bluuebunny/crossref_metadata_2025 huggingface dataset:
The amino-terminal 29- and 72-Kd fragments of fibronectin mediate selective monocyte recruitment
Authors: Lohr KM , Kurth CA , Xie DL , Seyer JM , Homandberg GA
Published: October 2019

Proteolytic fragments of fibronectin (Fn) can possess properties not inherent to intact Fn. Previously, only mixtures of low molecular weight Fn fragments, and the 120-Kd fibroblastic cell-binding segment, but not intact Fn, were shown to be selectively chemotactic for human monocytes (MOs). In order to determine if other structural domains of Fn were responsible, we tested six Fn fragments. The amino-terminal 72- Kd fragment at 1.5 microns was about 75% as potent as zymosan-activated serum (ZAS). Its amino-terminal 29-Kd degradation product at 1.0 micron was about one third as potent as ZAS. Checkerboard analysis confirmed chemotaxis. Complexing gelatin to 72-Kd fragments reduced MO chemotaxis by 28% to 30%. Reducing disulfide bonds in 29- and 72-Kd segments had no effect. A synthetic peptide containing the thrombin cleavage site between the 29- and 50-Kd segments of the 72-Kd fragment was chemotactic. The 50-, 190/170-, 35-, and 160/150/120-Kd fragments, and intact Fn were not chemotactic for MOs. The data suggest that the 72-Kd fragment and its 29-Kd subfragment are additional Fn fragments that mediate selective MO chemotaxis. We speculate that proteinases present at inflammatory sites can liberate such fragments that selectively recruit MOs.
Molecular and hematologic characterization of Scottish-Irish type (epsilon gamma delta beta)zero thalassemia
Authors: Trent RJ , Williams BG , Kearney A , Wilkinson T , Harris PC
Published: October 2019

The DNA deletion associated with an example of (epsilon gamma delta beta)zero thalassemia (Scottish-Irish type) was characterized. The deletion is approximately 205 kb in length and involves the epsilon, G gamma, A gamma, delta, and beta globin genes. The breakpoint is located 263 bp 3′ to exon 3 of the beta globin gene. An LI (KpnI) repeat element approximately 320 bp in size is found at the 3′ end of the novel DNA sequence. Different clinical phenotypes for three heterozygous neonates suggest that the deletion alone does not predict severity of (epsilon gamma delta beta)zero thalassemia at this age.
Transplantation potential of peripheral blood stem cells induced by granulocyte colony-stimulating factor
Authors: Molineux G , Pojda Z , Hampson IN , Lord BI , Dexter TM
Published: October 2019

The major effect of granulocyte colony-stimulating factor (G-CSF) is to induce neutrophilia in previously untreated animals or after chemotherapy or marrow transplantation in humans, primates and rodents. In addition, it has been reported that migration of committed progenitor cells to the blood occurs during G-CSF therapy. In this article, by using sex mismatched transplants and a molecular probe for Y-chromosome specific DNA sequences, we show that among the peripheral blood cells during G-CSF therapy are substantial numbers of primitive stem cells capable of (1) reconstituting the hematopoietic system in the long term, and (2) making a contribution to the lymphoid populations of the thymus, in radiation ablated recipients. These data suggest that blood from patients treated with G-CSF may provide a convenient source of the most primitive stem cells for autologous or allogeneic bone marrow transplantation.
Clonal B-cell populations in patients with idiopathic thrombocytopenic purpura
Authors: van der Harst D , de Jong D , Limpens J , Kluin PM , Rozier Y , van Ommen GJ , Brand A
Published: October 2019

Idiopathic thrombocytopenic purpura (ITP) may be associated with other autoimmune diseases and the development of lymphoproliferative malignancies. In Sjogren's disease, Graves' disease, and essential mixed cryoglobulinemia, which are also associated with the development of B-cell neoplasia, clonal B-cell expansions have been detected. Eleven patients with ITP were investigated for the presence of a clonal excess (CE) using kappa-lambda flow cytometry and DNA analysis for rearrangement of immunoglobulin heavy and light chain genes in blood and/or spleen lymphocytes. In 10 of 11 patients, clonal B-cell populations were found by one or both tests. In three of these patients, oligoclonal B-cell populations were suggested by the combined findings. In all four patients with a small paraproteinemia, the isotype was confirmed by either flow cytometry or DNA rearrangement analysis. Our data suggest that the oligoclonal expansions are not restricted to CD5+ B cells, as in the majority of patients this subset was below the detection level of flow cytometry or DNA rearrangement analysis. None of the patients developed clinical manifestations of malignant lymphoma during a follow-up period of 10 to 44 months after sampling. We conclude that clonal excess populations of B cells are not a unique feature of malignant lymphoma, but may occur in autoimmune diseases, suggesting a benign (oligo)clonal B-cell proliferation.
In vitro drug sensitivity of cells from children with leukemia using the MTT assay with improved culture conditions
Authors: Pieters R , Loonen AH , Huismans DR , Broekema GJ , Dirven MW , Heyenbrok MW , Hahlen K , Veerman AJ
Published: October 2019

The knowledge about drug resistance in childhood leukemias and acute lymphoblastic leukemia (ALL) in general is limited. This is because of the lack of a suitable in vitro drug sensitivity assay, which is in part due to low in vitro ALL cell survival. We recently adapted the highly efficient 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay to test cells from ALL patients and showed that its results were comparable with those of the DiSC assay, up to now the most valid but laborious assay. In this study, in vitro drug sensitivity was assessed in cells from 82 children with leukemia, 79 of whom had ALL, with the MTT assay. Dose response curves were obtained for 6-mercaptopurine, 6-thioguanine (6-TG), prednisolone (Pred), daunorubicin (DNR), vincristine (VCR), cytosine arabinoside (Ara-C), L- asparaginase (L-Asp), mafosfamide, and mustine. A cytotoxic effect of methotrexate could be detected in only a few cases. Large interindividual differences in drug sensitivity were detected. Compared with leukemia cells from newly diagnosed patients, leukemia cells from relapsed patients were significantly more in vitro resistant to 6-TG, Pred, Ara-C, mafosfamide and mustine but not to DNR, VCR, and L-Asp. Improvements of culture medium and methods to increase MTT reduction were studied. From 10 components tested, addition of insulin and bovine serum albumin to serum-containing medium improved ALL cell survival. Addition of succinate did not increase the amount of MTT reduction. We conclude that the in vitro MTT assay highly facilitates large-scale studies on drug resistance of ALL patients that can lead to rational improvements in existing treatment protocols.
Platelet-derived neutrophil adherence-inhibiting factor in humans
Authors: Iwabuchi K , Yamashita T
Published: October 2019

The effect of constituents of human platelets on leukocyte adherence was examined. Adherence-inhibiting factors (AIFs), which strongly inhibited neutrophil adherence to glass, were present in both cytosol and granule fractions of human platelets. On the Superose 6 gel chromatography (Pharmacia LKB Biotechnology, Uppsala, Sweden), the granular AIF was eluted as a single active peak (2,600 Kd), whereas cytosolic AIFs were eluted at two different positions (2,600 and 480 Kd). When platelets were stimulated by thrombin, granular AIF was released extracellularly without releasing a cytosolic marker. Using DE32 anion exchange chromatography and Superose 6 gel filtration, granular AIF was completely purified. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis suggests that granular AIF consists of two subunits with molecular masses of approximately 340 and 190 Kd. Purified granular AIF inhibited human neutrophil adherence to glass, plastic, and type IV collagen-coated plastic, whereas it did not affect monocyte adherence. These results suggest that granular AIF inhibits neutrophil adherence not only via nonspecific adsorption sites, but also via type IV collagen receptors.
Human neutrophils release the Leu-8 lymph node homing receptor during cell activation
Authors: Berg M , James SP
Published: October 2019

The Leu-8 molecule, the human homologue of the murine MEL-14 peripheral lymph node homing receptor, is expressed on neutrophils in both species and may be important in localization of cells to sites of inflammation. Most circulating human neutrophils express the Leu-8 molecule, and activation of neutrophils with phorbol myristate acetate causes a rapid decline in Leu-8 membrane fluorescence staining within 15 minutes. Northern blot analysis of total cellular RNA from neutrophils demonstrated two species of Leu-8 messenger RNA, a major one of 2.4 kb and a minor one of 1.9 kb. Because two different Leu-8 cDNA clones were obtained from human lymphocytes that were predicted to encode both transmembrane and phosphatidylinositol (PI)-anchored forms of the molecule, experiments were conducted to determine whether Leu-8 is anchored to neutrophils by a PI-anchor. There was a slight decrease in expression of Leu-8 on neutrophils when they were treated with PI- specific phospholipase C (PI-PLC). However, Leu-8 was abundant on neutrophils obtained from a patient with paroxysmal nocturnal hemoglobinuria. To determine the fate of the Leu-8 molecule during cell activation, neutrophils were labeled with 125I-anti-Leu-8. During activation antibody was rapidly lost from the cell surface and was not internalized, suggesting that Leu-8 is released from the cell membrane during cell activation. When cell extracts of neutrophils were compared with extracts of lymphoid cells by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and immunoblotting, the Leu-8 species expressed on neutrophils had a significantly higher and more variable relative mobility (70 to 120 Kd for neutrophils v 70 Kd for Jurkat T cells). In addition, Leu-8 molecules were detected in the supernatants of activated neutrophils. These results indicate that human neutrophils express a high-molecular-weight form of the Leu-8 molecule that has a conventional transmembrane anchor and is rapidly released from the membrane during activation. The loss of the Leu-8 membrane glycoprotein during activation may be a mechanism for rapid alteration of neutrophil adhesion characteristics.
Relative oral efficacy and acute toxicity of hydroxypyridin-4-one iron chelators in mice [see comments]
Authors: Porter JB , Morgan J , Hoyes KP , Burke LC , Huehns ER , Hider RC
Published: October 2019

The relationship between the oral efficacy and the acute toxicity of hydroxypyridin-4-one iron chelators has been investigated to clarify structure-function relationships of these compounds in vivo and to identify compounds with the maximum therapeutic safety margin. By comparing 59Fe excretion following oral or intraperitoneal administration of increasing doses of each chelator to iron-overloaded mice, the most effective compounds have been identified. These have partition coefficients (Kpart) above 0.3 in the iron-free form with a trend of increasing oral efficacy with increasing Kpart values (r = .6). However, this is achieved at a cost of increasing acute toxicity, as shown by a linear correlation between 59Fe excretion increase per unit dose and 1/LD50 (r = .83). A sharp increase in the LD50 values is observed for compounds with Kpart values above 1.0, suggesting that such compounds are unlikely to possess a sufficient therapeutic safety margin. Below a Kpart of 1.0, acute toxicity is relatively independent of lipid solubility. All the compounds are less toxic by the oral route than by the intraperitoneal route, although iron excretion is not significantly different by these two routes. At least five compounds (CP51, CP94, CP93, CP96, and CP21) are more effective orally than the same dose of intraperitoneal desferrioxamine (DFO) (P less than or equal to .02) or orally administered L1(CP20) (P less than or equal to .02).
A new method for isolation of reticulocytes: positive selection of human reticulocytes by immunomagnetic separation
Authors: Brun A , Gaudernack G , Sandberg S
Published: October 2019

A method for isolating pure reticulocytes from leukocyte-depleted blood of normal persons is presented. The separation was achieved using an immunomagnetic technique. A monoclonal mouse antibody against human transferrin receptor was bound to magnetic beads conjugated with sheep antimouse antibody. The recovery of reticulocytes from peripheral blood was 15% to 42%. Blood used for isolation of reticulocytes could be stored for 4 days at 22 degrees C without altering the yield of reticulocytes. At 37 degrees C incubation, the reticulocytes matured rapidly and the transferrin receptor was found to have a half-life of 16 hours. The activity of several enzymes and the amount of creatine and hemoglobin A1C were measured both in the reticulocytes and peripheral blood. Of the enzymes, porphobilinogen deaminase had the best discriminatory power with a ratio of 8.8 between reticulocytes and peripheral red blood cells. The ratio for creatine was 16.7. The ability to isolate pure human reticulocytes, released after normal erythropoiesis, will offer new possibilities in the study of these cells.
Restoration of phagocyte function by interferon-gamma in X-linked chronic granulomatous disease occurs at the level of a progenitor cell
Authors: Ezekowitz RA , Sieff CA , Dinauer MC , Nathan DG , Orkin SH , Newburger PE
Published: October 2019

Phagocytes from X-linked chronic granulomatous disease (X-CGD) patients are deficient in their ability to generate superoxide because of a defective gene that encodes a heavy chain of cytochrome b, a critical component in the superoxide-generating pathway. Previously we have shown that a single in vivo treatment of selected X-CGD patients with interferon-gamma (INF-gamma) resulted 14 days later in near-normal levels of superoxide generation by phagocytes. The effect persisted for 28 days. This prolonged effect suggested that the lymphokine affected progenitor cells. In this study, we examined progenitor-derived colonies from the peripheral blood from this unusual X-CGD kindred. Progenitor-derived colonies examined before treatment were unable to generate superoxide as visualized by lack of nitro blue tetrazolium (NBT) reduction compared with normal controls. By contrast, colonies derived 7 days after a single INF-gamma injection were able to generate superoxide as shown by increased NBT reduction. Colonies harvested 21 days after treatment contained only rare cells capable of NBT reduction. Our results indicate that INF-gamma can reprogram the myeloid progenitor cells to express a partially corrected phenotype. This corrected phenotype is later expressed in daughter cells.
Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients
Authors: Kolb HJ , Mittermuller J , Clemm C , Holler E , Ledderose G , Brehm G , Heim M , Wilmanns W
Published: October 2019

Three patients with hematologic relapse after bone marrow transplantation for chronic myelogenous leukemia were treated with interferon alpha and transfusion of viable donor buffy coat. All had complete hematologic and cytogenetic remission, which persisted 32 to 91 weeks after treatment. In two patients graft-versus-host disease developed and was treated by immunosuppression. These results are an example of adoptive immunotherapy without cytoreductive chemotherapy or radiotherapy in human chimeras.
Lack of evidence for infection of or effect on growth of hematopoietic progenitor cells after in vivo or in vitro exposure to human immunodeficiency virus
Authors: Molina JM , Scadden DT , Sakaguchi M , Fuller B , Woon A , Groopman JE
Published: October 2019

The pathogenesis of the hematologic abnormalities commonly observed in patients with acquired immunodeficiency syndrome (AIDS) is incompletely understood. We report here that in vitro growth of myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells from six patients with AIDS was not significantly different from that of normal human immunodeficiency virus (HIV) seronegative donors: 25.3 +/- 5 CFU-GM per 5 x 10(4) low density marrow cells and 33.5 +/- 5 BFU-E were observed in AIDS patients versus 32.7 +/- 5 CFU-GM and 42.1 +/- 5 BFU-E in controls. Furthermore, no HIV-DNA in individual colonies (CFU-GM and BFU-E) could be detected using the polymerase chain reaction (PCR) technique, although HIV-1 DNA was detected in peripheral blood mononuclear cells from the same patients. Similarly, normal bone marrow cells exposed in vitro to different isolates of HIV or recombinant purified HIV-1 envelope glycoprotein (gp) 120 did not exhibit any difference in growth of CFU-GM or BFU-E as compared with mock exposed bone marrow cells. HIV- 1 DNA could not be detected by the PCR technique in individual colonies derived from HIV exposed marrow. This study suggests that committed myeloid and erythroid progenitors from AIDS patients are responsive to hematopoietic growth factors in vitro and do not appear to contain HIV- 1 DNA. Also, HIV or its envelope gp did not alter the growth of hematopoietic progenitor cells in vitro. No evidence of HIV infection of progenitor cells could be demonstrated. Impaired hematopoiesis in patients with AIDS may not be related to direct effects of HIV on committed progenitor cells.
Interleukin-1 beta (IL-1 beta) induces thrombocytosis in mice: possible implication of IL-6
Authors: Kimura H , Ishibashi T , Shikama Y , Okano A , Akiyama Y , Uchida T , Maruyama Y
Published: October 2019

We administered recombinant human interleukin-1 beta (IL-1 beta), the common mediator of inflammation process, to C57B1/6 male mice (0.5 microgram, every 12 hours over five times) intraperitoneally and consequently induced a remarkable thrombocytosis. Day 1 was designated as the following day of the last injection in the morning. A significant thrombocytosis was observed on days 1 through 5 with a peak on day 2 (162 +/- 9 x 10(4)/mm3) compared with the control mice injected with heated IL-1 beta (101 +/- 11 x 10(4)/mm3). A striking increase in mean size of marrow megakaryocytes was noted on days 1 and 2. The incorporation of 75Se-selenomethionine into circulating platelets as a measure of platelet production was about 2.3 times higher in IL-1 beta-treated mice than in control mice. To determine which factor(s) is responsible for elicited thrombocytosis, the in vitro studies and bioassays for several hematopoietic factors were performed. IL-1 beta by itself did not stimulate megakaryocytopoiesis in vitro, suggesting that the thrombocytosis is attributed to other factor(s) via IL-1 beta stimulation. Serum colony-stimulating factor (CSF) activity after a single IL-1 beta (0.5 microgram) injection, monitored by colony assay with 10% tested serum, peaked at 3 hours. Formed colonies were mostly granulocyte (G) and granulocyte-macrophage (GM)-types, and studies using rabbit anti-mouse GM-CSF serum or using human marrow as target cells showed that the CSF activity of the tested serum consisted of, at least, GM-CSF and G-CSF. Addition of IL-3 concomitantly with the tested serum gave rise to a greater number of megakaryocytic colonies. Serum IL-3, monitored by IL-3-dependent cell line 32D clone 5, and erythropoietin activities were not detected at serum level in IL-1 beta-treated mice. Serum IL-6 assay by IL-6- dependent mouse hybridoma cell line MH-60.BSF2 showed high levels of the tested serum with a peak at 2.5 hours with no detection at 10 hours after the injection. Heated IL-1 beta caused an increase of neither IL- 6 nor CSF activities. Our data suggest that the thrombocytosis induced by IL-1 beta is mediated by IL-6 or a combination of IL-6 and other cytokine(s), and that IL-6 may play a regulatory role in platelet production in vivo.
Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways
Authors: van Deventer SJ , Buller HR , ten Cate JW , Aarden LA , Hack CE , Sturk A
Published: October 2019

Endotoxemia was evoked by bolus injection of Escherichia coli endotoxin (2 ng/kg body weight) in six healthy subjects to investigate the early kinetics of cytokine release in relation to the development of clinical and hematologic abnormalities frequently seen in gram-negative septicemia. The plasma concentration of tumor necrosis factor (TNF) increased markedly after 30 to 45 minutes, and reached a maximal level after 60 to 90 minutes. In each volunteer, the initial increase of plasma interleukin 6 (IL-6) concentrations occurred 15 minutes after the initial TNF increase, and maximal IL-6 concentrations were reached at 120 to 150 minutes. A transient increase in body temperature and pulse rate occurred simultaneously with the initial TNF and IL-6 increases, whereas a significant decrease in blood pressure occurred after 120 minutes. These changes were proportional to the changes in TNF and IL-6 concentrations. Coagulation activation, as assessed by a rise of prothrombin fragments and thrombin-antithrombin III complexes, was noted after 120 minutes, in the absence of activation of the contact system. A two- to sixfold increase in the concentrations of tissue plasminogen activator (t-PA) and von Willebrand factor antigen indicated endothelial cell activation. This increase started at 120 and 90 minutes, respectively. The release of t-PA coincided with activation of the fibrinolytic pathway, as measured by plasmin-alpha 2-antiplasmin complexes. The fibrinolytic activity of t-PA was subsequently offset by release of plasminogen activator inhibitor, observed 150 minutes after the endotoxin injection, and reaching a peak at 240 minutes. No complement activation was detected. These results show that in humans endotoxin induces an early, rapidly counteracted fibrinolytic response, and a more long-lasting activation of thrombin by a mechanism other than contact system activation. In addition, our data suggest that endotoxin-induced leukopenia and endothelial cell activation are mediated by TNF.
Free protein S levels are elevated in familial C4b-binding protein deficiency
Authors: Comp PC , Forristall J , West CD , Trapp RG
Published: October 2019

In plasma, 40% of the protein S is free and functions as a cofactor for the anticoagulant effects of activated protein C. The remaining 60% of protein S is complexed to C4b-binding protein and is functionally inactive. A family with hereditary C4b binding protein deficiency has been identified with C4b-binding protein levels in an affected father and daughter of 37 micrograms/mL and 23 micrograms/mL, respectively; these values are significantly below the normal range for this protein of 180 micrograms/mL +/- 44 micrograms/mL (mean +/- 2 SD). The total protein S (free + bound) is normal in these individuals (23.2 micrograms/mL and 17.8 micrograms/mL, respectively; normal 19.1 micrograms/mL +/- 6.0 micrograms/mL). The free protein S levels are markedly increased at 22.5 micrograms/mL and 17.4 micrograms/mL, respectively (normal 5.9 micrograms/mL +/- 2.4 micrograms/mL). This experiment of nature shows that total protein S levels in plasma are not affected by the absence of C4b-binding protein and that chronic elevation of free protein S is not associated with increased hemorrhagic tendencies.
Regulatory control of the terminal complement proteins at the surface of human endothelial cells: neutralization of a C5b-9 inhibitor by antibody to CD59
Authors: Hamilton KK , Ji Z , Rollins S , Stewart BH , Sims PJ
Published: October 2019

Functionally inhibitory antibody to the plasma membrane complement inhibitor CD59 has been used to investigate control of the terminal complement proteins at the endothelial cell surface. Antibodies against purified human erythrocyte CD59 (polyclonal anti-CD59 and monoclonal antibodies [MoAbs] 1F1 and 1F5) were found to bind specifically to monolayers of cultured human umbilical vein endothelial cells, and by Western blotting to recognize an 18- to 21-Kd endothelial protein. When bound to the endothelial monolayer, anti-CD59 (immunoglobulin G or Fab fragment) potentiated membrane pore formation induced upon C9 binding to C5b-8, and augmented the C5b-9-induced cellular responses, including stimulated secretion of von Willebrand factor and expression of catalytic surface for the prothrombinase enzyme complex. Although potentiating endothelial responses to the terminal complement proteins, anti-CD59 had no effect on the response of these cells to stimulation by histamine. Taken together, these data suggest that human endothelial cells express the CD59 cell surface inhibitor of the terminal complement proteins, which serves to protect these cells from pore- forming and cell-stimulatory effects of the C5b-9 complex. These data also suggest that the inactivation or deletion of this cell surface regulatory molecule would increase the likelihood for procoagulant changes in endothelium exposed to complement activation in plasma.
Monoclonal antibody to the interferon-inducible protein Leu-13 triggers aggregation and inhibits proliferation of leukemic B cells
Authors: Evans SS , Lee DB , Han T , Tomasi TB , Evans RL
Published: October 2019

Interferon (IFN)-alpha inhibits DNA synthesis stimulated by low molecular weight B-cell growth factor (BCGF) in hairy cells in vitro, suggesting that the therapeutic efficacy of IFN-alpha in hairy cell leukemia (HCL) involves growth inhibition of malignant B cells. Evidence that the 16-Kd cell surface protein Leu-13 mediates an antiproliferative signal in T lymphocytes and is IFN-inducible in endothelial cells prompted us to examine the expression and functional role of this molecule in leukemic B cells. Leu-13 density, determined by flow cytometry, was upregulated in vitro and in vivo by IFN-alpha on malignant B cells from patients with HCL, chronic lymphocytic leukemia, and prolymphocytic leukemia. Monoclonal anti-Leu-13 triggered homotypic aggregation of leukemic B cells via an adhesion pathway that was not inhibited by antibodies to leukocyte function associated antigen-1 (LFA- 1) or intercellular adhesion molecule-1 (ICAM-1). Moreover, anti-Leu-13 potentiated the inhibitory effects of IFN-alpha on BCGF-stimulated DNA synthesis, assessed by [3H]-thymidine and [3H]-deoxyadenosine incorporation into DNA. These results indicate that Leu-13 is part of a novel IFN-inducible signaling pathway which may modify the growth and adhesive properties of leukemic B cells under physiologic or therapeutic conditions.
t(3;21)(q26;q22): a recurring chromosomal abnormality in therapy- related myelodysplastic syndrome and acute myeloid leukemia
Authors: Rubin CM , Larson RA , Anastasi J , Winter JN , Thangavelu M , Vardiman JW , Rowley JD , Le Beau MM
Published: October 2019

We have identified an identical reciprocal translocation between the long arms of chromosomes 3 and 21 with breakpoints at bands 3q26 and 21q22, [t(3;21)(q26;q22)], in the malignant cells from five adult patients with therapy-related myelodysplastic syndrome (t-MDS) or acute myeloid leukemia (t-AML). Primary diagnoses were Hodgkin's disease in two patients and ovarian carcinoma, breast cancer, and polycythemia vera in one patient each. Patients had been treated with chemotherapy including an alkylating agent for their primary disease 1 to 18 years before the development of t-MDS or t-AML. We have not observed the t(3;21) in over 1,500 patients with a myelodysplastic syndrome or acute myeloid leukemia arising de novo or in over 1,000 patients with lymphoid malignancies. We have previously reported that the t(3;21) occurs in Philadelphia chromosome-positive chronic myelogenous leukemia (CML). Thus, the t(3;21) appears to be limited to t-MDS/t-AML and CML, both of which represent malignant disorders of an early hematopoietic precursor cell. These results provide a new focus for the study of therapy-related leukemia at the molecular level.
Granulocyte-macrophage colony-stimulating factor synergizes with interleukin-6 in supporting the proliferation of human myeloma cells [see comments]
Authors: Zhang XG , Bataille R , Jourdan M , Saeland S , Banchereau J , Mannoni P , Klein B
Published: October 2019

The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the growth of multiple myeloma (MM) was investigated in 21 patients with MM. In 17 patients with proliferating myeloma cells in vivo, recombinant GM-CSF significantly increased the endogenous-IL-6-mediated spontaneous myeloma cell proliferation occurring in 5-day cultures of tumor cells in vitro (P less than .01). Furthermore, GM-CSF was detected in 5-day culture supernatants of myeloma bone marrow cells. This endogenous GM-CSF was produced by the myeloma bone marrow microenvironment but not by myeloma cells and contributed to the spontaneous myeloma-cell proliferation observed in 5-day cultures. In fact, this proliferation was partially blocked (67%) by anti-GM-CSF monoclonal antibodies. The stimulatory effect of rGM-CSF was mediated through IL-6 because it was abrogated by anti-IL-6 monoclonal antibodies. rGM-CSF did not reproducibly increase the endogenous IL-6 production in short-term cultures of bone marrow cells of MM patients. Using an IL-6-dependent myeloma cell line (XG-1 cell line), rGM-CSF was shown to act directly on myeloma cells stimulating by twofold their IL- 6 responsiveness. rGM-CSF did not induce any IL-6 production in XG-1 cells, nor was it able to sustain their growth alone. Although no detectable GM-CSF levels were found in the peripheral or bone marrow blood of MM patients, it is possible that GM-CSF, produced locally by the tumoral environment, enhances the IL-6 responsiveness of myeloma cells in vivo in a way similar to that reported here in vitro.
Impaired transendothelial migration by neonatal neutrophils: abnormalities of Mac-1 (CD11b/CD18)-dependent adherence reactions
Authors: Anderson DC , Rothlein R , Marlin SD , Krater SS , Smith CW
Published: October 2019

In order to evaluate the functions of lymphocyte function antigen-1 (LFA-1) (CD11a/CD18) and Mac-1 (CD11b/CD18) on neonatal neutrophils, we examined neutrophil adhesion to and migration through human umbilical vein endothelial cell (HUVEC) monolayers in vitro. Transendothelial migration of adult neutrophils was greatly enhanced by preincubation of HUVEC with interleukin-1 (IL-1). This migration was significantly inhibited by monoclonal antibodies (MoAbs) against LFA-1 (CD11a) and Mac-1 (CD11b) subunits. Migration of neonatal neutrophils was markedly diminished compared to adult neutrophils, and MoAbs against LFA-1 further reduced migration. In contrast, anti-Mac-1 MoAb was not inhibitory. Adhesion of adult neutrophils was significantly enhanced by prestimulation of HUVEC with IL-1, and was significantly inhibited by MoAbs against LFA-1. Adhesion of neonatal neutrophils was near adult levels and comparably inhibited by anti-LFA-1 MoAb. In addition, adhesion of neonatal and adult neutrophils to purified ICAM-1 in artificial planar membranes was comparable and almost completely inhibited by anti-LFA-1 MoAb. Chemotactic stimulation induced Mac-1- dependent adhesion of adult neutrophils to endothelial cells, purified intercellular adherence molecule-1 (ICAM-1) and protein-coated glass. In marked contrast, adhesion of neonatal neutrophils to these substrates was not significantly increased by chemotactic stimulation. These findings indicate that diminished transendothelial migration by neonatal neutrophils is related to abnormal interactions of Mac-1 with ICAM-1 and possibly other endothelial ligands. These functional deficits may contribute to impaired inflammation and infectious susceptibility in human neonates.
Human immunodeficiency virus infection of human bone marrow stromal fibroblasts
Authors: Scadden DT , Zeira M , Woon A , Wang Z , Schieve L , Ikeuchi K , Lim B , Groopman JE
Published: October 2019

The human immunodeficiency virus (HIV) preferentially infects CD4 positive T cells and monocytes. Other human cell types have been reported to be infectable with HIV, including cells of mesenchymal origin. In this report, we show that both primary human bone marrow stromal fibroblasts and an immortalized human stromal fibroblast line are susceptible to HIV infection. These cells are capable of passing HIV to cells of lymphoid or myeloid lineage, and may thereby act as a reservoir of virus. This in vitro system may be a useful model for assessing the pathophysiology of hematopoietic dysfunction in AIDS patients.
Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood
Authors: Hantgan RR , Hindriks G , Taylor RG , Sixma JJ , de Groot PG
Published: October 2019

We have investigated the molecular basis of thrombus formation by measuring the extent of platelet deposition from flowing whole blood onto fibrin-coated glass coverslips under well-defined shear conditions in a rectangular perfusion chamber. Platelets readily and specifically adhered to fibrin-coated coverslips in 5 minute perfusion experiments done at either low (300 s-1) or high (1,300 s-1) wall shear rates. Scanning electron microscopic examination of fibrin-coated coverslips after perfusions showed surface coverage by a monolayer of adherent, partly spread platelets. Platelet adhesion to fibrin was effectively inhibited by a monoclonal antibody (MoAb) specific for glycoprotein (GP) IIb:IIIa. The dose-response curve for inhibition of adhesion by anti-GPIIb:IIIa at both shear rates paralleled that for inhibition of platelet aggregation. Platelet aggregation and adhesion to fibrin were also blocked by low concentrations of prostacyclin. In contrast, anti- GPIb reduced adhesion by 40% at 300 s-1 and by 70% at 1,300 s-1. A similar pattern of shear rate-dependent, incomplete inhibition resulted with a MoAb specific for the GPIb-recognition region of von Willebrand factor (vWF). Platelets from an individual with severe von Willebrand's disease, whose plasma and platelets contained essentially no vWF, exhibited defective adhesion to fibrin, especially at the higher shear rate. Addition of purified vWF restored adhesion to normal values. These results are consistent with a two-site model for platelet adhesion to fibrin, in which the GPIIb:IIIa complex is the primary receptor, with GPIb:vWF providing a secondary adhesion pathway that is especially important at high wall shear rates.
Properties of factor VIIa/tissue factor complexes in an umbilical vein model
Authors: Almus FE , Rao LV , Fleck RA , Rapaport SI
Published: October 2019

An umbilical vein model was designed in which washed vein segments are filled with a reaction mixture containing factor VIIa, Ca(+)+, and a substrate, either 3H-factor IX or 3H-factor X. The vein wall provides the tissue factor (TF) for factor VIIa/TF complexes that activate the substrates as measured by activation peptide release. The model was developed to study TF induced on venous endothelium in situ. However, unlike previous studies with TF expressed on cultured umbilical vein endothelial cells, factors IX and X were activated without first having to expose the vein wall to a perturbing stimulus. Histologic studies revealed that washing the vein and mixing the reaction mixture before subsampling had disrupted the endothelium. Immunostaining with anti-TF antibodies revealed no staining of endothelium but intense staining in extensions of Wharton's jelly penetrating fenestrations of the muscularis media of the vein. Thus, the model provided data on factor VIIa/TF formed, not on endothelium, but within the mucoid connective tissue of Wharton's jelly. It is known that factor VIIa/TF formed with TF in suspension or with TF expressed on the surface of cultured cells activates factor X more rapidly than factor IX. In contrast, in the umbilical vein model, when each substrate was present in an 88 nmol/L concentration, factors IX and X were activated at equivalent rates (mean activation rate for factor IX, 18.8 +/- 3.6 nmol/L/h; for factor X, 17.8 +/- 2.9 nmol/L/h; n = 9 paired vein segments). These data strengthen the evidence that factor VIIa/TF activation of factor IX represents a key initial reaction of coagulation in tissues. These results also show that data obtained with factor VIIa/TF complexes formed on the surface of cultured cells need not hold for factor VIIa/TF complexes formed in extracellular matrix.
A clinical, hematologic, and immunologic analysis of 21 HTLV-II- infected intravenous drug users [published erratum appears in Blood 1990 Nov 1;76(9):1901]
Authors: Rosenblatt JD , Plaeger-Marshall S , Giorgi JV , Swanson P , Chen IS , Chin E , Wang HJ , Canavaggio M , Hausner MA , Black AC
Published: October 2019

Infection with human T-cell leukemia virus type II (HTLV-II) has been associated with rare chronic T-cell malignancies and has recently been demonstrated in a significant proportion of American intravenous drug abusers (IVDA). Identification of an HTLV-II-infected cohort of IVDA has allowed analysis of the HTLV-II carrier state. We analyzed clinical, hematologic, and immunologic parameters in 21 HTLV-II- infected IVDA, two HTLV-I-infected IVDA, and 20 uninfected control IVDA identified by serologic screening and by analysis of peripheral blood mononuclear cell (PBMC) DNA by polymerase chain reaction (PCR). An elevated absolute lymphocyte count was observed in 4 of 21 HTLV-II- infected IVDA, 1 of 2 HTLV-I-infected IVDA, and 1 of 20 control IVDA. CD8+ T-cell elevation was observed in three of four HTLV-II IVDA with lymphocytosis and one of two HTLV-I-infected IVDA. Activation of CD8+ T cells in HTLV-II-infected IVDA was suggested by an overall increase in CD8+/HLA-DR+ lymphocytes. Cell fractionation and analysis by PCR of HTLV-II-infected carrier blood showed high levels of HTLV-II provirus in unfractionated PBMC and purified T cells and little or no detectable HTLV-II DNA in B cells or monocytes, indicating that T cells were the most likely target of infection in vivo. The frequency of HTLV-II- infected cells was estimated at approximately 1 in 500 cells or less using dilution analysis by PCR of PBMC DNA. Most HTLV-II-infected IVDA are asymptomatic and have no overt hematologic or immunologic abnormalities, although some manifest benign lymphocytosis.
Selective depletion of CD8+ T lymphocytes for prevention of graft- versus-host disease after allogeneic bone marrow transplantation
Authors: Champlin R , Ho W , Gajewski J , Feig S , Burnison M , Holley G , Greenberg P , Lee K , Schmid I , Giorgi J
Published: October 2019

The effects of selectively depleting CD8+ cells from donor bone marrow were assessed in 36 patients receiving transplantation from an HLA- identical sibling as treatment for leukemia. Donor bone marrow underwent ex vivo treatment using anti-Leu-2 monoclonal antibody and complement. Patients received cyclosporine post-transplant for 6 months. Thirty-three patients had initial engraftment. Three failed to have hematologic recovery, and one patient with initial engraftment had late graft failure. The actuarial incidence of grade greater than or equal to 2 acute graft-versus-host disease was 28% +/- 18% and was usually confined to the skin. Of 33 patients with engraftment, 32 were complete chimeras and one had mixed chimerism. The tempo of hematologic and immunologic recovery was comparable with that reported with transplantation of unmodified bone marrow, although CD4+ and CD8+ T cells recovered at comparable rates. The actuarial rate of leukemia relapse was 11% +/- 10%, occurring in three patients with acute leukemia but in none of 13 patients transplanted for chronic myelogenous leukemia. Actuarial survival was 57% +/- 17% at 2 years. These data indicate that after transplantation of marrow depleted of CD8+ cells, engraftment with prompt hematologic and immunologic recovery generally occurs, with a relatively low rate of acute graft- versus-host disease. Graft failure remains a problem despite retention of CD4+ cells within the donor marrow. The lack of leukemia relapse in patients with chronic myelogenous leukemia suggests retention of a graft-versus-leukemia effect, at least for this malignancy.
Comparison of high-dose therapy and autologous bone marrow transplantation for T-cell and B-cell non-Hodgkin's lymphomas
Authors: Vose JM , Peterson C , Bierman PJ , Weisenburger DD , Linder J , Harrington D , Vaughan WP , Kessinger A , Armitage JO
Published: October 2019

Advanced T-cell non-Hodgkin's lymphoma in adults has been found to have a poor outlook with conventional chemotherapy. To see if this extends to patients treated with high dose therapy and autologous hematopoietic stem cell transplantation, we reviewed the results with this treatment approach at our institution. From October, 1983, to May, 1988, 41 patients who underwent high-dose therapy and autologous hematopoietic stem cell transplant for recurrent non-Hodgkin's lymphoma were re- biopsied before transplantation to determine their immunophenotype. Seventeen of these patients were found to have a T-cell lymphoma, and 24 had a B-cell lymphoma. All patients were included in the intermediate or high grade non-Hodgkin's lymphoma categories, and none were histologically transformed from a low grade lymphoma. Analysis of the response to autologous transplantation in these two patient populations revealed a slightly better complete response rate for patients with T-cell lymphoma (ie, 59% versus 42%, P = NS). The actuarial 2-year survival was 35% in the T-cell group compared with 30% in the B-cell group (P = NS). The 2-year disease-free survival was 28% for the T-cell and 17% for the B-cell patients. Our results with autologous transplantation for salvage therapy revealed equivalent long- term survival and disease-free survival in both relapsed T- and B-cell non-Hodgkin's lymphoma.
Effects of recombinant human granulocyte colony-stimulating factor on the hematologic recovery and survival of irradiated mice
Authors: Tanikawa S , Nose M , Aoki Y , Tsuneoka K , Shikita M , Nara N
Published: October 2019

We studied the effects of intraperitoneal injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF) according to various administration schedules on the recovery of spleen colony- forming units (CFU-S) and peripheral blood counts, and on the survival of irradiated mice. The sooner and more frequently the mice were injected with rhG-CSF after irradiation, the more enhanced the recovery of CFU-S in bone marrow was obtained on day 7. Twice-daily injections of rhG-CSF from day 0 to day 2 significantly enhanced the recovery of platelets and hematocrit, but two injections of rhG-CSF on only day 0 did not. Twice-daily injections of rhG-CSF from day 0 to day 6 enhanced the recovery of platelets more effectively than twice-daily injections of rhG-CSF from day 1 to day 7, and increased the survival of irradiated mice more effectively than any other examined administration schedules. Twice-daily injections of rhG-CSF from day 0 to day 6 were significantly effective in enhancing the survival of mice irradiated with 8.5-, 9.0-, and 9.5-Gy x-rays, although not effective after irradiation of 10.5-Gy x-rays.
High-dose etoposide and cyclophosphamide without bone marrow transplantation for resistant hematologic malignancy
Authors: Brown RA , Herzig RH , Wolff SN , Frei-Lahr D , Pineiro L , Bolwell BJ , Lowder JN , Harden EA , Hande KR , Herzig GP
Published: October 2019

Seventy-five patients with resistant acute leukemia or lymphoma received high-dose cyclophosphamide and etoposide to explore the activity of this combination in resistant hematologic malignancies, and to determine the maximum doses of these drugs that can be combined without bone marrow transplantation. Etoposide was administered over 29 to 69 hours by continuous infusion corresponding to total doses of 1.8 g/m2 to 4.8 g/m2. Cyclophosphamide, 50 mg/kg/d, was administered on 3 or 4 consecutive days total 150 to 200 mg/kg ideal body weight). At all dose levels myelosuppression was severe but reversible. Mucosal toxicity was dose-limiting with the maximum tolerated dose level combining etoposide 4.2 g/m2 with cyclophosphamide 200 mg/kg. Continuous etoposide infusion produced stable plasma levels that were lower than would be achieved after administration by short intravenous infusion, and this could explain our ability to escalate etoposide above the previously reported maximum tolerated dose. There were 28 complete (35%) and 12 partial (16%) responses. Median duration of complete response (CR) was 3.5 months (range 1.1 to 20+). Seventeen of 40 patients (42%) with acute myelogenous leukemia (AML) achieved CR, including 6 of 20 (30%) with high-dose cytosine arabinoside resistance. We conclude that bone marrow transplantation is not required after maximum tolerated doses of etoposide and cyclophosphamide. This regimen is active in resistant hematologic neoplasms, and the occurrence of CR in patients with high-dose cytosine arabinoside-resistant AML indicates a lack of complete cross-resistance between these regimens.
Family studies and prenatal diagnosis in severe von Willebrand disease by polymerase chain reaction amplification of a variable number tandem repeat region of the von Willebrand factor gene
Authors: Peake IR , Bowen D , Bignell P , Liddell MB , Sadler JE , Standen G , Bloom AL
Published: October 2019

We have previously demonstrated within intron 40 of the von Willebrand factor (vWF) gene a region of ATCT repeats that was shown to vary in length between two different DNA clones from unrelated individuals. The polymerase chain reaction (PCR) was used to examine the variability in length of this variable number tandem repeat (VNTR) in 53 normal individuals, using primers to DNA sequence flanking the repeat region. Overall, eight different length allelic bands were seen. These were individually sequenced and shown to contain from 6 to 14 ATCT repeats (a nine-repeat band was not seen). Seventy-five percent of individuals were shown to be heterozygous for this vWF.VNTR, and family studies showed Mendelian inheritance with allelic frequencies from 1% (vWF.VNTR [8] and vWF.VNTR [14]) to 39% (vWF.VNTR [7]). In the family of a patient with type III severe von Willebrand disease (vWD), vWF.VNTR results mirrored the phenotypic data and results with previously reported intragenic vWF restriction fragment length polymorphisms (RFLP). The patient was shown to be a compound heterozygote. In a family with a child with severe type III vWD, prenatal diagnosis by vWF.VNTR analysis on DNA obtained by chorionic villus sampling at 10 weeks gestation during a subsequent pregnancy indicated a severely affected fetus. This diagnosis was confirmed by fetal blood sampling at 18 weeks.
Origin of mutations in two families with X-linked chronic granulomatous disease
Authors: Francke U , Ochs HD , Darras BT , Swaroop A
Published: October 2019

The most common X-linked recessive form of chronic granulomatous disease (X-CGD) is characterized by the absence of cytochrome b558 in neutrophils. In a rare variant form of X-CGD, cytochrome b558 is present but not functional. The gene (locus symbol CYBB) was localized to band Xp21 by studies of patients with small chromosome deletions. The gene was cloned based on its location and found to encode the 91-Kd subunit of the cytochrome b558 complex. Most female carriers for X-CGD can be identified by their X-inactivation mosaicism; on average 50% of their neutrophils express the mutant phenotype and fail to reduce nitroblue tetrazolium (NBT). In 2 of 4 families studied, the maternal grandmothers had normal NBT tests, suggesting either nonrandom X- inactivation or new mutations. Restriction fragment length polymorphism analysis using closely linked flanking markers or the NsiI polymorphism detected by the CYBB probe itself, allowed us to identify the X chromosome carrying the mutation as derived from a healthy NBT-positive maternal grandfather. The mothers of the affected boys must have received a paternal X chromosome carrying a new mutation, consistent with the maternal grandmothers' normal NBT tests. In all of eight potential carriers studied, the results of the NBT and DNA marker testing were in complete agreement. Prenatal diagnosis by DNA testing can be performed in early gestation obviating the need for fetal blood sampling.