bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiinstitution study of non-Hodgkin's lymphomas using frozen section immunoperoxidase: the Southeastern Cancer Study Group experience
Authors: Borowitz MJ , Newby S , Brynes RK , Cousar JB , Whitcomb CC , Crissman JD , Byrne GE Jr , Collins RD
Published: October 2019

This report describes the experience of the Southeastern Cancer Study Group (SECSG) with a transport medium used for immunologic phenotyping of non-Hodgkin’s lymphomas. In a 2-mo pilot study, portions of 53 specimens of non-Hodgkin's lymphoma from four member institutions of the SECSG and affiliated community hospitals were sent by regular mail to a central laboratory. Immunologic phenotyping was carried out using a frozen section immunoperoxidase technique. In 48 of the cases, a clear-cut immunologic phenotype was obtained. Thirty-four tumors were of B cell origin and 7 had T cell markers. Six of the remaining lymphomas had neither B nor T cell markers, and the seventh had both. In 12 cases, phenotyping was also carried out at the originating institution using conventional cell suspension techniques; agreement between the two methods was excellent. The immunologic results were correlated with histopathologic diagnosis standardized using the Working Formulation for non-Hodgkin’s lymphomas. It was found that the low grade tumors were all B cell, but that the intermediate grade tumors were very heterogeneous immunologically. About one-fourth of the diffuse, intermediate grade or miscellaneous tumors had T cell markers. Our results indicate that immunologic phenotyping may be performed satisfactorily on transported material, making multiinstitution studies on the prognostic significance of immunologic phenotype in non- Hodgkin’s lymphomas feasible.
Evidence for the presence of CFU-E with increased in vitro sensitivity to erythropoietin in sickle cell anemia
Authors: Pennathur-Das R , Alpen E , Vichinsky E , Garcia J , Lubin B
Published: October 2019

To investigate the cellular events that accompany erythroid hyperplasia, we studied several effects of erythropoietin (Epo) on marrow CFU-E in sickle cell anemia (SCA). We measured CFU-E number, CFU- E growth as a function both of Epo exposure time and of Epo concentration, and suppression of Epo-induced CFU-E formation by anti- Epo antiserum. With 0.5 U Epo/ml, the number of CFU-E was elevated in SCA (1,087 +/- 520) compared to normal (430 +/- 130). CFU-E were formed even when Epo was immediately neutralized by a 1/150 dilution of anti- Epo. After 40 hr of Epo exposure, only 2% of total CFU-E were expressed in normal marrow, whereas 12%-40% of CFU-E were expressed in SCA. Inhibition of CFU-E growth required at least 1/50 dilution of anti-Epo in SCA and a 1/300 dilution in normal marrow. In contrast to normal, a small number (5%-20%) of CFU-E were expressed in the absence of added Epo in SCA, and this pool required a 1/150 dilution of anti-Epo for inhibition. The Epo dose-response curve in SCA revealed a peak in colony formation around 0.1 U Epo/ml and 0.5 U Epo/ml, whereas only one peak at 0.5 U Epo/ml was seen in normals. These data strongly suggest that, in response to the demands of chronic erythroid hyperplasia in SCA, a pool of CFU-E is present characterized by increased in vitro sensitivity to Epo.
Aberrant sialylation of granulocyte membranes in chronic myelogenous leukemia
Authors: Baker MA , Taub RN , Whelton CH , Hindenburg A
Published: October 2019

Peripheral blood granulocytes from patients with chronic myelogenous leukemia (CML) were studied for accessibility of membrane sialic acid and galactose residues to sodium borohydride-3H radiolabeling after oxidation with sodium metaperiodate (PI/B3H4) or with galactose oxidase (GO/B3H4). Granulocytes from untreated patients with chronic myelogenous leukemia showed increased radiolabeling with PI/B3H4, and decreased labeling with GO/B3H4 when compared to normal granulocytes. Granulocytes from leukemic patients receiving chemotherapy showed normal labeling patterns. Gel electrophoresis of membrane extracts showed that the changes in PI/B3H4 and GO/B3H4 reactivity of CML cells were distributed over all membrane protein bands. Our data suggest that CML granulocyte membrane proteins are aberrantly sialylated, with decreased accessibility of galactose residues, and that these changes may be reversed by clinical drug treatment.
Cell membrane shape control--effects of chloromethyl ketone peptides
Authors: Alhanaty E , Sheetz MP
Published: October 2019

The shape of the human erythrocyte is normally maintained in vivo as a biconcave disc for 120 days. In vitro, the cell shape can be altered readily by amphipathic compounds; however, given time and an energy source, the cells can recover the discoid morphology. An active shape control mechanism is postulated to regulate erythrocyte shape. The shape recovery process is a necessary element in reversing perturbations of shape and is basic to our understanding of how membrane shape is altered. We report here that the process of shape recovery from crenation is dramatically accelerated upon pretreatment of the cells with micromolar (20–100 microM) concentrations of chloromethyl ketone peptides [such as N-alpha-tosyl-L-phenylalanine- chloromethyl ketone (tos-pheCH2Cl)]. Such pretreatments do not appear to affect cellular viability, as judged by their normal biconcave disc shape, their sensitivity to crenators, their lactic acid production, or the ATP-dependent shape change of the purified membranes. Treatment with high concentrations of tos-pheCH2Cl does cause normal cells to become stomatocytic by an energy-requiring process, i.e., it requires glucose, incubation at 37 degrees C, and will not occur in ATP-depleted cells. We suggest that the chloromethyl ketone peptides affect a metabolic process that is associated with the hexose monophosphate (HMP) shunt. Through the alteration of the HMP shunt metabolism, they modify an active stomatocytic process in the erythrocyte that can correct for the perturbation caused by crenators. Implications of these findings for analogous phenomena in cultured cells are discussed.
Interdigitative coupling of presumptive hematopoietic stem cells to macrophages in endocloned marrow colonies
Authors: Lambertsen RH
Published: October 2019

Hematopoietic marrow colonies were studied ultrastructurally to investigate stromal cell-hematopoietic cell interactions during early hematopoiesis. An elaborate interdigitative coupling of mature marrow macrophages to morphologically undifferentiated colony cells is described. This coupling was found in undifferentiated and granulocytic colonies and established a physical linkage of the macrophage and undifferentiated cell. The spaces between the coupled cells contained an electron-dense material and appeared to be modified by the uptake or release of cytoplasmic vesicles of the macrophage. Based on available evidence that hematopoietic stem cell proliferation is controlled at a local, or stromal, level, and the finding that this interaction occurs in very early hematopoietic colonies, it is suggested that interdigitative couplings represent a mechanism of stem cell regulation. In addition, the observations indicate macrophages to be actively involved with the lodgement of morphologically undifferentiated hematopoietic cells in the marrow space.
Prognostic significance of immunologic phenotype in hairy cell leukemia
Authors: Jansen J , Schuit HR , Hermans J , Hijmans W
Published: October 2019

Hairy cell leukemia (HCL) is a usually chronic B cell lymphoproliferative disorder. To evaluate the prognostic significance of the various heavy and light chain determinants of the surface immunoglobulins (slg), we analyzed the clinical data and immunologic phenotype of 64 patients with HCL. Sixty-two of the 64 patients showed slg, which was invariably of only one light chain type (kappa 33, lambda 29). The actuarial survival of the cases expressing kappa-light chains was significantly better than those with lambda-light chains (p less than 0.002). This difference persisted when only cases with gamma or alpha gamma heavy chains were considered. No differences between the kappa and lambda-subgroups were discovered with respect to parameters of clinical importance. The various heavy chain classes of slg did not correlate significantly with the survival time. These results suggest that the immunologic phenotype, in particular the light chain type, may be a prognostic factor in patients with HCL.
The use of radiolabeled and fluorescein-labeled antiglobulins in assays to predict platelet transfusion outcome
Authors: Ware R , Reisner EG , Rosse WF
Published: October 2019

We used both radiolabeled and fluorescein-labeled antiglobulins in assays to detect antibodies against platelets in multiply transfused patients to determine the value of these tests in predicting the outcome of platelet transfusion in such patients. In 15 allosensitized patients, we studied 68 single-donor platelet transfusions, 43 (63%) of which had a poor outcome, defined as a corrected count increment (CCI), less than 10,000. The results obtained with either test were significantly correlated with the CCI following transfusion (p less than 0.001), but the assay using the radiolabeled antiglobulin had slightly better sensitivity, specificity, and predictive value. When the assays were used in combination, there was again significant correlation with the CCI of the transfusion, p less than 0.001. When both assays predicted failure of the transfusions, 31/31 (100%) such transfusions resulted in a CCI of less than 10,000, and when both assays predicted success of the transfusions, 14/15 (93%) such transfusions resulted in a CCI of greater than 10,000. Both assays are useful in predicting the outcome of the platelet transfusions; when the assay results were concordant, almost total predictive accuracy was obtained.
Predictive factors for acute graft-versus-host disease in patients transplanted with HLA-identical bone marrow
Authors: Bross DS , Tutschka PJ , Farmer ER , Beschorner WE , Braine HG , Mellits ED , Bias WB , Santos GW
Published: October 2019

To identify predictive parameters for incidence and severity of acute graft-versus-host disease (GVHD), 136 patients, transplanted with histocompatible marrow as therapy for aplastic anemia and hematologic malignancies, were examined using univariate and multivariate analyses. The risk of GVHD increased in patients with acute lymphocytic leukemia (p less than 0.05), in sex-mismatched donor-recipient pairs (p less than 0.01), and in patients older than 23.7 yr (p less than 0.05). No other commonly observed factors appeared to have any relationship to GVHD except the presence of certain alleles. The presence of a Cw4 allele or of the Bw21 specificities B49 and B50 were associated with significantly increased risks of GVHD (p less than 0.05), whereas the presence of Aw19 (or the related specificities A29, Aw30, Aw31 , Aw32, Aw33 ) was associated with a significantly decreased risk (p less than 0.01). Using these factors, a regression equation can be constructed that estimates the risk of a given patient to develop clinically significant acute GVHD.
A Hodgkin cell-specific antigen is expressed on a subset of auto- and alloactivated T (helper) lymphoblasts
Authors: Andreesen R , Osterholz J , Lohr GW , Bross KJ
Published: October 2019

A Hodgkin cell-specific antigen detected by the monoclonal antibody Ki- 1 was found on T helper lymphocytes after activation by autologous and allogeneic stimulator cells. About 50% of lymphoblasts generated by auto- and alloactivation reacted with the antibody. In contrast, only less than 6% of lymphoblasts stimulated with Con-A, phytohemagglutinin (PHA), or protein A, and none of lymphoblasts activated by oxidative mitogenesis, expressed this antigen. Among several permanent cell lines tested, the K562, MOLT-4, HL-60, and EBV transformed B lymphoblastoid cells reacted with the Ki-1 antibody. The results may indicate possible relationships between the autoreactive subset of T lymphocytes and the pathogenesis of Hodgkin's disease.
Lineage infidelity following exposure of T lymphoblasts (MOLT-3 cells) to 5-azacytidine
Authors: Smith LJ , McCulloch EA
Published: October 2019

The appearance on single leukemic blast cells of markers of at least two different lineages has been termed lineage infidelity. MOLT-3 cells, a continuous line of human T lymphoblasts, express T cell markers as defined immunologically with monoclonal antibodies. Following a single exposure to 5-azacytidine, other markers, usually associated with non-T cell lineages, appeared transiently. A stable clone with lineage infidelity was obtained by selection from colonies expressing novel markers. Marker expression followed reproducible kinetics during growth. 5-Azacytidine-treated MOLT-3 subclones may be useful models in the study of lineage infidelity and gene expression.
Effects of thalassemia and microcytosis on the hematologic and vasoocclusive severity of sickle cell anemia
Authors: Steinberg MH , Rosenstock W , Coleman MB , Adams JG , Platica O , Cedeno M , Rieder RF , Wilson JT , Milner P , West S
Published: October 2019

The characteristic clinical heterogeneity of sickle cell anemia (HbSS) may be, in part, a result of its interactions with alpha-thalassemia. Although alpha-thalassemia clearly affects some hematologic features of HbSS, its role in modulating the vasoocclusive severity of disease is not clear. To further explore this relationship, we examined the incidence of painful episodes, acute chest syndrome, aseptic bone necrosis, and leg ulcers in 3 patient groups with sickle cell disease: (1) 2,147 patients over age 2 yr, stratified according to mean corpuscular volume (MCV); (2) 183 patients selected on the basis of microcytosis and elevated HbA2, on whom globin biosynthesis studies were done; and (3) 125 patients who had alpha-globin genotype assigned by restriction endonuclease gene mapping. When patients were stratified by MCV, there was a reciprocal relationship between HbA2 levels and MCV, reflecting the presence of patients with beta o and alpha- thalassemia in the low MCV groups. The erythrocyte indices and HbA2 levels in patients classified as HbSS-alpha-thalassemia, by either globin synthesis studies or gene mapping, were very similar to those previously reported by others. Neither microcytosis, beta o, or alpha- thalassemia appeared to provide any clear protection from the vasoocclusive complication evaluated, and the prevalence of aseptic necrosis was increased in patients with microcytosis over age 20 yr and in groups with alpha-thalassemia. The effects of a reduced MCV and mean corpuscular hemoglobin concentration (MCHC), of possible benefit by themselves, when accompanied by a reduction in hemolysis and rise in hemoglobin concentration, as in HbSS-alpha-thalassemia, may cause sufficient rise in blood viscosity in critical vascular beds to impair blood flow and negate any amelioration of vasoocclusive complications in HbSS.
Oxidative mechanisms utilized by human neutrophils to destroy Escherichia coli
Authors: Passo SA , Weiss SJ
Published: October 2019

Serum-opsonized bacteria are efficiently ingested and killed by neutrophils within the phagocytic vacuole, where they are exposed to an array of reactive oxygen metabolites and toxic lysosomal components. Although bacteria may be destroyed by oxygen-independent mechanisms alone, many types of bacteria are not killed effectively unless they are attacked by oxygen metabolites. However, the apparent inability of extracellular scavengers, or inhibitors, of oxygen metabolites to gain access to the phagocytic vacuole makes this system difficult to evaluate. Therefore, we investigated the ability of neutrophils triggered with phorbol myristate acetate to destroy unopsonized E. coli in a serum-free model system. Neutrophils incubated with phorbol myristate acetate at a cell-to-bacteria ratio of 1:4 caused a greater than 95% reduction in colony-forming units (CFU) of E. coli in 60 min at 37 degrees C. Destruction of E. coli by the stimulated neutrophils was dependent on neutrophil number, stimuli concentration, and the incubation period. The neutrophil-mediated bactericidal effect was stimulated by superoxide dismutase, but was inhibited by catalase, azide, or compounds known to scavenge hypochlorous acid. Although stimulated neutrophils can generate long-lived endogenous N- chloroamines , these compounds did not play a direct role in destruction of E. coli in our model system. However, in the presence of exogenous iodide, endogenous N- chloroamines exerted a powerful bactericidal effect. Finally, neutrophils triggered with opsonized zymosan could also mediate E. coli destruction by a qualitatively similar process. Thus, we have demonstrated that neutrophils have the potential to utilize the myeloperoxidase system to generate bactericidal quantities of a species with characteristics similar to, if not identical with, hypochlorous acid.
A new variant of dominant type II von Willebrand's disease with aberrant multimeric pattern of factor VIII-related antigen (type IID)
Authors: Kinoshita S , Harrison J , Lazerson J , Abildgaard CF
Published: October 2019

A new type II variant form of von Willebrand's disease has been recognized in a mother and daughter who have bleeding manifestations typical of von Willebrand's disease. Laboratory findings include consistently prolonged bleeding times, with normal levels of factor VIII procoagulant and antigen, but decreased ristocetin cofactor activity. Electrophoresis in SDS 1.5% agarose gel and reaction with 125I-labeled anti-factor VIII-related antigen rabbit IgG, followed by autoradiography, revealed that both plasma and platelets lack the large multimers of factor VIII-related antigen. In 2.5% gel, the propositus plasma lacked the normal “triplet” pattern. In 3.0% gel, a 5-band pattern was observed in normal, type IIA, and type IIB plasma, whereas type IIC plasma revealed a 2-band pattern. The patient's plasma revealed a 4-band pattern distinctly different from normal or other type II variants. We suggest that this new variant be labeled type IID, until a more appropriate nomenclature is developed.
Differential binding of plasminogen to crosslinked and noncrosslinked fibrins: its significance in hemostatic defect in factor XIII deficiency
Authors: Sakata Y , Mimuro J , Aoki N
Published: October 2019

In spontaneous fibrinolysis of an alpha 2-plasmin inhibitor-deficient plasma clot or tissue-type plasminogen activator-induced fibrinolysis in a purified system without alpha 2-plasmin inhibitor, the lysis was faster when factor XIII-mediated crosslinking of fibrin to fibrin did not occur. During the initial period, the binding of plasminogen to fibrin steadily increased with incubation time. The initial level and subsequent increase of the binding, which may be critical for the subsequent development of fibrinolysis, were more remarkable when fibrin was not crosslinked. The amount of glu- or lys-plasminogen bound to noncrosslinked fibrin was around 4 or 1.5 times larger than the amount of the respective plasminogen bound to crosslinked fibrin. Plasmin was also found to be bound to noncrosslinked fibrin twice as much as the amount bound to crosslinked fibrin. Structural changes induced by crosslinking of fibrin alpha-chain may reduce either the affinity or the number of available complementary sites to lysine binding sites of plasmin(ogen), thereby decreasing the binding of plasmin(ogen) to fibrin. These results suggest that an increased affinity of noncrosslinked fibrin for plasmin(ogen) is contributory to the accelerated fibrinolysis observed in factor XIII deficiency, in addition to an absence of crosslinking of alpha 2-plasmin inhibitor to fibrin.
T lymphocyte repopulation and differentiation after bone marrow transplantation. Early shifts in the ratio between T4+ and T8+ T lymphocytes correlate with the occurrence of acute graft-versus-host disease
Authors: Gratama JW , Naipal A , Oljans P , Zwaan FE , Verdonck LF , de Witte T , Vossen JM , Bolhuis RL , de Gast GC , Jansen J
Published: October 2019

Acute graft-versus-host disease (GVHD), a major complication of allogeneic bone marrow transplantation (BMT), is probably mediated by T lymphocytes present in the marrow graft. In this study, the repopulation of the peripheral blood with T4+ and T8+ T cells was investigated during the period preceding the occurrence of acute GVHD. Twenty-four allogeneic and 11 autologous BMT recipients were monitored from day 4 post-BMT onward by the use of monoclonal antibodies, indirect immunofluorescence, and flow cytometry. The recipients of allogeneic transplants received methotrexate as GVHD prophylaxis. Similar recovery patterns for T4+ and T8+ T cells were found following autologous and allogeneic BMT. However, lymphoid repopulation occurred at a clearly faster rate after autologous BMT. T4+ T cells were the first to reappear in the peripheral blood, followed by T8+ T cells 4–7 days later. The T8+ T cell reconstitution occurred at an even faster rate in patients who were to develop grade II-IV GVHD, as compared with those with grade O-I GVHD, thus leading to an earlier decrease in the T4/T8 ratio. Of 10 patients with a T4/T8 ratio less than 2.5 at day 19, 9 developed grade II-IV GVHD and 1 showed no GVHD. Of 14 patients with a ratio greater than 2.5 at that time, only 2 developed grade II-IV and 12 grade O-I GVHD (p less than 0.001). In the 11 patients developing grade II-IV GVHD, the T4/T8 ratio decreased to values less than 2.5 before the first clinical symptoms of GVHD in 9; it coincided in one and occurred later in another patient. Thus, early monitoring of the T4/T8 ratio can distinguish patients at risk of developing grade II-IV GVHD.
Expression of human B cell-associated antigens on leukemias and lymphomas: a model of human B cell differentiation
Authors: Anderson KC , Bates MP , Slaughenhoupt BL , Pinkus GS , Schlossman SF , Nadler LM
Published: October 2019

A series of monoclonal antibodies that define B cell restricted and associated antigens was utilized in an attempt to characterize tumors of B lineage and to relate these tumors to B cell differentiative stages. Antigens that were previously shown to be B cell restricted on normal B lymphocytes were similarly expressed only on B cell malignancies. In contrast, antigens that were B cell associated were also found on tumors of other lineages. Moreover, on the basis of cell surface phenotypes, tumors of B cell origin were divided into three major subgroups, which corresponded to the level of differentiation of the malignant tumor cell: pre-B cell stage (non-T acute lymphoblastic leukemia and chronic myelocytic leukemia in lymphoid blast crisis); the mid-B cell stage (chronic lymphocytic leukemia, poorly differentiated lymphomas); and secretory B cell stage (large cell lymphomas and plasma cell tumors). A hypothetical model is derived that relates the malignant B cell to its normal cellular counterpart on the basis of cell surface expression of this panel of B cell-restricted and B cell- associated antigens.
Autoantibodies against platelet glycoprotein Ib in patients with chronic immune thrombocytopenic purpura
Authors: Woods VL Jr , Kurata Y , Montgomery RR , Tani P , Mason D , Oh EH , McMillan R
Published: October 2019

The present studies provide direct evidence that some patients with chronic immune thrombocytopenic purpura (ITP) have autoantibodies reactive with platelet glycoprotein Ib ( GPIb ). Microtiter wells coated with a monoclonal antibody that recognized GPIb were reacted with either platelet extract or a control cell extract. After washing and incubating with test plasma, well-bound IgG was quantitated using radioactive anti-IgG. When compared to plasma from normal subjects, plasma from 3 of 106 patients with chronic ITP had significantly increased quantities of IgG bound to microtiter wells reacted with platelet extracts. Negative results were obtained with the remaining 103 patients with chronic ITP and 59 patients with a variety of other platelet disorders. Plasma from two of the three positive patients precipitated a protein from 125I-surface-labeled platelet extract with a molecular weight similar to GPlb . One of the three patients with anti- GPlb antibody also had demonstrable autoantibodies to the platelet glycoprotein llb / llla complex.
Prolongation of sickle cell survival by dimethyl adipimidate is compromised by immune sensitization
Authors: Guis MS , Lande WM , Mohandas N , Pennathur-Das R , Preisler H , Lubin BH , Mentzer WC
Published: October 2019

The effect of dimethyl adipimidate (DMA), an amino-reactive crosslinking reagent with demonstrated antisickling properties in vitro, on the survival of 51Cr-labeled autologous sickle cells was evaluated in five adult males with sickle cell anemia. The survival of cells pretreated with 5 mmol/L DMA (pH 7.4), normal (t1/2 28–33 days) in four subjects and near-normal (t1/2 20 days) in the fifth, was considerably longer than that usually observed in sickle cell disease. In fact, the effect of DMA on the survival of sickle cells in vivo equals or exceeds that of any other agent tested to date. In three subjects, the survival of a second infusion of DMA-treated red cells was much shorter (t1/2 1.8, 3, 4.5 days) than in the initial study. An antibody was detected in the serum of these subjects that was directed to DMA-treated red cells. Modification of the immunogenicity of treated cells will be required before further consideration of DMA for use in the therapy of sickle cell anemia.
Human hepatoma cells secrete single chain factor X, prothrombin, and antithrombin III
Authors: Fair DS , Bahnak BR
Published: October 2019

The human hepatoma cell line, Hep G2, was analyzed for the ability to synthesize and secrete several coagulation proteins. Using specific radioimmunoassays, factor X, prothrombin, and antithrombin III were present in 8-day culture supernatants at 62, 405, and 1,220 ng/mL, respectively. Factor IX was not detected, either in supernatants or in cell extracts. Intrinsically labeled factor X was secreted as a single- chain polypeptide of 66,000 daltons, as measured by sodium dodecylsulfate-polyacrylamide gels under nonreduced and reduced conditions. Immunoblots of Hep G2 supernatants and normal human plasma also indicate the presence of single-chain factor X. These findings support the hypothesis of a postsecretion proteolytic cleavage of factor X into the two-chain form. Prothrombin and antithrombin represented their plasma protein counterparts structurally, with molecular weights of 73,000 and 61,000, respectively. Secreted factor X, prothrombin, and antithrombin III were biologically active, as determined in coagulation or chromogenic assays, and all three activities were neutralized by monospecific antibodies. Vitamin K increased the quantity of prothrombin secreted by twofold, without affecting the rate of secretion over a five-day culture period, and had an apparent transient inhibitory effect on secretion of antithrombin III. Warfarin caused a three to fourfold decrease in the rate and quantity of secreted prothrombin, but did not affect intracellular concentrations. The intracellular and extracellular concentrations and rate of secretion of antithrombin III were not modulated by warfarin. These data suggest that the Hep G2 cell line may provide a useful model for assessing the regulation of biosynthesis and secretion of human coagulation proteins.
The response of human neutrophils to a chemotactic tripeptide (N-formyl- methionyl-leucyl-phenylalanine) studied by microcinematography
Authors: Maher J , Martell JV , Brantley BA , Cox EB , Niedel JE , Rosse WF
Published: October 2019

A quantitative microcinematographic technique for the measurement of cellular motion in response to chemical influences is described. The data are analyzed by using a magnetic digitizer connected to a computer, permitting comparison of several characteristics of such motion. When human granulocytes are placed in a gradient of formyl- methionyl-leucyl-phenylalanine (fMLP) (concentration 10(-6)-10(-8) mol/L) against buffer, the motion of the cells is increased in velocity (chemokinesis) and is directed toward the chemoattractant, as measured by the orientation of the vector of motion. This directionality is confirmed by positive values for the McCutcheon index and analysis of directed linear displacement. Concentrations of fMLP below 10(-9) mol/L did not result in chemokinesis or chemotaxis, presumably due to insufficient stimulus. Concentrations of 10(-6) mol/L fMLP and greater resulted in marked stimulation of the cell surface but reduced directionality and velocity of motion compared with cells in a more optimal gradient. Motion of the unstimulated cells tended to be ortholinear , and this was not increased by the presence of a gradient of fMLP. Hence, as previously shown, the Random Walk analysis, which does not give weight to the direction of motion, is not useful in the analysis of the response of these cells to a chemotaxin in this system.
Beta O-39 thalassemia gene: a premature termination codon causes beta- mRNA deficiency without affecting cytoplasmic beta-mRNA stability
Authors: Humphries RK , Ley TJ , Anagnou NP , Baur AW , Nienhuis AW
Published: October 2019

The function of a beta O-thalassemia globin gene with a premature termination codon at position 39 was studied in tissue culture cells using plasmid expression vectors. The thalassemic globin gene was isolated by molecular cloning from the bone marrow DNA of an Italian patient with severe thalassemia. Sequences upstream of the normal beta- globin gene and the beta O-39 globin gene were removed to 127 nucleotides (nt) 5′ to the start site of transcription, thereby eliminating uncharacterized DNA sequences but preserving promoter function. To create a hybrid gene differing from the normal by only the single nt change in codon 39, the truncated 5′ end of the beta O-39 gene was recombined with the 3′ end of the normal gene. The beta O-39 substitution resulted in a 6–14-fold reduction in cytoplasmic RNA accumulation in transfected monkey kidney or HeLa cells. This decrease in mRNA did not appear to be due to instability, as the beta-mRNA present 24–36 hours after transfection was stable for up to 24 hours in the presence of actinomycin D. In the presence of actinomycin D, the globin mRNA precursor disappeared, suggesting that globin gene transcription had been effectively blocked. We also examined a second globin mRNA with a premature termination codon; this RNA that arises from incorrect splicing of the transcript of a beta + -24 thalassemia gene was as stable as the correctly spliced species. Thus, the presence of a premature termination codon does not necessarily alter cytoplasmic mRNA stability, nor does cytoplasmic instability account for the quantitative deficiency of beta-globin mRNA. Our observations suggest that a premature termination codon alters intranuclear stability and/or nuclear to cytoplasmic transport of the beta-globin mRNA.
TdT-positive acute leukemia with monocytoid characteristics: clinical, cytochemical, cytogenetic, and immunologic findings
Authors: Cuttner J , Seremetis S , Najfeld V , Dimitriu-Bona A , Winchester RA
Published: October 2019

Thirteen patients with acute leukemias that were difficult to classify by the use of cytochemical staining and terminal deoxyribonucleotidyl transferase (TdT) activity are reported. The phenotype of the leukemic cells was characterized by the presence of mature or early monocyte lineage antigens and intense Ia antigen expression detected by monoclonal antibodies, terminal deoxytransferase activity, and cytochemical features, including both Sudan black B and periodic acid- Schiff activity. The mean age of this group of patients was 60 years. Five patients had leukemia occurring after chemotherapy or radiotherapy of a prior malignant disease, and two patients had a refractory anemia prior to development of acute leukemia. These patients had a low response rate to chemotherapy. This series of leukemia appears to form a distinct nosologic entity, representing a leukemic transformation among early cells of the monocyte lineage, resulting in a predominant neoplastic cell that is less mature than either the French-American- British M4 acute myelomonocytic leukemia or M5 acute monoblastic leukemia. The presence of terminal deoxytransferase activity was interpreted as indicating the primitive state of the cells in the differentiation sequence, rather than as implying any significance with respect to lineage.
Characterization of immature T cell subpopulations in neonatal blood
Authors: Griffiths-Chu S , Patterson JA , Berger CL , Edelson RL , Chu AC
Published: October 2019

A series of monoclonal antibodies directed against T cell differentiation antigens was used to identify circulating T cells in normal human neonates. Twenty-five cord blood samples, taken after cesarean or vaginal delivery, and 16 venous blood samples from normal adult controls were examined using monoclonal antibodies in an indirect immunofluorescence technique. The percentage of circulating OKT3 positive (pan-T cell) cells was significantly lower in the neonatal blood (52.8%) compared with the adult controls (74.9%) (P less than .001). Of the cord mononuclear cells, 58% showed reactivity with OKT10 (common thymocyte antigen) compared with only 7% in adult controls (P less than .001). The helper:suppressor T cell ratio was lower in cord blood (1:2) as compared with 1:3 for adult blood (P less than .005) as observed in these studies. These figures reflect the presence of a significant population (25%) of immature T cells in cord blood that expresses simultaneously both helper (OKT4) and suppressor (OKT8) phenotypes. Twenty-four percent of the T cells in cord blood also expressed OKT6 antigen (cortical thymocyte), a feature not found in adult blood. Double-labeling studies characterized a previously undescribed blood T cell phenotype, which was simultaneously OKT6 and OKT3 (pan-T cell) positive; of the cells reactive with OKT3, 43% also were positive with OKT6. This study reveals the presence of immature populations of T cells in normal human neonatal blood exhibiting phenotypes characteristic of normal developing thymocytes and a previously undescribed cell phenotype.
The beta-adrenergic receptor adenylate cyclase complex of Rauscher murine erythroleukemia cells and its response to erythropoietin-induced differentiation
Authors: Sytkowski AJ , Kessler CJ
Published: October 2019

Rauscher murine erythroleukemia cells, grown continuously in vitro, undergo erythroid differentiation in response to the hormone erythropoietin. Therefore, they serve as an important model system with which to examine critical biochemical aspects of this developmental process. Intact, uninduced Rauscher cells possess a functional beta- adrenergic receptor-adenylate cyclase complex. The adrenergic agonists, isoproterenol, epinephrine, and norepinephrine, exhibited activation constants (Kact) of 0.1, 0.5, and 20 mumol/L, respectively. Thus, the beta-receptor-cyclase complex of Rauscher cells is apparently one of the most sensitive of all erythroid cells reported thus far. The epinephrine-stimulated cyclic adenosine monophosphate (cAMP) response was inhibited by propranolol, alprenolol, and hydroxybenzylpindolol, with inhibition constants (KI) of 3.8, 2.2, and 0.1 nmol/L, respectively. Using [125I]-iodohydroxybenzylpindolol as ligand, uninduced Rauscher cells were shown to possess 1,100 receptors/cell, with an equilibrium dissociation constant (KD) of 400 pmol/L. Erythropoietin, but not dimethylsulfoxide, induction caused a specific increase in receptor density to 3,300/cell on differentiating Rauscher cells. This is the first demonstration of membrane receptor regulation by erythropoietin that may be important in the complex interplay of hormonal effects during erythropoiesis.
Characterization of erythropoietin produced by IW32 murine erythroleukemia cells
Authors: Choppin J , Lacombe C , Casadevall N , Muller O , Tambourin P , Varet B
Published: October 2019

IW32 is a recently described murine erythroleukemia cell line that produces an erythropoietic factor similar to erythropoietin by in vivo and in vitro bioassays and without species specificity. Biochemical characteristics of IW32 erythropoietic factor and sheep or mouse plasma erythropoietins were compared. Murine colonies derived from erythroid colony-forming units (CFU-E) in plasma clot culture were used as the bioassay system. Both IW32 erythropoietic factor and sheep plasma erythropoietin were stable in the pH range of 3 to 10, after exposure to denaturing agents (8 mol/L urea, 4 mol/L guanidine hydrochloride, 1% sodium dodecyl sulfate), to a reducing agent (0.1 mol/L 2- mercaptoethanol) and to an oxidizing agent (5 mmol/L sodium metaperiodate). Only the combination of 0.1 mol/L 2-mercaptoethanol and 1% sodium dodecyl sulfate resulted in a significant loss of activity. IW32 erythropoietic factor and murine plasma erythropoietin were similarly precipitated by ethanol and ammonium sulfate. IW32 erythropoietic factor eluted as a single major peak after gel exclusion chromatography, with an estimated molecular weight of 45,000 daltons. Results were identical using supernatants from cultures in the presence of and absence of fetal calf serum. The supernatant of IW32 cells cultured without serum induced erythroid colonies after seven days on normal human bone marrow nonadherent mononuclear cells cultured in serum-free conditions. All these results made it very likely that IW32 cells produce an authentic erythropoietin. This cell line would be very useful for the study of murine erythropoietin.
Association of an interleukin abnormality with the T cell defect in Hodgkin's disease
Authors: Ford RJ , Tsao J , Kouttab NM , Sahasrabuddhe CG , Mehta SR
Published: October 2019

The cellular immune defect in untreated Hodgkin's disease (HD) has long been recognized. This defect appears to be responsible for at least some of the morbidity and ultimately the mortality associated with the disease. In recent years, many studies have shown that the T cell component of the immune response is the apparent site where the defect in HD exists and where the immunoregulatory abnormalities that may account for the deficit are observed. The discovery of the lymphokines and monokines, comprising the human interleukin system, has elucidated some aspects of the regulatory control of the functional pathways involved in T lymphocyte activation and proliferation. The interleukin system can therefore provide the framework to dissect immunodeficiency states, such as that seen in HD. The present study indicates that HD patients' interleukin 1 (IL1) response appears to be normal, as is their T cell proliferative response to exogenous IL2. Interleukin 2 production by HD patients' peripheral blood mononuclear cells, however, is decreased when compared with age/sex-matched controls. The inability to generate IL2 after appropriate stimulation may reflect either a primary cellular defect or a regulatory defect, such as excessive immunosuppression, giving rise to the characteristic T cell hyporesponsiveness seen in HD.
Platelet metabolism during storage of platelet concentrates at 22 degrees C
Authors: Kilkson H , Holme S , Murphy S
Published: October 2019

The development of methods for storing platelet concentrates (PCs) at 22 degrees C for transfusion has been predominantly empiric, with minimal knowledge of metabolic events occurring during storage. It is known that a decrease in pH due to accelerated production of lactic acid in hypoxic conditions is a major cause for loss of platelet viability. In the current studies, we have measured metabolic parameters such as O2 and glucose consumption rates and CO2 and lactic acid production rates. We have also determined the O2 and CO2 transport capacities of various containers and the buffering capacity of plasma. The O2 consumption rate was 1.10 +/- 0.16 (SD) nmol/min/10(9) platelets. In well-oxygenated systems, lactic acid formation was 1.74 +/- 0.12 nmol/min/mL PC for PCs with a platelet count of 1 to 2 X 10(9)/mL; and 0.52 mol of glucose was consumed per 1 mol lactic acid produced. In a completely oxygen-free system, lactic acid production increased 5–8-fold. These calculations suggest that 85% of energy generation is derived through oxidative metabolism and that glucose may not be the primary substrate for this metabolism. Bicarbonate concentration, initially 22.1 +/- 1.6 mEq/L, decreased 1.41 +/- 0.18 nEq/min/mL PC for PCs with counts 1 to 2 X 10(9) platelets/mL. The loss of bicarbonate was caused by displacement by lactic acid and as a consequence of spontaneous CO2 efflux from the container. CO2 production, 2.3 +/- 0.4 nmol/min/10(9) platelets, was derived from oxygen consumption and the CO2 liberated from bicarbonate as it was consumed. A rapid fall in pH to levels below 7.0 (22 degrees C) took place when the bicarbonate concentration fell below 5 mEq/L as lactate concentrations reached 20 to 25 mmol/L. A further increase in lactate concentration from 25 mmol/L to 40 mmol/L correlated with a further fall in pH to 5.8. Thus, the ultimate storage life of a PC is determined by continuous lactate production and the fixed buffering capacity of plasma and by the glucose concentration of the PC. With knowledge of these parameters, methods for predicting pH as a function of time, platelet count, and O2 and CO2 transport capability of the container have been developed as guidelines for future work.
Microtubule coils in spread blood platelets
Authors: White JG , Sauk JJ
Published: October 2019

The fate of the circumferential bundle of microtubules in activated platelets has been a subject of disagreement. Thin sections of stimulated platelets fixed at multiple intervals following exposure to aggregating agents have revealed that the circumferential band is constricted into a tight ring around centrally concentrated organelles. However, studies of detergent-resistant platelet cytoskeletons fixed and either negatively stained or critical point dried after activation on polylysine-coated grids have revealed that microtubule rings disappear, leaving only fragments in the peripheral cytoplasm of spread cells. The present study has employed immunofluorescence on glass slides and the whole mount technique with detergent extraction and either negative staining or critical point drying to evaluate the fate of microtubules in surface-activated platelets treated with or without the microtubule stabilizing agent, taxol. Significant numbers of microtubule coils were visible in control and taxol-treated platelets stained indirectly with a fluorescein-coupled antibody to tubulin 30 to 60 minutes after surface activation on glass. Coils of microtubules were also visible in dendritic forms and in significant numbers of spread platelets on negatively stained or critical point dried whole mounts in the electron microscope. The findings support the concept that microtubule disassembly is not an integral step in early phases of platelet activation.
Characteristic expression of glycosphingolipid profiles in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60
Authors: Nojiri H , Takaku F , Tetsuka T , Motoyoshi K , Miura Y , Saito M
Published: October 2019

Changes of glycosphingolipids (GSLs) in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60 cells were investigated by high-performance thin-layer chromatography (HPTLC), with special reference to morphological and functional changes, such as phagocytosis and nitroblue tetrazolium (NBT) reduction. Nine molecular species of neutral GSLs and 13 or more species of sialo-GSLs, ie, gangliosides, were detected on the HPTLC chromatograms for untreated HL-60 cells. The major components were ceramide dihexoside (CDH), GM3, and sialo-paragloboside (SPG). When HL- 60 cells were induced to differentiate into both myeloid mature cells and macrophage-like cells in vitro, no new molecular species of GSLs specific for one of the cell differentiations was induced, but distinctive quantitative changes in the GSL composition were definitely observed between the two cell differentiations. During the myeloid differentiation induced by either dimethylsulfoxide (DMSO) or retinoic acid (RA), CDH, paragloboside (PG), and gangliosides having longer sugar moieties characteristically increased with a concomitant decrease of GSLs with shorter sugar chains, such as ceramide monohexoside (CMH) and GM3, and the GSL composition profile of myeloid differentiation- induced HL-60 cells became more similar to that of normal human granulocytes. However, some marked differences were noted between the induced HL-60 cells and the normal granulocytes, especially in the ganglioside compositions. These differences might reflect either some deficiency in the in vitro myeloid differentiation or some leukemic properties of HL-60 cells. In marked contrast to the change of GSL composition during myeloid differentiation, a remarkable increase of GM3, with a concurrent marked decrease of CDH, was observed in the process of cell differentiation into macrophage-like cells with 12-O- tetradecanoyl-phorbol-13-acetate (TPA), which suggested an increase in the biosynthesis of GM3. These results demonstrate that HL-60 cells express distinct GSL profiles, depending not only on maturation stages but also on differentiation directions.
The polymerization of nickel (II) hemoglobin S under aerobic conditions
Authors: Alston K , Park CM , Rodgers DW , Edelstein SJ , Nagel RL
Published: October 2019

Sickle hemoglobin (Fe(II)HbS) reconstituted with nickel(II)protoporphyrin IX yields an artificial hemoglobin (Ni(II)HbS), the first heme-substituted hemoglobin shown to mimic the polymerization of deoxyHbS. Unlike Fe(II)HbS, Ni(II)HbS does not bind oxygen and therefore polymerizes under aerobic conditions. While the polymer solubility coefficient (Csat) for Ni(II)HbS is elevated about 2 g/100 mL compared with that for deoxy Fe(II)HbS, hemoglobin concentration in the polymer phase is the same. Electron micrographs of thin sections of embedded Ni(II)HbS reveal 20-nm-diameter fibers indistinguishable from those seen with deoxygenated native HbS. Nickel(II)HbS can be used in studies on the sickling process and on antisickling agents that could not previously be done or were difficult to execute because of the need for an anaerobic environment.
Richter's syndrome with different immunoglobulin light chains and different heavy chain gene rearrangements
Authors: van Dongen JJ , Hooijkaas H , Michiels JJ , Grosveld G , de Klein A , van der Kwast TH , Prins ME , Abels J , Hagemeijer A
Published: October 2019

In a patient with Richter's syndrome, the chronic lymphocytic leukemia (CLL) expressed lambda, mu, and delta immunoglobulin (lg) chains and the non-Hodgkin lymphoma (NHL) kappa, mu, and delta lg chains. The difference in lg light chain expression suggests that the CLL and NHL are independent malignancies, or that the oncogenic event occurred in a B cell differentiation stage after the heavy chain gene rearrangements but before the selection of the light chain. Analysis of DNA by Southern blotting revealed that the lg heavy chain genes of the two malignancies were rearranged in a different way. We therefore conclude that in this patient the NHL cannot be regarded as a progression of the CLL but should most likely be considered as an independent B cell malignancy, which arose in a susceptible host.