of glutamate, glutamine, and aspartate
Authors: Maede Y , Kasai N , Taniguchi N
Published: October 2019

We have found a dog family in which there were five cases of increased red cell glutathione corresponding to four to five times the normal concentration without any clinical signs. In the present study, we mainly examined the concentrations of free amino acids in the erythrocytes, plasma, and urine of two of the dogs, and we demonstrated that the concentrations of glutamate, aspartate, and glutamine in their erythrocytes increases to 92, 63, and 13 times the mean value in the normal blood, respectively. There were no changes observed in the other amino acids as compared to normal, although the glycine and histidine in the erythrocyte showed slight increases. The concentrations of amino acids in the plasma and urine of the dogs were almost equal to normal ones. The activities of some of the enzymes involved in the glutathione metabolism in the erythrocytes from the two dogs were all within the normal range. The increased level of glutathione could be explained by the fact that the feedback inhibition of gamma-glutamyl cysteine synthetase by glutathione was released by the high levels of glutamate in the erythrocytes.
Pipobroman therapy of polycythemia vera
Authors: Najman A , Stachowiak J , Parlier Y , Gorin NC , Duhamel G
Published: October 2019

Between 1971 and 1981, 74 patients with polycythemia vera were treated with pipobroman using a high-dose induction, low-dose maintenance regimen. Complete remission was achieved in 51 of 54 previously untreated patients (94.4%) and in 17 of 20 patients (85%) previously treated wih radioactive phosphorus (32 p) and busulfan. The earliest modifications were noted on day 16, and on the average, blood counts were normal by day 45. Thirty percent of the patients relapsed, the mean duration of the remission being 17.5 mo. Following recurrence pipobroman was consistently effective in the same doses but the mean duration of the next remissions was 10 mo. Transient leukopenia and thrombocytopenia occurred in 8% and 7% of patients, respectively, during initial phase, and anemia was noted in 3 patients. Macrocytosis was noted in 20% of patients during maintenance phase. Three cases of acute leukemia and 3 cases of osteomyelosclerosis were recorded, all occurring in patients who had previously received 32 p and/or busulfan. No hematologic malignancies were seen among patients treated with pipobroman alone; follow-up exceeded 6 yr for 20 patients and the median follow-up period was 3.6 yr. Pipobroman appears safer than other alkylating agents; it is as effective as 32 p and works more quickly. Longer follow-up will be required to evaluate the drug's oncogenic potential, which is still not known.
The effect of pharmacologic inhibition of phospholipid methylation on human platelet function
Authors: Shattil SJ , Montgomery JA , Chiang PK
Published: October 2019

Human platelets are capable of synthesizing their major membrane phospholipid, phosphatidylcholine, by a methylation pathway. This involves the sequential transfer of methyl groups from S-adenosyl-L- methionine (AdoMet) to phosphatidylethanolamine, and in the process, AdoMet is converted to S-adenosylhomocysteine (AdoHcy). The activity of this methylation pathway is decreased upon stimulation of platelets by various agonists. We inhibited methylation reactions pharmacologically to see whether this inhibition plays any role in the process of platelet activation. Two inhibitors of AdoHcy hydrolase, 3-deaza- adenosine and 3-deaza-(+/-)aristeromycin (500 microM each), were effective in increasing platelets levels of AdoHcy and preventing turnover of AdoMet. Also, these compounds were equipotent in inhibiting platelet phospholipid methylation. However, while 3-deaza-adenosine potentiated platelet aggregation and 14C-serotonin release induced by epinephrine or adenosine diphosphate (ADP) (p less than 0.01), 3-deaza- aristeromycin had no such effect. Neither compound affected platelet responses to thrombin or collagen. Inhibition of methylation reactions was not the only biochemical effect of 3-deaza-adenosine since it also blunted significantly the elevation of platelet cyclic adenosine monophosphate (AMP) levels induced by prostaglandin E1 (p less than 0.02). Therefore, these studies demonstrate that inhibition of platelet phospholipid methylation, per se, has no discernable effect on the function of human platelets. The methylation pathway, though active in platelets, does not appear to be primarily involved in membrane events responsible for platelet activation.
The immunologic phenotyping of bone marrow biopsies and aspirates: frozen section techniques
Authors: Wood GS , Warnke RA
Published: October 2019

Techniques were developed for the preparation of frozen sections from undecalcified bone marrow biopsy cores and bone marrow aspirates. Selected indirect antibody and biotin-avidin detection systems, employing immunoperoxidase or immunofluorescent labels, were studied to determine which were best suited for bone marrow frozen section immunohistopathology. Methods employing murine hybridoma monoclonal antibodies illustrate the immunophenotyping of representative lymphoid neoplasms, involving bone marrow. The results of immunohistologic staining were comparable to those of fluorescence-activated cell sorter (FACS) analysis of cell suspensions. The advantages and limitations of immunohistologic techniques are discussed as they relate to immunophenotypic studies previously feasible only with bone marrow cell suspensions. Frozen section immunohistologic techniques serve as useful adjuncts to the conventional evaluation of bone marrow aspirates and biopsies.
Survival in Hodgkin's disease: the role of immunocompetence and other major risk factors
Authors: Faguet GB , Davis HC
Published: October 2019

The prognostic value of age, sex, symptoms, histopathology, lymphocyte count, skin tests, immunocompetence (lymphocyte response profile to a spectrum of PHA concentrations), and stage was evaluated in 35 previously untreated patients with Hodgkin's disease by multiple linear regression and logistic analysis. Immunocompetence exhibited the highest correlation (R = 0.537) with survival status and was required as a common denominator for deriving best sets of two or more variables. In addition, immunocompetence contributed to all other variables combined (p = 0.023), whereas stage did not (p = 0.116). Immunocompetence, age, symptoms, and histopathology generated a highly discriminant (R = 0.784) model not improved by stage or by other variables (p greater than or equal to 0.159). The utility and generalizability of this model are shown by a correct classification of 91.2% of cases according to expected versus actual survival status and by a predicted correlation (R) of 0.71, respectively, neither improved by sex, lymphocyte count, skin tests, or stage. In comparison, the conventional triad of stage, symptoms, and histopathology correctly classified only 70.6% of cases and showed actual and predicted correlations with survival status of R = 0.550 and R = 0.51. We conclude that immunocompetence is a powerful discriminant risk factor in Hodgkin's disease that exerts a pivotal role on survival and serves as a basis for models of greater discriminant power and generalizability than the conventional stage-based evaluation triad. Immunocompetence-based models are expected to provide a more discriminating basis for clinical evaluation, prediction of prognosis, and treatment selection for patients with Hodgkin's disease.
The use of the synthetic peptide, Gly-Pro-Arg-Pro, in the preparation of thrombin-degranulated rabbit platelets
Authors: Harfenist EJ , Guccione MA , Packham MA , Mustard JF
Published: October 2019

The method for preparing thrombin-degranulated platelets has been modified to avoid the use of plasmin or successive treatments with small amounts of thrombin, while still achieving more than 90% release of platelet amine storage granule contents. It was necessary to prevent the fibrinogen released from the platelets during thrombin treatment from forming an insoluble fibrin mesh that could trap the platelets and hinder their deaggregation. To accomplish this we have treated rabbit platelets with 0.73 U/ml of thrombin for 1 min in the presence of the synthetic peptide, Gly-Pro-Arg-Pro, which prevents the polymerization of fibrin molecules. We have demonstrated that it also prevents 125I, initially added as 125I-fibrinogen, from associating with the platelets in a form that was not removed by centrifuging and washing during the preparation of thrombin-degranulated platelets, and we infer that products formed from the fibrinogen released from the platelets would also be prevented from associating with them. Thrombin-degranulated platelets prepared by this method have lost 92% of their granule contents and they can be washed and resuspended. These platelets aggregate normally upon stimulation with thrombin, adenosine diphosphate (ADP), or arachidonate. Thus, Gly-Pro-Arg-Pro is useful in preparing thrombin-degranulated platelets for studying platelet reactions without the complicating effects of released materials such a ADP and fibrinogen.
Effect of HLA Bw4/Bw6 compatibility on platelet transfusion responses of refractory thrombocytopenic patients
Authors: McElligott MC , Menitove JE , Duquesnoy RJ , Hunter JB , Aster RH
Published: October 2019

Platelet transfusions from donors matched for cross-reactive antigens have been shown to be effective in providing hemostasis in alloimmunized thrombocytopenic patients. A significant number of these transfusions, however, fail to provide posttransfusion platelet recoveries. We investigated incompatibility in the Bw4/bw6 system as a possible explanation for these failures. The Bw4/Bw6 system is a biallelic antigen system closely associated with HLA-B. HLA-B locus antigens that are cross-reactive frequently differ in their Bw4/Bw6 specificity. Posttransfusion platelet recoveries from 21 alloimmunized thrombocytopenic patients homozygous for Bw4 or Bw6 and transfused with both Bw4/Bw6 compatible and incompatible platelets were analyzed. The mean 1-hr posttransfusion recovery was 84% following Bw4/Bw6-compatible platelets versus 52% with Bw4/Bw6-incompatible platelets (p less than 0.02). Twenty-four hours following transfusion, mean recoveries were 44% and 24%, respectively, (p less than 0.01). A subgroup of 8 patients (38%) was identified who had markedly lower responses following Bw4/Bw6- incompatible transfusions as compared to Bw4/Bw6-compatible transfusions (mean recoveries: 1 hr--compatible 100%, incompatible 27%, p less than 0.001; 24 hr--compatible 45%, incompatible 7%, p less than 0.01). These data suggest that the Bw4/Bw6 antigen system has clinical significance for some patients requiring platelet transfusion therapy and, when appropriate, should be considered in the selection of donors.
Chronic leukemia with a hybrid surface phenotype (T lymphocytic/myelomonocytic): leukemic cells displaying natural killer activity and antibody-dependent cellular cytotoxicity
Authors: Schlimok G , Thiel E , Rieber EP , Huhn D , Feucht H , Lohmeyer J , Riethmuller G
Published: October 2019

A patient with chronic leukemia exhibited uncommon clinical features, such as hypergammaglobulinemia and activation of intravascular coagulation after low-dose irradiation of the enlarged spleen. By light and electron microscopy, the leukemic cells resembled large granular lymphocytes. The following markers were simultaneously expressed on their surface: receptors for sheep erythrocytes and the Fc part of IgG; common T-cell antigens as revealed by a heteroantiserum (HuTLA) and monoclonal antibodies (OKT3, T411); antigens shared by cytotoxic/suppressor T cells (OKT8, T811) as well as myelomonocytic antigens defined by the OKM1 and M522 monoclonal antibodies. The leukemic cells showed both spontaneous (NK) and antibody-dependent (ADCC) cytotoxicity, but they did not suppress B-cell differentiation in vitro.
Increased heat sensitivity of red blood cells in hereditary elliptocytosis with acquired cobalamin (vitamin B12) deficiency
Authors: Schoomaker EB , Butler WM , Diehl LF
Published: October 2019

Structural membrane proteins were studied from erythrocytes (RBC) of a patient with a nonhemolytic form of hereditary elliptocytosis (HE) who developed a microcytic anemia with fragmented RBC while cobalamin (B12) deficient. Evidence is presented for qualitative changes in the patient's RBC membranes not related to a loss of structural proteins. Sensitivity of RBC to heat treatment was studied as well as quantitative changes in proteins by densitometry of 1% SDS--10% PAGE gels. Fractions of RBC of various sizes from the patient while B12 deficient all possessed a marked degree of heat sensitivity when compared to RBC from the patient after B12 repletion, normal family members, HE controls, B12-deficient controls, anemic controls, and normal controls. Because loss of spectrin (bands 1 + 2) from heat- sensitive RBC membranes in hereditary pyropoikilocytosis has been reported, the amount of spectrin relative to band 3 was measured. No decrease in the ratio of bands (1 + 2)/3 was found. In addition, no chromatographically abnormal membrane proteins were found by SDS-PAGE of the patient's RBC while B12 deficient. Our findings indicate that B12 deficiency results in an abnormal membrane with enhanced instability in some forms of HE. Since protein loss was not found, we conclude that an alteration in membrane protein interaction may be involved.
Phagocytosis and intracellular killing by peripheral blood monocytes of patients with monocytic leukemia
Authors: van Furth R , Leijh PC , van Zwet TL , van den Barselaar MT
Published: October 2019

This article concerns a study on the endocytic functions of circulating monocytes from 12 patients with acute or chronic monocytic leukemia. The results show that phagocytosis and intracellular killing of Staphylococcus aureus are impaired in only two patients and that the opsonic activity of the serum of all patients is normal. With respect to the intracellular killing of ingested Staphylococcus aureus, an interesting phenomenon was found in that the cells of patients with monocytic leukemia proved to be in a state of activation, as shown by the finding that patients' monocytes with normal phagocytosis killed about 64% of the ingested bacteria in the absence of extracellular stimulation by serum factors. When extracellular serum was present, the mean killing index rose to only 69%. This is unlike the situation seen in monocytes from healthy donors, where no killing occurs in the absence of extracellular serum and extracellular stimulation by serum factors is mandatory for optimal intracellular killing.
T-cell subpopulations identified by monoclonal antibodies after human marrow transplantation. I. Helper-inducer and cytotoxic-suppressor subsets
Authors: Atkinson K , Hansen JA , Storb R , Goehle S , Goldstein G , Thomas ED
Published: October 2019

Peripheral blood helper-inducer and cytotoxic-suppressor T-cell subpopulations in patients receiving marrow transplants for the treatment of acute leukemia or severe aplastic anemia were quantitated on the fluorescence-activated cell sorter (FACS) using the monoclonal antibodies OKT4 and OKT8, respectively. The relative (percent) and absolute number of OKT4+ cells were severely and persistently depleted for up to 2.7 yr posttransplant. In contrast, the percent and absolute number of OKT8+ cells began to recover within the first 60 days of transplant and subsequently remained at normal or high levels for periods of up to 7.3 yr. There was no significant difference in percent or absolute numbers of OKT8+ cells for patients with or without acute graft-versus-host disease (GVHD). The reversal of the normal OKT4:OKT8 ratio (2:1) occurred regardless of whether the recipient was given an allogeneic, syngeneic, or autologous transplant and regardless of whether or not acute or chronic GVHD developed. The reversed ratio was due in the first 3 mo posttransplant to low numbers of OKT4+ cells and later to a combination of low numbers of OKT4+ and high numbers of OKT8+ cells. Normalization and then an increase in the number of OKT8+ cells correlated with increasing time posttransplant and not with resolution of acute GVHD.
Protein A adsorption of acute myelogenous leukemia serum induces in vitro blast lysis
Authors: Miller WJ , Branda RF , Hurd DD , Wachsman W , Nelson NL , Jacob HS
Published: October 2019

We studied cytotoxic activity of acute myelogenous leukemia (AML) sera for AML blasts before and after immunoadsorption with Staphylococcus aureus, Cowan I (SAC), which contains protein A. We found in vitro that incubation with treated AML sera reduced viability to 42.7% of control (p less than 0.01) for autologous and 21.0% of control (p less than 0.01) for allogeneic blasts. Normal peripheral blood cells were not killed by treated AML sera. Wood 46 strain of Staphylococcus aureus, which does not contain protein A, did not significantly reduce AML blast viability (94.8%, p greater than 0.4), while Sepharose-bound protein A reduced viability to 63.8% (p less than 0.01). Cytotoxicity does not appear to be complement-mediated, byt cytotoxic activity is trypsin-sensitive and is contained in the immunoglobulin fraction. This model for study of the tumoricidal action of protein A adsorption should be useful for predicting utility of plasma adsorption as a therapeutic adjunct in the future.
Studies on the interaction between GP-18-0-deficient neutrophils and vascular endothelium
Authors: Buchanan MR , Crowley CA , Rosin RE , Gimbrone MA Jr , Babior BM
Published: October 2019

A patient whose neutrophils lack the glycoprotein gp-180 shows an increased susceptibility to bacterial infections. Neutrophils from this patient migrate abnormally both in vivo and in vitro. To examine the basis for this abnormality in migration, a study was carried out on the interaction of gp-180-deficient neutrophils with artificial surfaces and with human endothelial cell cultures. Compared with normal neutrophils. gp-180-deficient neutrophils showed decreased adhesion to cold-insoluble globulin-coated plastic surfaces, and their ability to spread on this substratum was greatly impaired. In contrast, gp-180- deficient neutrophils interacted in a normal fashion with endothelial monolayers, attaching to their surfaces and migrating between cell junctions to spread between the monolayers and the subjacent plastic. A normal interaction with endothelium in vivo was implied by the finding that the rise in the neutrophil count in response to epinephrine, an index of the marginated pool, was normal in the gp-180-deficient patient. We conclude that the abnormal function of gp-180-deficient cells is unlikely to be caused by a faulty interaction with the vascular endothelium. We postulate instead that these cells migrate poorly in vivo because of an abnormal interaction with extravascular connective tissue matrix constituents.
Direct quantitation of platelet-associated IgG by electroimmunoassay
Authors: Kunicki TJ , Koenig MB , Kristopeit SM , Aster RH
Published: October 2019

An electroimmunoassay was applied to the quantitation of platelet- associated IgG (PAIgG). Protein solubilized by Triton X100 from washed platelets was electrophoresed at pH 5.0 in agarose gels containing carbamoylated rabbit anti-human IgG (pI approximately equal to 5.0). Because the rabbit antibody is immobilized under these conditions, while PAIgG is freely mobile, rocket precipitates were produced, the heights of which were directly proportional to the amount of antigen (PAIgG) present. By this method, PAIgG for normal individuals was found to be 4.3 +/- 1.7 fg/platelet (mean +/- 2 SD; n = 35). Increased PAIgG levels (direct assay) were found in 27 of 29 patients with a diagnosis of clinically active idiopathic thrombocytopenic purpura (ITP), ranging from 10.5 to 101.5 fg/platelet. Moderately elevated PAIgG was found in 8 of 10 patients in an early stage of recovery from ITP (range 7.5–9.5 fg/platelet) and in 3 of 6 patients with apparent nonimmune thrombocytopenia (range 14.5–24.0 fg/platelet). Electroimmunoassay for PAIgG can be performed on patients with platelet counts as low as 2000/microliters, yields results in less than 24 hr, is highly reproducible, and appears to provide a useful tool for the evaluation of patients with immunologically mediated thrombocytopenia.
Induction of human platelet fibrinogen receptors by epinephrine in the absence of released ADP
Authors: Peerschke EI
Published: October 2019

The ability of epinephrine to expose platelet fibrinogen receptors independent of released ADP was assessed using aspirin-treated, gel-filtered platelets. Similar to ADP-induced aggregation, platelet aggregation in response to epinephrine was accompanied by fibrinogen binding. Ten micromolar epinephrine induced a maximum number of platelet fibrinogen receptors in the absence of significant 14C-serotonin release. As indicated by Scatchard analysis, receptors exposed by both epinephrine and ADP had similar affinities for fibrinogen, but epinephrine induced approximately 30% fewer receptors than did ADP. This appears to correlate with the lesser degree of primary aggregation observed with this agent. Studies using phentolamine, a specific alpha-adrenergic antagonist, apyrase, or creatine phosphate/creatine kinase indicate that the exposure of platelet fibrinogen receptors by epinephrine was specific for platelet alpha-adrenergic receptor stimulation and was not the result of released ADP.
Causes of initial remission induction failure in acute myelogenous leukemia
Authors: Estey EH , Keating MJ , McCredie KB , Bodey GP , Freireich EJ
Published: October 2019

One-hundred and sixty-one of 378 previously untreated patients with acute myelogenous leukemia (AML) failed to enter complete remission with a combination of anthracycline, cytosine arabinoside, vincristine, and prednisone between 1973 and 1979. Thirty-six of the failing patients (22%) were considered chemotherapy failures. As in the past, the remainder failed largely because of death from infection. However, despite the routine use of prophylactic platelet transfusions, hemorrhage was a major cause of death in 33%. Thirty-seven percent of the fatal infections were due to fungi and the incidence of fungal infection was as high during the second week of treatment as later. Age greater than 50 yr predisposed to fatal infection but not chemotherapy failure, while the presence of an antecedent hematologic disorder increased the risk of both fatal fungal infection and resistance to chemotherapy. Patients with an initial white blood cell count of greater than or equal to 25000/microliter were more likely to die of hemorrhage at all times during treatment. Improvement in supportive care remains crucial if improved complete rates are to be forthcoming in previously untreated patients.
Relation of human neutrophil phorbol ester receptor occupancy and NADPH- oxidase activity
Authors: Tauber AI , Brettler DB , Kennington EA , Blumberg PM
Published: October 2019

Phorbol esters are potent stimulants of the respiratory burst of the human neutrophil as assessed by superoxide (O2-) generation in whole cells and by NADPH-oxidase activity in a broken-cell 27,000-g particulate fraction. Phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) stimulate production of O2- by human neutrophils with ED50 concentrations of 3.9 +/- 2.1 and 41.7 +/- 7.1 nM, respectively. The relation of biologic activity to receptor occupancy was assessed with binding studies of PMA and PDBu. Phorbol ester binding revealed a single high affinity phorbol ester receptor present at 7.6 x 10(5) sites/cell. The binding affinities for PMA and PDBu, 4.9 nM and 38.4 nM, respectively, agreed quantitatively with that of biologic potencies. Because of the high concentration of phorbol ester receptors (up to 125 nM) and the large amount of nonspecific binding at high cell density, apparent discrepancies between ED50′s for NADPH-oxidase and whole cell O2- generation were noted. With the use of low cell concentrations, quantitative agreement between intact cell production of O2-, NADPH-oxidase activity, and receptor binding was found. These results further support the identity of the NADPH-oxidase as the enzymatic source of respiratory burst O2- production in human neutrophils.
Effect of modulators of erythropoiesis on the hemoglobinization of human erythroid cell cultures
Authors: Clarke BJ , Brickenden AM , Ives RA , Chui DH
Published: October 2019

A rapid spectrophotometric assay capable of detecting the hemoglobin content of 1000 mature erythrocytes has been utilized to quantitate the total hemoglobin synthesized by the progeny of circulating human erythroid progenitors in both the plasma clot and methylcellulose culture systems. The pronounced variation in the effect of different erythropoietin preparations on the hemoglobin content of cultured human peripheral blood bursts, previously described in a subjective manner, has been objectively quantitated. Further experiments demonstrated that both lymphocyte conditioned media and dexamethasone substantially increased the total hemoglobin synthesized by the progeny of cultured erythroid progenitors. The elevated amount of hemoglobin present in erythroid cultures containing either lymphocyte conditioned media and/or dexamethasone was due to both increased colony numbers and colony size. Assay of the total hemoglobin content per erythroid culture is an accurate, sensitive, measure of erythropoiesis in vitro and should be a valuable adjunct to the enumeration of BFU-E-derived erythroid colonies.