bluuebunny/crossref_metadata_2025 huggingface dataset:
Amidolytic assay of human factor XI in plasma: comparison with a coagulant assay and a new rapid radioimmunoassay
Authors: Scott CF , Sinha D , Seaman FS , Walsh PN , Colman RW
Published: October 2019

The traditional coagulant assay for plasma factor XI suffers from a relatively high coefficient of variation, the need for rare congenitally deficient plasma, and a poor correlation between precision and sensitivity. We have developed a simple functional amidolytic assay for factor XI in plasma using the chromogenic substrate PyrGlu-Pro-Arg- p-nitroanilide (S-2366). After inactivation of alpha 1-antitrypsin, CI inhibitor, and other plasma protease inhibitors with CHCI3, plasma was incubated with kaolin, in the absence of added calcium, which limited the enzymes formed to those dependent on contact activation. Soybean trypsin inhibitor was used to minimize the action of kallikrein on the substrate. Once the reaction was complete, corn trypsin inhibitor was used to inactive factor XIIa, the enzyme generated by exposure of plasma to negatively charged surfaces, which had activated the factor XI. The assay is highly specific for factor XI, since plasma totally deficient in that zymogen yielded only 1%-3% of the enzymatic activity in normal plasma under identical conditions. The requirements for complete conversion of factor XI to XIa in plasma within 60 min were, respectively, factor XII, 0.6 U/ml, and high molecular weight kininogen, 0.2 U/ml. Prekallikrein was not an absolute requirement for complete activation but did accelerate the reaction. The intraassay coefficient of variation was 3.4%, and the mean of 35 normal plasmas was 1.00 U +/- 0.24 SD. In addition, a new rapid radioimmunoassay was devised using staphylococcal protein A as the precipitating agent for a complex of factor XI antigen with monospecific rabbit antibody. The mean was 1.01 U +/- 0.30 SD. The correlation coefficients for amidolytic versus coagulant and amidolytic versus radioimmunoassay were r = 0.95 for the former and 0.96 for the latter. Thus, a simple, accurate amidolytic assay and a radioimmunoassay have been devised for measuring factor XI in plasma that correlate well with the coagulant activity of factor XI, as determined in our laboratory.
Mechanisms for the oxidation of reduced gluthathione by stimulated granulocytes
Authors: Sagone AL Jr , Husney RM , O'Dorisio MS , Metz EN
Published: October 2019

We have reported previously that human granulocytes have an irreversible fall in their endogenous reduced soluble sulfhydryls following zymosan stimulation. In the present study, we demonstrate that stimulated granulocytes release one or more reactive oxygen species (ROS) with the capacity to oxidize reduced glutathione (GSH). One or more of these compounds is stable enough to be detected in the supernatant. The formation of these stable oxidants appears to require H2O2 and heme or a heme-containing enzyme. However, once formed, the compound reacts with GSH without these factors. The ROS is not superoxide or hydroxyl radical, since neither superoxide dismutase nor the hydroxyl scavengers, mannitol and benzoic acid, change the rate of the reaction. Methionine has recently been demonstrated to be oxidized to a sulfoxide by a reactive oxygen species that is dependent on H2O2 and heme for its production. We found that methionine could directly react with the same ROS that degrades GSH. The ROS also has the capacity to oxidize iodide and fix halogen to proteins. Our data indicate that stimulated granulocytes release a ROS with the capacity to oxidize GSH, react with methionine, and oxidize and fix I- to protein. The compound, therefore, appears dependent on H2O2 and the myeloperoxidase system for its production, and is either hypochlorous acid (HOCI) or a compound derived from HOCI, such as a chloramine. The capacity of GSH to react with this ROS suggests an additional role for this tripeptide in cellular protection against oxidant injury.
A model of intramedullary hematopoietic microenvironments based on stereologic study of the distribution of endocloned marrow colonies
Authors: Lambertsen RH , Weiss L
Published: October 2019

Hematopoietic colonies were studied in the marrow of alternate fraction- irradiated mice by light microscopic stereology to investigate the microenvironmental organization of marrow. Separate analyses of the relative colony cell density of undifferentiated, granulocytic, erythrocytic, and macrophage colonies in four marrow zones were carried out at 3, 4, and 5 days postirradiation (PI) for all colonies, all periarterial colonies, and all non-periarterial colonies. The results demonstrate a differential colony cell distribution that does not appear to be due to a preferential distribution of certain colony types around arteries. Undifferentiated colony cells showed a consistent predilection for endosteal and periarterial regions, with the majority of colony cells occurring along bone. Erythrocytic colony cells proliferated initially in intermediate and central marrow zones and along arteries. Granulocytic colony cells occurred in all areas at 3 days PI, but increased in density along bone thereafter. Macrophage colony cells occurred in all zones at 4 days PI, but at 5 days were concentrated in subosteal and central regions. Macrophage colonies also occurred periarterially. To explain these findings and the organization of normal bone marrow, we present a detailed model of the microenvironmental organization of intramedullary hematopoiesis. This model portrays the stroma as engendering distinct microenvironments for stem cell replication, stem cell commitment, and early progenitor cell proliferation.
Ph1-positive acute lymphoblastic leukemia with a 14q+ chromosome abnormality
Authors: Maekawa T , Sonoda Y , Taniwaki M , Misawa S , Abe T , Takino T
Published: October 2019

A 13-yr-old Japanese female with acute lymphoblastic leukemia (ALL) that was associated with a Philadelphia chromosome (Ph1) as well as a 14q+ chromosome abnormality is reported. The cell surface phenotype of leukemic cells was determined to be non-T, non-B ALL on the basis of positive Ia-like antigen, terminal deoxynucleotidyl transferase activity, and lack of receptors for sheep erythrocytes, surface immunoglobulin, or intracytoplasmic mu-chain immunoglobulin. The combination of both a Ph1 and a 14q+ has not been reported previously in patients with ALL.
Refractory dysmyelopoietic anemia and acute leukemia
Authors: Rosenthal DS , Moloney WC
Published: October 2019

One hundred and seventeen consecutive patients with refractory dysmyelopoietic anemia (RDA) were followed and studied over 6 yr. All RDA cases had at least two marrow cell lines involved with dysplasia and usually all three. Morphologically, the series could be divided into 55 cases that had primarily “erythroid” hyperplasia and 62 cases with primarily “myeloid” dysplasia. There was a significantly higher number of acute leukemias in the myeloid group, 21/62 (33.9%), than in the erythroid group, 7/55 (12.7%). Of the 117 cases, 64 had marrow culture studies performed using implanted diffusion chambers (DC) in an irradiated rat host. There was a highly statistically significant correlation between the development of leukemia and abnormal growth in DC during the RDA stage.
The availability of therapeutic hydroxocobalamin to cells
Authors: Hall CA , Begley JA , Green-Colligan PD
Published: October 2019

Hydroxocobalamin (OH-Cbl), when used to treat vitamin B12 deficiency, is better retained by the body than is cyanocobalamin (CN-Cbl), but the availability to cells has not been studied systematically. In a series of experiments, we compared the uptake and internalization of OH-Cbl and CN-Cbl bound to transcobalamin II (TCII) by a human cell model, the HeLa cell. TCII-OH-Cbl was: (1) taken up in larger amounts per unit time, (2) the greater uptake was not a consequence of more effective attachment to receptors of TCII-Cbl nor to a more rapid regeneration of receptors, (3) the difference was expressed during the phase of internalization of TCII-Cbl, (4) with CN-Cbl, the stages of binding to receptors plus internalization were more readily reversed, and (5) larger amounts of OH-Cbl were internalized and converted to active coenzyme forms of Cbl. When injected into a healthy person, 200 micrograms of OH-Cbl was better retained in the circulation than 200 micrograms of CN-Cbl. When added in vitro in equivalent amounts, more OH-Cbl was bound to nonspecific plasma proteins. This greater and broader binding neither enhanced nor interfered with the uptake of Cbl by cells, which was determined by the amount of Cbl bound physiologically to TCII. It was concluded that OH-Cbl is a more efficient form of treatment of the common types of Cbl deficiency, principally because of the better retention, which requires less frequent injections, but also because of greater availability to cells.
Therapy of severe aplastic anemia with anti-human thymocyte globulin and androgens: the effect of HLA-haploidentical marrow infusion
Authors: Doney K , Dahlberg SJ , Monroe D , Storb R , Buckner CD , Thomas ED
Published: October 2019

Fifty-four patients with severe aplastic anemia were treated with horse anti-human thymocyte globulin (ATG) and androgens. Thirty of these patients also received an infusion of HLA-haploidentical marrow cells. Only those patients having evidence of hematologic recovery within 3 mo after ATG therapy were considered responders to the immunosuppressive regimen. Of 53 patients evaluable for response, 21 had complete or partial responses and 7 had minimal improvement by defined criteria. The remaining patients did not respond or died. Factors correlated with response to therapy included a short duration of aplasia and a high admission granulocyte count. Thirty-six patients (66.7%) are surviving between 18 and 43 mo, and 18 have died. Deaths were due to hemorrhage and/or infection. Short duration of aplasia and high granulocyte counts also correlated with survival, as did younger age. Four patients with complete or partial responses had a recurrence of severe aplasia 6–17 mo after their first course of ATG. Three of these patients were retreated with ATG (and oxymetholone in two cases). All three had second responses to therapy, but two of the three have had second relapses. The fourth patient responded to oxymetholone alone, but died after a second relapse. Mismatched marrow infusion had no effect on the incidence of response or survival.
Regulation of normal human blood neutrophilic, macrophagic, and eosinophilic committed stem cell proliferation by autologous blood T lymphocyte subsets
Authors: Chikkappa G , Phillips PG
Published: October 2019

The influence of T lymphocyte (T cell) subsets on the proliferation of the blood neutrophilic, macrophagic, and eosinophilic cluster and colony (aggregate) forming cells (CFC) was evaluated. The T cells and the CFC-enriched B-null cells (mononuclear cells depleted of monocytes and T cells) and null cells (B-null) cells depleted of B lymphocytes) were separated from the blood of normal individuals. The population enriched for OKT4 (T4 cells) monoclonal antibody-binding T helper/inducer cells and for OKT8 (T8 cells) monoclonal antibody- binding T suppressor/cytotoxic cells were separated from the T cells or mononuclear cells depleted of monocytes by negative enrichment using an immunoadherence “panning” method. These separated cell populations were cultured separately, and the B-null or null cells were cocultured with the T, T4, and T8 cells in agar medium in the presence and absence of an exogenous source of colony-stimulating activity (CSA). The cultures were evaluated at day 14. The B-null and null cells, but not the T cells or subsets, contained CFC. The number of CFC proliferated from the B-null or null cells increased significantly (p less than 0.001) in cocultures with unseparated T and T8 cells in a dose-dependent manner. The T4 cells neither promoted nor inhibited the CFC growth. The T8 population was a better promoter (p less than 0.01) of CFC growth than the unseparated T cells. This suggests that the CFC promoting effect of the unseparated T cells is probably due to the influence of the T8 subset present within the T cells. The proportion of the neutrophilic, macrophagic, and eosinophilic CFC that proliferated in these cultures was comparable under the influence of the T and T8 cells. The results of these studies indicate that the T8 population, which is the suppressor in the classical immune system, promotes the growth of the blood CFC. We speculate that the T cells are involved in the regulation of granulopoiesis in vivo.
A novel phospholipid in irreversibly sickled cells: evidence for in vivo peroxidative membrane damage in sickle cell disease
Authors: Jain SK , Shohet SB
Published: October 2019

In individuals with sickle cell disease, a variable number of irreversibly sickled cells (ISC) is present that may contribute to the pathophysiology of sickle cell anemia. The present study was undertaken to determine the possible role of membrane lipid peroxidation in the genesis of ISC. After 24 hr of simple aerobic incubation, sickle cells accumulated 2–3 times more malonyldialdehyde (MDA), an end product of lipid peroxidation, than normal cells. To assess the possibility of peroxidative damage in ISC in vivo, ISC were separated from sickle blood using Stractan density gradients. Lipid extracts of the untreated ISC-enriched fraction of sickle blood showed significant fluorescence and contained a novel phospholipid:MDA adduct that was not seen in control cells. Taken together, these observations suggest that ISC have previously undergone lipid peroxidative damage and the accumulation of MDA in vivo.
Autoantibodies against the platelet glycoprotein IIb/IIIa complex in patients with chronic ITP
Authors: Woods VL Jr , Oh EH , Mason D , McMillan R
Published: October 2019

Chronic idiopathic thrombocytopenic purpura (ITP) is caused by an antibody reactive with platelet-associated antigens. The present studies provide direct evidence that some patients with chronic ITP have autoantibodies against the platelet glycoprotein (GP) IIb/IIIa complex. Microtiter wells, coated with a monoclonal antibody (2G12) specific for GPIIb/GPIIIa were reacted with GPIIb/GPIIIa contained in a platelet extract. Control wells containing the same antibody were reacted with a cell extract containing no GPIIb/GPIIIa. After washing, the wells were reacted with patient or control plasma, and IgG binding was detected using 125I-Fab2-anti-human IgG. Assay values were expressed as binding ratios (cpm GPIIb/GPIIIa wells/cpm control wells). Plasma from 5 of 56 patients with chronic ITP had ratios (1.36–3.14) greater than 3 standard deviations above the mean (+/- SD) of control plasmas--0.93 +/- 0.12. Elevated values were also noted in two patients with anti-P1A1 antibody (ratios greater than 30) and in one patient with Hodgkin's disease and an ITP-like syndrome (ratio 1.53). Normal values were noted in 34 patients with a variety of immune and nonimmune diseases. Plasma from two of the positive ITP patients was reacted with 125I-surface-labeled platelets and, after solubilization, the IgG and bound antigen were precipitated with Staph-A. Autoradiographs from SDS- PAGE electrophoresis of the Staph-A-bound proteins shows two radioactive bands consistent in size with GPIIb and GPIIIa.
Characterization of glucocorticoid receptors in animal lymphoblastic disease: correlation with response to single-agent glucocorticoid treatment
Authors: Bell R , Cotter S , Lillquist A , Sallan S , McCaffrey R
Published: October 2019

The clinical significance of initial DEAE chromatography of glucocorticoid binders in lymphoblastic disease was evaluated in an animal model. Domestic cats and dogs with lymphoblastic disease were treated with prednisone, 2 mg/kg/day, for 14 days, and the outcome of therapy was correlated with DEAE chromatograms of glucocorticoid binders, using 3H-triamcinolone as ligand. Six of 30 animals had a single-peak low-salt binder species, similar to that seen in a subset of human leukemia, and none of these responded. Of the 29 animals with chromatograms identical to normal tissues, 6 had a complete response and another 11 a partial response. This distribution of responders is statistically significant (p = 0.02). Thus, the leukemia-associated single-peak DEAE species appears to be associated with glucocorticoid resistance, as defined by clinical responsiveness. In contrast, the two- peak normal pattern is a necessary, but insufficient, criterion for defining responsive disease.
Evidence that platelet density depends on the alpha-granule content in platelets
Authors: van Oost BA , Timmermans AP , Sixma JJ
Published: October 2019

The relation between platelet buoyant density and beta-thromboglobulin (beta-TG), a marker for platelet alpha-granule content, was assessed by three independent approaches. (1) Platelets were separated on iso- osmolar discontinuous Stractan density gradients into five fractions, ranging in density from 1.061 g/ml to 1.091 g/ml (20 degrees C). The beta-TG content (mean +/- SD, n = 17) increased with the platelet density from 27.8 +/- 8.6 micrograms beta-TG/10(9) cells (20% less- dense platelets) up to 65.6 +/- 15.5 micrograms beta-TG/10(9) cells (15% most-dense platelets). (2) Activation of platelets in platelet- rich plasma with thrombin, adenosine diphosphate, collagen, or epinephrine resulted in a decreased density of the platelets. This was only seen when there was simultaneous secretion of beta-TG. (3) The less-dense and the more-dense platelet fractions, after isolation by density gradient centrifugation, were separately treated with thrombin. After complete degranulation, the density distribution of the originally less-dense and more-dense platelets were identical and were much narrower than the density distribution of resting platelets.
Monoclonal antibodies differentiating between monocytic and nonmonocytic variants of AML
Authors: Linch DC , Allen C , Beverley PC , Bynoe AG , Scott CS , Hogg N
Published: October 2019

Blast cells from 70 cases of acute myeloid leukemia (AML), with a preponderance of monocytic variants (M4 and M5), were stained with a panel of 10 monoclonal antibodies (MoAb) that react with normal myeloid cells. Two McAb, UCHMI and UCHALF, stained cells from nearly every case of M4 and M5 leukemia, but not cells from cases of M1/M2 leukemia. Two other antibodies, E11 and UC45, were also specific for the monocytic leukemias, but stained cells from a lesser proportion of cases. The antibodies TG1, OKM1, Smo, DA2, 28, and 5.5 stained a differing proportion of cases in both the M1/M2 and M4/M5 subtypes. Routine morphology and alpha-naphthyl acetate esterase (ANAE) staining were largely in agreement with the antibody findings, though the interpretation of the fluoride sensitivity of ANAE was apparently misleading in 10% of cases. It is concluded that these antibodies, particularly UCHMI and UCHALF, will be of considerable value in the more precise subtyping of AML and that some of this panel of antibodies will be valuable tools for the investigation of normal monocytic differentiation.
Increased tissue factor activity of monocytes/macrophages isolated from canine renal allografts
Authors: Rothberger H , Barringer M , Meredith J
Published: October 2019

Kidney allografting was performed in a group of ten beagles, and viable leukocytes infiltrating the transplanted organs were isolated during episodes of acute rejection 5 or 6 days postoperatively. These infiltrate populations, consisting predominantly of lymphocytes and monocytes/macrophages, were found to have significantly increased amounts of procoagulant activity relative to control leukocytes isolated from circulating blood and lymph. Using nonspecific esterase staining in an agar microclot assay, procoagulant activity in the infiltrate leukocytes was found to reside in monocytes/macrophages rather than other coisolated cell types. By contrast, control monocytes from blood had no activity in this microclot assay. Procoagulant activity in the infiltrate cells was characterized as tissue factor. Increased amounts of this activator of the extrinsic pathway, as found in infiltrate monocytes/macrophages, may initiate clotting reactions and fibrin deposition within allografts.
Chronic myelomonocytic leukemia according to FAB classification: analysis of 35 cases
Authors: Solal-Celigny P , Desaint B , Herrera A , Chastang C , Amar M , Vroclans M , Brousse N , Mancilla F , Renoux M , Bernard JF
Published: October 2019

Thirty-five patients who fulfilled the FAB diagnosis criteria of chronic myelomonocytic leukemia (CMML), i.e., myelodysplastic features, monocytosis over 10(9)/liter, bone marrow monocyte infiltration, blast cells less than 5% in the peripheral blood and less than 30% in the bone marrow, are analyzed. CMML appears as an entity distinct from myelodysplastic and myeloproliferative disorders. Splenomegaly, anemia, thrombocytopenia, leukocytosis with monocytes and granulocytic cells in all stages of development, increased blood and urine lysozyme levels without renal failure, and polyclonal hyperimmunoglobulinemia are its main clinical and biologic features. With conventional cytotoxic drugs (6-mercaptopurine, hydroxyurea), the prognosis of CMML appears poor (median survival 475 days). None of the clinical hematologic or biologic parameters tested had a significant effect on prognosis. As other chemotherapy trials seemed necessary, we recently administered small doses of cytosine-arabinoside (ARA-C) to six patients over several consecutive days and obtained a complete remission in four. These preliminary results must be confirmed by larger series using the diagnostic criteria proposed by the FAB cooperative group.
A functional assay of protein C in human plasma
Authors: Sala N , Owen WG , Collen D
Published: October 2019

A three-step spectrophotometric assay was developed for measuring functional protein C (PC) in human plasma. The assay is based on: (1) adsorption of citrated platelet-poor plasma on barium citrate and elution of the vitamin K-dependent factors with EDTA; (2) activation of PC by incubation of the mixture of vitamin K-dependent factors with a complex of thrombin and its endothelial cell cofactor, thrombomodulin; (3) addition of antithrombin III and heparin to the system to inhibit thrombin and other coagulation enzymes generated during incubation and measurement of the activated PC with a synthetic (chromogenic) substrate. The assay appears to be specific for PC because: (a) PC- depleted plasma (by immunoadsorption) is inactive; (b) addition of purified PC to PC-depleted plasma reconstitutes its activity; and (c) no enzymatic activity is generated in the absence of the thrombin- thrombomodulin complex. Mixtures of a normal plasma pool with PC- depleted plasma yielded an amount of enzymatic activity proportional to the fraction of normal plasma. Using this as a standard curve, the amount of PC in the plasma of 23 normal subjects was 97% +/- 15%. The within-assay coefficient of variation was 3.5% and the between-assay coefficient 6.5%. A linear correlation (r = 0.86) was found between PC as measured with the functional assay and with a radioimmunoassay. In 3 patients with congenital PC deficiency, the functional PC level was 37% +/- 9% and the antigen level 64% +/- 11%. It is concluded that the present assay may be used for reliable and accurate estimation of activatable PC in human plasma.
Cellular and molecular mechanisms of the bone marrow sparing effects of the glucose chloroethylnitrosourea chlorozotocin
Authors: Byrne P , Tew K , Jemionek J , MacVittie T , Erickson L , Schein P
Published: October 2019

1,(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and other chloroethylnitrosourea anticancer agents in clinical use produce severe and cumulative bone marrow toxicity. Chlorozotocin, a glucose analogue, has demonstrated reduced hematologic toxicity while retaining full antitumor activity. The biochemical-pharmacologic properties of chlorozotocin and CCNU were compared in human bone marrow. After a 2-hr incubation with a 0.1-mM drug concentration, total cellular uptake of chlorozotocin in whole marrow was 2.47 +/- 0.80 pmole/10(4) cells and was not significantly different compared to the uptake of 1.94 +/- 0.53 pmole/10(4) cells with CCNU. The quantitative alkylation of bone marrow DNA by chlorozotocin, 22.8 +/- 1.2 pmole/mg DNA, was equivalent to that produced by CCNU, 22.9 +/- 0.5 pmole/mg DNA. Bone marrow was separated into 14 fractions by centrifugal elutriation. CCNU uptake was found to be greater than that of chlorozotocin in 3 fractions that were primarily composed of lymphocytes, monocytes, and normoblasts. Chlorozotocin uptake was greater than CCNU in 6 fractions that contained primarily mature and immature myeloid cells as well as the highest CFU-GM activity. The two drugs produced a comparable degree of DNA strand breakage and DNA-protein cross-linking as measured by alkaline elution of pooled fractions of elutriated bone marrow. DNA interstrand crosslinking was not found with either drug. The most significant finding of this study is the differences in the site of drug alkylation by chlorozotocin and CCNU in bone marrow chromatin. Endonuclease digestions with MCN, DNase I, and DNase II showed nonrandom alkylation of specific regions of chromatin by the two drugs: CCNU demonstrated a preferential binding to the transcriptionally active regions of chromatin, whereas chlorozotocin predominantly alkylated the transcriptionally inactive regions. These data suggest that the lethal damage of nitrosourea alkylation in human bone marrow is principally expressed in transcriptionally active regions of chromatin.
Erythropoietin production in a primary culture of human renal carcinoma cells maintained in nude mice
Authors: Hagiwara M , Chen IL , McGonigle R , Beckman B , Kasten FH , Fisher JW
Published: October 2019

The present studies report erythropoietin (Ep) production in primary cultures of a human renal carcinoma from a patient with erythrocytosis that has been serially transplanted to BALB/c nude mice. The levels of erythropoietin in the culture media were estimated using the exhypoxic polycythemic mouse assay (EHPCMA), fetal mouse liver erythroid colony- forming technique (FMLC), and a radioimmunoassay (RIA). The spent culture media of the exponentially growing cells contained less than 10 mU/ml of Ep measured by RIA. However, after the cells became confluent, Ep levels (RIA) in the spent media showed a marked increase to approximately 300 mU/ml. Ep levels estimated using the FMLC and EHPCMA were approximately 2/3 and 1/10, respectively, of those measured by RIA. Rabbit antiserum to highly purified human urinary Ep (70,400 U/mg protein) was utilized for immunocytochemical (peroxidase-antiperoxidase method) localization of Ep in the cultured cells. Very few of the cells in exponential growth exhibited Ep-like immunoreactivity, whereas intense Ep-like immunoreactivity was observed in the cytoplasm of the cells maintained in culture for a prolonged period after reaching confluency. The most intense staining was observed in some of the cells forming domes. The domes developed after the cells reached confluency, and their numbers increased with increasing time in confluent culture, in parallel with the increase in Ep levels in the spent media. This primary cell culture system of a renal cell carcinoma maintained in nude mice, which produces immunologically and biologically active Ep, may provide a useful model for studies of the mechanism of Ep production.
A randomized study of the efficacy of consolidation therapy in adult acute nonlymphocytic leukemia
Authors: Cassileth PA , Begg CB , Bennett JM , Bozdech M , Kahn SB , Weiler C , Glick JH
Published: October 2019

The Eastern Cooperative Oncology Group conducted a randomized study to determine the efficacy of consolidation therapy in prolonging the duration of complete remission (CR) in adults with acute nonlymphocytic leukemia (ANLL). Induction chemotherapy with daunorubicin, cytosine arabinoside, and 6-thioguanine (DAT) yielded CR in 65% of 283 patients with ANLL, aged 16–69. For patients aged 60–69, the CR rate was 58%. Of 184 patients in CR, 146 patients were then randomized to receive either maintenance therapy with weekly cytosine arabinoside and 6-thioguanine alone (69 patients) or two courses of reduced doses of DAT 1 mo apart, before commencing the same maintenance program (77 patients). Consolidation therapy resulted in hematologic toxicity, but was not lethal in any of the eligible patients. Patients receiving consolidation plus maintenance therapy experienced a longer CR duration (40 wk) and disease-free survival at 2 yr (28%) than did those patients receiving maintenance therapy alone (34 wk and 14%, respectively). These differences are not statistically significant. These results suggest that approaches to consolidation therapy employing reduced doses of the induction therapy regimen can have, at best, only a small benefit. For consolidation therapy to provide substantial improvement in CR duration, intensive regimens with non-cross-resistant drugs will be required.
Induction of proliferation of purified human myeloid progenitor cells: a rapid assay for granulocyte colony-stimulating factors
Authors: Griffin JD , Sullivan R , Beveridge RP , Larcom P , Schlossman SF
Published: October 2019

The proliferation and differentiation of granulocyte and monocyte progenitor cells (CFU-C) in vitro is dependent on the presence of a group of closely related glycoproteins termed colony-stimulating factors (CSF). In order to investigate the interaction of these factors with CFU-C, we purified CFU-C from the peripheral blood of chronic myeloid leukemia patients with an immune rosette technique using specific monoclonal antibodies (mean 74-fold enrichment, 45% cloning efficiency). Colony formation by purified CFU-C demonstrated an absolute dependence on an exogenous source of CSF. Liquid culture of small aliquots of enriched CFU-C with CSF-containing medium resulted in a rapid, time- and concentration-dependent induction of DNA synthesis as measured by 3H-thymidine incorporation. This specific CSF induction of DNA synthesis by enriched CFU-C was used to develop a microassay system for CSF activity. CSF activity could be reproducibly quantitated in 24–48 hr. The proliferating cells in this assay system were shown to be myeloid progenitor cells by examining the morphology of their progeny and by determining the surface antigen phenotype of the responding cells (Ia+, T3-, B1-, Mo1-). This microassay provides a quantitative assessment of CSF activity that may be useful in the purification of human CSF and in the generation of monoclonal antibodies to CFU-C surface structures.
Platelet-associated IgG in patients with lymphoma
Authors: Berkman AW , Kickler T , Braine H
Published: October 2019

Levels of platelet-associated IgG (PA-IgG) were studied in 72 patients with Hodgkin–s (HD) and non-Hodgkin–s lymphoma (NHL). Thirty-nine percent of patients with HD and 20% of patients with NHL had elevated PA-IgG levels. There was a positive correlation between disease activity and the presence of PA-IgG in HD and NHL. In patients with HD, PA-IgG strongly correlated with extent of disease and may serve as a marker of disease activity. PA-IgG may have facilitated platelet destruction in 5 of 11 thrombocytopenic patients with HD and increased PA-IgG and in 2 patients with HD and increased PA-IgG who developed severe thrombocytopenia when treated with chemotherapy.
Lack of T cell antigen expression on hairy cells of B cell origin after in vitro exposure to PHA
Authors: Han T , Dadey B , Pollard C , Barcos M , Bloom ML , Minowada J , Ozer H
Published: October 2019

The malignant monoclonal population in hairy cell leukemia (HCL) has been variously ascribed to be of myeloid, B, or even T cell origin. Recent data have been interpreted as suggesting that hairy cells (HC) may concomitantly or serially express both B and T surface determinants, a phenomenon which, if verified, would be unique among the lymphoproliferative malignancies. Data described here, however, demonstrate that (1) at least the majority of HCL are phenotypically of B cell derivation, and (2) the initial B cell phenotype is retained and solely expressed on cultured as well as phytohemagglutinin (PHA) activated monoclonal malignant HC.
Speciation in the baboon and its relation to gamma-chain heterogeneity and to the response to induction of HbF by 5-azacytidine
Authors: DeSimone J , Schroeder WA , Shelton JB , Shelton JR , Espinueva Z , Huynh V , Hall L , Zwiers D
Published: October 2019

In the baboon (Papio species), the two nonallelic gamma-genes produce gamma-chains that differ at a minimum at residue 75, where isoleucine (I gamma-chain) or valine (V gamma) may be present. This situation obtains in baboons that are sometimes designated as Papio anubis, Papio hamadryas, and Papio papio. However, in Papio cynocephalus, although the I gamma-chains are identical with those in the above mentioned types, the V gamma-chains have the substitutions ala----gly at residue 9 and ala----val at residue 23. The V gamma-chains of P. cynocephalus are called V gamma C to distinguish them from the V gamma A-chains of P. anubis, etc. A single cynocephalus animal has been found to have only normal I gamma-chains and I gamma C-chains (that is, glycine in residue 9, valine in 23, and isoleucine in 75). When HbF is produced in response to stress with 5-azacytidine, P. anubis baboons respond with greater production than do P. cynocephalus, and hybrids fall between. Minimal data on P. hamadryas and P. papio suggest an even lower response than P. cynocephalus. As HbF increases under stress, the ratio of I gamma to V gamma-chains changes from the value in the adult or juvenile baboon toward the ratio in the newborn baboon. However, it does not attain the newborn value. The V gamma A and V gamma C-genes respond differently to stress. In hybrids, the production of V gamma A- chains exceeds that of V gamma C-chains. A controlling factor in cis apparently is present and may be responsible for the species-related extent of total HbF production. It may be concluded that the more primitive the cell in the erythroid maturation series that has been subjected to 5-azacytidine, the more active is the I gamma-gene.
Polymorphic and monomorphic HLA-DR determinants on human hematopoietic progenitor cells
Authors: Falkenburg JH , Jansen J , van der Vaart-Duinkerken N , Veenhof WF , Blotkamp J , Goselink HM , Parlevliet J , van Rood JJ
Published: October 2019

The expression of monomorphic Ia-like antigens and polymorphic (allotypic) HLA-DR determinants on CFU-GM, BFU-E, CFU-E, and CFU-GEMM was studied in bone marrow and peripheral blood cells from normal healthy individuals. Using various polyclonal and monoclonal anti-Ia- like antibodies, the presence of HLA-DR backbone antigens was shown on all hematopoietic progenitor cells (HPC) studied, both in complement- dependent cytotoxicity assays and in fluorescence-activated cell sorting (FACS). The expression of allotypic determinants was demonstrated on all HPCs, using the HLA-DR typing sera anti-HLA-DR1, 2, 3, 4, 5, and 7. The Class II antigen MT-2 was also shown on all HPCs, using both monoclonal and alloantisera, whereas the MB-1 (DC-1) determinant could not be demonstrated on HPCs. This might open the possibility of removing MB-1-positive malignant cells from the graft in autologous bone marrow transplantation.
A morphologic and immunologic study of the large granular lymphocyte in neutropenia with T lymphocytosis
Authors: Chan WC , Check I , Schick C , Brynes RK , Kateley J , Winton EF
Published: October 2019

We report four patients with expansion of a unique population of lymphocytes that is consistently associated with neutropenia. Two patients also had rheumatoid arthritis and autoantibodies. The lymphocytes contained many cytoplasmic azurophilic granules, which possessed strong acid phosphatase activity. Multiple cytoplasmic parallel tubular arrays were observed ultrastructurally. These granular lymphocytes showed the T suppressor/cytotoxic cell phenotype (E+, OKT3+, OKT8+, OKT4-, OKM1-, OKI1-) and exhibited antibody-dependent cell-mediated cytotoxic activity but little or no natural killer cytotoxicity. They did not respond to recall antigens, concanavalin A, or pokeweed mitogen, but the cells from one patient did respond to phytohemagglutinin. No in vitro suppressor cell activity on mitogenic responses of allogeneic cells and on mixed lymphocyte cultures could be demonstrated. There was no evidence of suppression of immunoglobulin synthesis in vivo. It is uncertain that the expansion of this subset of lymphocytes represents a leukemic process. Their constant association with neutropenia, however, raises the possibility that the increase in large granular lymphocytes and neutropenia might be pathogenetically related.
The elevation of blood levels of zinc protoporphyrin in mice following whole body irradiation
Authors: Walden TL , Draganac PS , Farkas WR
Published: October 2019

Elevation of zinc protoporphyrin (ZPP) levels in the blood has served as an indicator of lead poisoning and iron deficiency anemia for many years. We have discovered that sublethal doses of whole body irradiation with x-rays also elevates ZPP 2–3-fold over normal levels. The ZPP level does not begin to increase until days 12–14 postirradiation and peaks between days 18 and 20 before returning to normal levels between days 28 and 35. Increasing the radiation dose delays the onset of the rise in ZPP, but does not affect the magnitude of the elevation. At lethal doses, ZPP elevation is not observed. Neither of the two previously described mechanisms that cause elevations of ZPP, namely iron deficiency and inhibition of ferrochelatase, are responsible for the radiation-induced elevation of ZPP. The elevation of ZPP appears to be correlated with the recovery of the hematopoietic system from radiation injury.
Elevated low density lipoprotein receptor activity in leukemic cells with monocytic differentiation
Authors: Vitols S , Gahrton G , Ost A , Peterson C
Published: October 2019

The receptor-mediated degradation of 125I-low density lipoprotein (LDL) was compared in normal white blood cells and leukemic cells. The cells were isolated from the peripheral blood and bone marrow of healthy subjects and patients with newly diagnosed leukemia. The cells from most of the 40 consecutive patients with acute myelogenous leukemia showed markedly higher degradation rates as compared to mononuclear cells and granulocytes from peripheral blood and nucleated cells from the bone marrow of healthy individuals. Leukemic cells from patients with monocytic (FAB-M5) or myelomonocytic leukemia (FAB-M4) exhibited the highest degradation rates. The rate of receptor-mediated degradation of 125I-LDL was also high in leukemic cells from all three patients with chronic myelogenous leukemia in blast crisis, as well as in two of three patients with acute undifferentiated leukemia. In contrast, leukemic cells isolated from two patients with acute lymphoblastic leukemia showed low rates. In most cases, there was little difference in LDL receptor activity between leukemic cells isolated from peripheral blood and those from bone marrow. Hypocholesterolemia was a frequent finding in the leukemic patients. There was an inverse correlation between the plasma cholesterol level and the rate of receptor-mediated degradation of 125I-LDL by the leukemic cells. Studies are now in progress to investigate the possibilities of using LDL as a carrier of cytotoxic drugs in the treatment of leukemia.
Human platelet fibrinogen gamma chain structure
Authors: Mosesson MW , Homandberg GA , Amrani DL
Published: October 2019

Human plasma fibrinogen is produced by liver parenchymal cells. Such molecules contain two classes of gamma-chains (gamma A, gamma'), which differ with respect to their COOH-terminal sequences. When fibrin is crosslinked in the presence of factor XIIIa and Ca2+, three types of gamma-dimer are formed (gamma A-gamma A; gamma A-gamma'; gamma'- gamma'). A separate fibrinogen pool is located in platelet alpha- granules. We analyzed this fibrinogen to determine whether gamma'- chains were present to the same extent (7%) that they are found in plasma fibrinogen. Electrophoretic analysis (Laemmli system) of reduced samples of the clot that formed subsequent to release of fibrinogen from thrombin-stimulated washed platelets, revealed a single crosslinked gamma-dimer band in the gamma A-gamma A position. Material collected into EDTA-containing buffer and subsequently crosslinked in the presence of added factor XIII and Ca2+ also revealed a gamma A- gamma A dimer band. This finding was further investigated by Western blotting of reduced gel specimens that had been reacted with an anti- gamma-chain antibody followed by 125I-labeled protein A. A single type of gamma-chain, gamma A, was present in the fibrinogen from a Triton X- 100 or 10 M urea platelet lysate, or in noncrosslinked fibrin obtained from thrombin-treated platelets. Crosslinked reduced fibrin from thrombin-treated platelets or that prepared from the Triton lysate revealed a single type of gamma-dimer, gamma A-gamma A. We conclude that there are no gamma'-chains (less than 1%) in platelet fibrinogen. This structural difference from hepatic fibrinogen probably results from differences in the processing and/or regulation of the fibrinogen gamma-chain gene in megakaryocytes.
Abnormal expansions of polyclonal large to small size granular lymphocytes: reactive or neoplastic process?
Authors: Semenzato G , Pizzolo G , Ranucci A , Agostini C , Chilosi M , Quinti I , De Sanctis G , Vercelli B , Pandolfi F
Published: October 2019

Morphological, immunologic, and functional properties of peripheral blood cells from two patients with chronic proliferations of granular lymphocytes are described. Cells from both patients showed a heterogeneous pattern from both a morphological and immunologic standpoint, indicating a polyclonal, rather than a monoclonal, expansion of these cells. In fact, both large and small-to-medium-sized granular lymphocytes were observed, and different percentages of positivity were found in the analysis with a large panel of monoclonal antibodies. Serologic and histologic features support the hypothesis that this lymphocytosis could be secondary to bacterial or viral infections rather than a primary event, suggesting that these patients may have chronic reactive immunoregulatory disorders.
Fetal hemoglobin synthesis in erythroid cultures in hereditary persistence of fetal hemoglobin and beta o-thalassemia
Authors: Weinberg RS , Antonarakis SE , Kazazian HH Jr , Dover GJ , Orkin SH , Lenes AL , Schofield JM , Alter BP
Published: October 2019

To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3′ to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had “adult” and not “fetal” progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.
Collagen-factor VIII/von Willebrand factor protein interaction
Authors: Kessler CM , Floyd CM , Rick ME , Krizek DM , Lee SL , Gralnick HR
Published: October 2019

Factor VIII/von Willebrand factor (FVIII/vWF) protein interaction with collagen was studied by incubating plasma or purified FVIII/vWF with purified type I fibrillar collagen. Collagen adsorbed FVIII/vWF activities in a similar time and concentration-dependent manner from normal plasma, plasmas from classical and variant type von Willebrand's disease (vWD), and from purified FVIII/vWF. Incubation with denatured collagen or fibrin, produced in the presence or absence of fibronectin, showed no adsorption of FVIII/vWF. Examination of the multimeric structure of the remaining unadsorbed FVIII/vWF protein by agarose gel electrophoresis and autoradiography showed that the largest multimers had been adsorbed to the collagen. Studies of the adsorbed FVIII/vWF protein when eluted from collagen showed that it complemented the alterations in multimeric structure observed in the supernatants following collagen exposure. The multimeric structure of normal plasma following collagen adsorption resembled that of unadsorbed type IIb plasma; however, the collagen-adsorbed normal plasma did not produce enhanced ristocetin-induced platelet aggregation ( RIPA ). This phenomenon, therefore, must not be due solely to absence of large multimers from type IIb FVIII/vWF protein. The adsorbed multimers of FVIII/vWF protein may act as a subendothelial collagen-platelet bridge to promote primary hemostasis.
Stabilization of thrombin-activated porcine factor VIII:C by factor IXa phospholipid
Authors: Lollar P , Knutson GJ , Fass DN
Published: October 2019

The activation of porcine factor X by an enzymatic complex consisting of activated factor IX (factor IXa), thrombin-activated factor VIII:C (factor VIII:Ca), phospholipid vesicles, and calcium was studied in the presence of an irreversible inhibitor of factor Xa, 5-dimethylamino- naphthalene-1-sulfonyl-glutamyl-glycyl-arginyl- chloro met hyl ketone ( DEGR -CK). The formation of factor Xa was measured continuously by monitoring the increase in solution fluorescence intensity that occurs upon formation of DEGR -factor Xa. Omission of any component from the enzymatic complex reduced the reaction rate to a negligible level. In the presence of fixed excess factor IXa, the velocity of factor X activation was linearly dependent on the concentration of factor VIII:C, and thus, provided a plasma-free assay of factor VIII:C. Activation of factor VIII:C by 0.1 NIH U/ml thrombin in the presence of factor IXa, phospholipid vesicles, and calcium, followed at variable time intervals by the addition of factor X and DEGR -CK, was complete within 5 min, as judged by the fluorometric assay, and resulted in little or no loss of factor VIII:C activity over a period of 20 min; whereas, activation in the absence of either IXa or phospholipid vesicles decreased the half-life of factor VIII:C to approximately 5 min. Analysis of 125I-factor VIII:C-derived activation peptides by sodium dodecyl sulfate polyacrylamide gel radioelectrophoresis revealed identical results, regardless of whether factor IXa and/or phospholipid vesicles were included in the activation, suggesting that the lability of factor VIII:Ca is not due to a major alteration of its primary structure. We conclude that the activated porcine factor VIII:C molecule is stabilized markedly because of its interaction with factor IXa and phospholipid.