bluuebunny/crossref_metadata_2025 huggingface dataset:
Demonstration of osteonectin mRNA in megakaryocytes: the use of the polymerase chain reaction
Authors: Villarreal XC , Grant BW , Long GL
Published: October 2019

Platelets have been shown to release osteonectin on thrombin stimulation. The origin of platelet osteonectin was unclear as it may have been synthesized by megakaryocytes or it may have been endocytosed from plasma as other platelet alpha-granule constituents are. Platelet osteonectin has a larger apparent molecular size than the bone species, although the molecular basis for this difference has not been elucidated. These two issues have been addressed here by (1) examining the potential for osteonectin biosynthesis in human megakaryocytes by demonstrating the presence of osteonectin mRNA in purified megakaryocytes, and (2) comparing the coding portion of osteonectin transcript in megakaryocytes to the size of its bone counterpart. Because of the limitations of cell population purity and in obtaining sufficient numbers of megakaryocyte cells for Northern analysis, we have used the polymerase chain reaction (PCR) to detect the presence of human osteonectin mRNA in megakaryocyte and megakaryocyte-depleted bone marrow cells. Isolation of RNA, cDNA synthesis, and PCR were performed on human osteosarcoma SaOS-2 cells, enriched megakaryocytes, and megakaryocyte-depleted cells. Restriction enzyme analysis of PCR DNA products confirmed the identity of the products as those encoding osteonectin for all three cell populations studied. In addition, the sizes of DNA indicate that osteonectin genomic DNA, nuclear RNA, or altered transcript were not amplified, and that the transcript from megakaryocytes is the same size as that from bone cells. These data suggest that the difference in protein size between platelet and bone osteonectin is due to posttranslational modification. To overcome the possibility that megakaryocyte signal originated from contaminating cells (less than 5% by cell count), all three cell populations were diluted to less than one cell per tube and PCR amplification was performed. Limiting dilution analyses demonstrated the presence of osteonectin mRNA in single megakaryocytes as well as in single cells from the cell population depleted of megakaryocytes, suggesting the capacity for osteonectin biosynthesis in all cells studied. The procedure we describe in this report can be used to examine specific characteristics of mRNA molecules in heterogeneous cell populations and in situations where only small quantities of cells can be obtained.
Sensitive detection of clonal antigen receptor gene rearrangements for the diagnosis and monitoring of lymphoid neoplasms by a polymerase chain reaction-mediated ribonuclease protection assay
Authors: Veelken H , Tycko B , Sklar J
Published: October 2019

This report describes a novel assay involving the polymerase chain reaction (PCR) and RNase protection for the rapid and sensitive detection of malignant lymphoid cells by nucleotide sequences within their individual rearranged gamma T-cell receptor (TCRG) genes. In this assay, clonal rearrangements are amplified from the DNA of diagnostic tumor specimens using a consensus V segment primer and a consensus J segment primer to which the promoter for T7 RNA polymerase has been appended. The PCR product from this amplification is transcribed into a radiolabeled RNA probe. Test RNA transcribed from the opposite DNA strand is synthesized by similar methods from TCRG genes of a subsequent biopsy specimen. The test RNA is hybridized with the probe, and mismatched nucleotide sequences in the RNA hybrids are digested by RNase A. Detection of fully protected probe by means of polyacrylamide gel electrophoresis and autoradiography indicates the presence of malignant cells in the test specimen. Dilution experiments with DNA of cell lines from acute lymphoblastic leukemias (ALLs) show that detection of one tumor cell among 10(5) normal bone marrow cells is usually possible. Residual disease was also successfully detected in several cases of ALL during clinical remission, including detection in one case at the 10(-5) level. The procedure described here may provide a simplified and rapid method for the sensitive diagnosis and monitoring of lymphoid malignancies. This procedure should be applicable to most antigen receptor genes, and unlike most comparable methods, requires neither analysis of nucleotide sequence nor synthesis of tumor-specific oligonucleotide probes or primers.
Isolation of c-kit receptor-expressing cells from bone marrow, peripheral blood, and fetal liver: functional properties and composite antigenic profile
Authors: Papayannopoulou T , Brice M , Broudy VC , Zsebo KM
Published: October 2019

To define the cellular targets for c-kit ligand (KL) and to study their functional properties and composite antigenic profile, we isolated cells expressing c-kit receptor (KR) from bone marrow (BM), peripheral blood, and fetal liver (FL) using immunoadherence to a recently obtained antibody (SR-1) against the human KR. Cells isolated by this approach (designated SR-1Ad) have the morphology of blasts and represent 1% to 4% of the original BM or FL populations. SR-1Ad cells from either source are highly enriched in progenitors (12% to 73%) and respond to KL in distinct patterns. In SR-1Ad cells from BM, the greatest impact of KL stimulation is on burst-forming units-erythroid (BFU-E), whereas in SR1-Ad cells from FL, the most significant KL effect is on a mixed erythroid/nonerythroid progenitor (erythroid/macrophage, colony-forming unit-mix [CFU-Mix]). When antibody SR-1 is continually present in culture, it neutralizes the effects of added KL. Furthermore, in the absence of added KL, it greatly diminishes the erythropoietin- and interleukin-3-dependent BFU- E growth in BM; whereas in FL, a wider spectrum of inhibition is observed, with CFU-Mix most severely curtailed. SR-1Ad cells coexpress other progenitor-associated antigens in a combination reflecting the dominant presence of erythroid progenitors (high expression of CD34, DR, CD38, and Ep-1; low expression of CD33). Several cytoadhesion molecules, ie, alpha L/beta 2 and alpha 4/beta 1 integrins, and intercellular adhesion molecule 1 and homing cell adhesion molecule 1, are also coexpressed. Our data provide new information on the isolation and characterization of KR expressing cells from normal, adult, and fetal hematopoietic tissues. On these biologically relevant target cells, the impact of ligand-induced stimulation or antibody-mediated ablation of KR function has been gauged.
Megakaryocytic maturation in murine long-term bone marrow culture: role of interleukin-6
Authors: Mei RL , Burstein SA
Published: October 2019

Megakaryocytic maturation was analyzed in long-term bone marrow cultures in the absence of added growth factors. Megakaryocytes could be observed for periods of up to 13 weeks in both the supernatant and stromal layer of these cultures. Using acetylcholinesterase staining for enumeration and sizing of megakaryocytes, and a novel rat antimurine platelet monoclonal antibody (MoAb) that detects only megakaryocytes in bone marrow, the number, volume, and ploidy of these cells were assessed microscopically and by flow cytometry. Correlation of these measurements with ambient interleukin-6 (IL-6) levels showed no relationship between IL-6 bioactivity and megakaryocyte number. Conversely, the relatively high IL-6 bioactivity present during the first 2 weeks of culture was correlated with increased megakaryocytic size and ploidy, while the relatively lower IL-6 bioactivity present after week 3 corresponded to decreased megakaryocytic size and ploidy. Addition of neutralizing anti-IL-6 MoAb decreased megakaryocytic size and ploidy at times when ambient IL-6 levels were relatively high, while the addition of exogenous IL-6 increased size and ploidy at times when endogenous IL-6 concentrations were low. The data show that long- term bone marrow cultures can be used as a means to evaluate megakaryocytic maturation in vitro, and suggest that, to some extent, IL-6 plays a role in the maturation process in this system.
Changes in splenic microcirculatory pathways in chronic idiopathic thrombocytopenic purpura
Authors: Schmidt EE , MacDonald IC , Groom AC
Published: October 2019

The spleen plays a central role in the pathogenesis of chronic idiopathic thrombocytopenic purpura (ITP); it produces massive quantities of antiplatelet antibodies, leading to accelerated phagocytosis of platelets. Lymphoid hyperplasia typically occurs in the spleen, characterized by large numbers of lymphatic nodules with active germinal centers. Whether changes in splenic microcirculatory pathways also occur is not known. We have studied this question by scanning electron microscopy of corrosion casts, comparing spleens removed from patients with ITP with normal spleens obtained from organ transplant donors. The casts demonstrate two major changes in microcirculatory pathways in ITP. Firstly, a striking proliferation of arterioles and capillaries is found in the white pulp and marginal zone (MZ), seen as extensive vascularization in 92.3% of lymphatic nodules (n = 191) versus 0.6% (n = 224) in normal spleens. Secondly, the marginal sinus, a series of flattened, anastomosing vascular spaces between the white pulp and MZ, is absent in 89.4% of lymphatic nodules versus 4.9% in normal spleens. The cause of these microcirculatory changes, which may not be exclusive to ITP, is presently unknown. Absence of the marginal sinus may affect distribution of blood flow through the MZ such that platelets spend increased amounts of time in the proximity of macrophages. In the presence of antiplatelet antibodies found in ITP spleens, this delayed transit would lead to greatly increased platelet destruction.
Infectious transmission of human T-cell lymphotropic virus type II in rabbits
Authors: Cockerell GL , Weiser MG , Rovnak J , Wicks-Beard B , Roberts B , Post A , Chen IS , Lairmore MD
Published: October 2019

To determine the susceptibility of rabbits to experimental infection with human T-cell lymphotropic virus type-II (HTLV-II), four separate groups of four weanling rabbits each were inoculated intravenously with lethally irradiated HTLV-II-infected human cell lines Mo-T (HTLV-IIMo- infected T cells), WIL-NRA (an Epstein-Barr virus [EBV]-transformed B- lymphoblastoid cell line infected with HTLV-IINRA), 729pH6neo (an EBV- transformed lymphoblastoid cell line transfected with a molecular clone of HTLV-IIMo), or G12.1 (HTLV-II-infected T cells from a Panamanian Guaymi Indian). Two additional groups of four rabbits each were similarly inoculated with control uninfected 729 or HuT 78 cells. Early and persistent seroconversion to HTLV-II core antigen p24, as determined by Western immunoblot, occurred in all HTLV-II-inoculated rabbits and was most intense in rabbits inoculated with G12.1 cells; seroreactivity to other HTLV-II gag or env antigens occurred later, with less intensity, or not in all inoculated rabbits. Peripheral blood mononuclear cells (PBMC) and other lymphoid cells from HTLV-II- inoculated rabbits produced minimal p24 in vitro, as determined by enzyme immunosorbent capture assay. Virus was more readily detected by polymerase chain reaction amplification of HTLV-II pol sequences; this occurred most frequently in rabbits inoculated with Mo-T cells, and most frequently in PBMC as compared with other tissues tested (bone marrow, brain, and liver). No evidence of disease occurred in HTLV-II- inoculated rabbits observed for as long as 24 weeks. All control rabbits remained negative for evidence of HTLV-II infection, as determined by the same procedures. These results provide the first evidence of HTLV-II infection in a species other than humans, and demonstrate the usefulness of the rabbit as an animal model to study the biologic response to different isolates of this human retrovirus.
Presenting features and prognosis of chronic lymphocytic leukemia in younger adults [see comments]
Authors: Montserrat E , Gomis F , Vallespi T , Rios A , Romero A , Soler J , Alcala A , Morey M , Ferran C , Diaz-Mediavilla J
Published: October 2019

We have analyzed 117 younger patients with chronic lymphocytic leukemia (CLL) (mean age, 44.5 years; SD, 4.8; range, 19 to 49; male/female ratio, 2.08) with three main objectives: (1) to see whether these patients have distinctive presenting clinical features; (2) to investigate the impact of the disease on survival; and (3) to analyze whether already well-known prognostic factors are also useful when applied to these patients. As compared with an older age population (greater than or equal to 50 years), there were no major differences in presenting features except for an increased proportion of males (2.08 v 1.21; P less than .025) and a higher hemoglobin level (13.47 +/- 2.70 g/dL v 12.84 +/- 2.77 g/dL; P less than .05) in the younger group. Median survival is 12.3 years (expected median from a control group, 31.2 years). Clinical stages, bone marrow patterns, blood lymphocyte counts, and its doubling time are all useful to separate different risk groups of patients. Whereas patients with favorable prognostic factors have a survival probability of about 80% 14 years after diagnosis, those with poor prognostic features have a median survival of less than 3 years. It is concluded that CLL in younger adults has no major distinctive presenting features and that known prognostic factors are useful to separate different risk groups of patients. These results should be of help in planning therapy for younger persons with CLL.
Ig VH gene expression among human follicular lymphomas
Authors: Bahler DW , Campbell MJ , Hart S , Miller RA , Levy S , Levy R
Published: October 2019

Thirty-six randomly selected cases of low grade follicular lymphoma (FL) were analyzed for Ig heavy chain variable region (VH) gene expression. Assignment to one of the six human VH gene families (VH1 to VH6) was made with a polymerase chain reaction-based technique using family-specific leader primers. The frequency of VH family use in FL was found to be similar to that reported for normal peripheral blood lymphocytes and is therefore also roughly proportional to VH family size. To evaluate expression within an individual family, all of the lymphoma VH genes from the middle size VH4 family were sequenced and compared with previously published sequences. Of these eight lymphoma VH sequences, six were most closely related to just two of the 10 known functional VH4 germline genes. Nonrandom usage by FL of the JH3, JH4, and JH5 joining segments was also observed. Nucleotide sequences were also determined for 10 randomly selected lymphoma VH genes from the large VH3 family. With one possible exception, none of these lymphoma VH sequences appear to represent any of the VH3 genes that may be preferentially used in the fetal repertoire.
Inhibitory effect of interleukin-4 on the in vitro growth of Ph1- positive acute lymphoblastic leukemia cells
Authors: Okabe M , Kuni-eda Y , Sugiwura T , Tanaka M , Miyagishima T , Saiki I , Minagawa T , Kurosawa M , Itaya T , Miyazaki T
Published: October 2019

We investigated the effect of recombinant human interleukin-4 (rhIL-4) on the in vitro growth of human leukemia cells in liquid culture and 3H- thymidine incorporation and found inhibitory effects on the growth of leukemic cells from patients with Ph1-positive acute lymphoblastic leukemia (Ph1 ALL) and three Ph1 ALL cell lines. However, no inhibitory effects were seen in Ph1-positive leukemic cell lines derived from patients with chronic myelogenous leukemia in blast crisis and various types of Ph1-negative leukemia cells, including B-lineage leukemia cells. In a flow cytometry assay of IL-4 receptor (IL-4R), all three Ph1-positive ALL cell lines showed the presence of IL-4R on their cell surfaces, and the IL-4-dependent inhibition on the growth of Ph1- positive ALL cells was abrogated by the addition of either monoclonal or polyclonal antibodies against rhIL-4. Other cytokines, including IL- 2, IL-3, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, and IL-6, showed no inhibitory effects on the growth of Ph1-ALL cells, but tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN)-alpha, -beta, and -gamma displayed slight inhibitory effects in a high concentration. The growth inhibition induced by rhIL- 4 in the Ph1-positive ALL cells was not abrogated by the addition of antibodies against either IFN-gamma or TNF-alpha. Furthermore, these cells showed no significant production of IFN-alpha, -beta, or -gamma or TNF-alpha after exposure to rhIL-4, thus indicating that the growth inhibition of Ph1-positive ALL cells by rhIL-4 is not associated with IL-4-stimulating production of these factors. rhIL-4 caused significant inhibition of the tyrosine kinase activity in these Ph1-positive ALL cells, similar to Herbimycin A, an inhibitor of tyrosine kinase that inhibited the tyrosine kinase activity in these cells. Our finding suggests that the clinical evaluation of rhIL-4 may offer promising therapeutic possibilities for patients with Ph1-positive ALL.
Improvement of mouse beta-thalassemia by recombinant human erythropoietin
Authors: Leroy-Viard K , Rouyer-Fessard P , Beuzard Y
Published: October 2019

Homozygous beta thalassemic mice received 50 U (1,660 U/kg) of recombinant human erythropoietin (rhEpo) 5 days a week for 2 weeks. Hemoglobin increased from 9.2 +/- 0.6 g/dL to 10.5 +/- 0.4 g/dL (P = .002) and hematocrit increased from 29.2% +/- 0.9% to 34.1% +/- 1.9% (P = .0014). The beta minor/alpha globin chain synthesis ratio increased slightly but significantly between day -4 (0.75 +/- 0.07) and day 4 (0.81 +/- 0.04) (P = .01) and reached a minimum ratio (0.67 +/- 0.03) on day 15 (P = .001), being parallel to reticulocyte counts and to the incorporated trichloracetic acid (TCA)-insoluble radioactivity, therefore parallel to the erythropoietic output in thalassemic mice, as in normal mice. Erythrocyte defects were improved in beta thalassemic mice treated by rhEpo: membrane-associated alpha globin was significantly decreased (P less than .01), thiol group reactivity of ankyrin was significantly improved (P less than .05), spectrin alterations were reduced, and deformability of mouse thalassemic red blood cells was normalized. These results provide experimental criteria for modulating globin chain imbalance necessary for the therapy of human beta thalassemia intermedia, and suggest that rhEpo might be of interest to improve the red blood cell mass and reduce erythrocyte alterations in this disease.
Treatment type and amount influenced human immunodeficiency virus seroprevalence of patients with congenital bleeding disorders
Authors: Gjerset GF , Clements MJ , Counts RB , Halvorsen AS , Thompson AR
Published: October 2019

Two hundred and eighty-two patients with congenital bleeding disorders received blood component replacement therapy between January 1979 and April 1985, were followed-up by the Puget Sound Blood Center's Hemophilia Care Program, and were tested for antibody to human immunodeficiency virus (HIV). Serologic results were obtained at least 1 year after the last exposure to volunteer donor products that were prepared before donor HIV screening or after the last exposure to concentrates produced before the manufacturer's use of treatment methods for inactivation of HIV. In all, 106 patients were anti-HIV positive. The risk of HIV infection was greater in patients with more severe bleeding tendencies, greater exposure to components, and exposure to lyophilized concentrates from large pools of donors. Of 100 patients with hemophilia A who only received cryoprecipitate from volunteer donors from Washington State (during the 6.3-year period), 14% had become anti-HIV positive. Of 27 patients receiving mostly cryoprecipitate but also being exposed to a single lot of concentrate during the same period, 13 (48%) were positive. Of 49 patients treated predominantly or solely with factor VIII concentrates during this period, 43 (88%) were anti-HIV positive. Of 29 patients with von Willebrand disease, four were anti-HIV positive, including 2 of 26 receiving only cryoprecipitate and two of three who had received a single dose of factor VIII concentrate. Of 19 patients who were treated solely with volunteer donor plasma, all remained anti-HIV negative. Of 47 patients exposed to factor IX concentrate, 28 (60%) were positive. Data relevant to the risk of HIV transmission subsequent to screening of the volunteer donor population were also obtained. Treatment records of 55 hemophilia A patients who have remained anti-HIV negative through at least June 1990 showed exposure to 71,173 screened donors from May 1985 through December 1989, and all 55 patients have remained anti-HIV negative.
A randomized controlled trial of recombinant interferon-alpha in chronic hepatitis C in hemophiliacs
Authors: Makris M , Preston FE , Triger DR , Underwood JC , Westlake L , Adelman MI
Published: October 2019

Chronic liver disease associated with hepatitis C virus (HCV) is an important cause of morbidity and mortality in hemophilia. We have used recombinant interferon alpha-2b (IFN alpha-2b) in a randomized controlled liver biopsy trial to treat hemophiliacs with chronic hepatitis. Eighteen patients entered the study, 16 of whom were subsequently shown to have antibodies to the HCV. All underwent liver biopsy at entry and were randomized to either treatment with self- administered IFN alpha-2b, 3 million units subcutaneously thrice weekly (n = 10) or no treatment (control group) (n = 8). Nine subjects had chronic active hepatitis, seven had chronic persistent hepatitis, and two had cirrhosis. Twelve months after entry into the study 17 patients underwent a second liver biopsy. All biopsies were coded, assessed, and scored according to the histologic severity of the liver disease. Ten patients were administered IFN for 1 year, and in four patients normalization of alanine aminotransferase (ALT) occurred compared with none in the untreated group. After the second liver biopsy, six of the eight initial no-treatment patients were treated with interferon 3 million units thrice weekly for 6 months, and normalization of ALT was seen in five patients. Biochemical relapse within 4 months of stopping IFN occurred in one of four patients treated for 1 year and in four of five patients treated for 6 months. IFN treatment was well tolerated. Although the histologic scores of the two groups were similar at entry into the study, after 12 months the biopsy appearances in the treated group were significantly improved compared with the controls (P less than .01). Histologic improvement was noted in the three interferon- treated human immunodeficiency virus antibody-positive patients and also in other patients who had no biochemical response. We conclude that low-dose recombinant IFN alpha is effective in normalizing transaminases and improving the histologic appearances in at least 50% of hemophiliacs with chronic hepatitis C.
Fibrinogen Ledyard (A alpha Arg16----Cys): biochemical and physiologic characterization
Authors: Lee MH , Kaczmarek E , Chin DT , Oda A , McIntosh S , Bauer KA , Clyne LP , McDonagh J
Published: October 2019

Fibrinogen Ledyard was discovered in a 10-year-old boy with a mild bleeding history. His father had the same defect and a bleeding history after surgery. Both patients were heterozygous. The plasma fibrinogen concentration was normal immunologically (335 mg/dL) and very low functionally (52 mg/dL). Purified fibrinogen Ledyard had a prolonged polymerization, which was somewhat corrected by addition of Ca2+ ions. High performance liquid chromatography (HPLC) analyses of the fibrinopeptides released by thrombin showed 1 mol of fibrinopeptide A (FPA) and 2 mol of fibrinopeptide B (FPB) released per mole of fibrinogen Ledyard. Steady-state kinetic parameters were evaluated for release of FPA by thrombin. When the concentration of fibrinogen Ledyard was corrected to 50% of total protein, because only 50% of fibrinogen Ledyard can release FPA, the kinetic constants were similar to those of control fibrinogen (Km = 7.5 mumol/L for A alpha chain, kcat = 54 s-1). This finding indicates that the cleavage site of the A alpha chain in these abnormal molecules may not interact with the catalytic site of thrombin. The three chains of fibrinogen Ledyard were isolated on reverse-phase C4-HPLC. The sequence of the amino terminus of A alpha chain showed that Arg in position 16 was replaced by Cys in the abnormal molecules. Approximately half of fibrinogen Ledyard (52%) was clotted by reptilase, suggesting that fibrinogen Ledyard may consist of 50% normal homodimers (A alpha Arg16 . A alpha Arg16) and 50% abnormal homodimers (A alpha Cys16 . A alpha Cys16). Abnormal molecules could form disulfide bond between the A alpha Cys16 residues. Thus, the abnormal molecules have a different structure that does not bind to thrombin. Probably the abnormality of polymerization of fibrinogen Ledyard results from the interaction of the abnormal molecules with normal fibrin monomers, so that the growth of fibrin protofibrils is inhibited. This abnormal fibrinogen supports adenosine diphosphate-induced platelet aggregation in a normal manner.
CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide- induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation
Authors: Shipp MA , Stefano GB , Switzer SN , Griffin JD , Reinherz EL
Published: October 2019

The common acute lymphoblastic leukemia antigen (CALLA, CD10), which is expressed on early lymphoid progenitors and neutrophils, is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, “enkephalinase”). The CD10 cell surface enzyme is known to hydrolyze a variety of biologically active peptides including met-enkephalin, formyl-met-leu- phe (f-MLP), and substance P. These three CD10/NEP substrates induce the migration and aggregation of neutrophils, suggesting that each of the peptides can function as a mediator of neutrophil inflammatory responses. Recently, inhibition of CD10/NEP was found to reduce the concentration of metenkephalin needed to activate human and invertebrate granulocytes by several orders of magnitude. Herein we show that f-MLP and substance P induce rapid changes in neutrophil morphology, migration, and adhesion molecule expression, including upregulation of Mo1 (CD11b/CD18) and shedding of LAM-1 (also known as LECAM-1, Leu8, or TQ-1, the human homologue of murine gp100MEL14). Importantly, these coordinated changes are potentiated by inhibition of cell surface CD10/NEP enzymatic activity. Neutrophil cell surface CD10/NEP enzymatic activity is also shown to be regulated by the activation state of the cell during the time period in which the enzyme has its most pronounced effects. These results suggest that in neutrophils, CD10/NEP functions to control responsiveness to multiple inflammatory peptides.
Anemia and mast cell depletion in mutant rats that are homozygous at "white spotting (Ws)" locus
Authors: Niwa Y , Kasugai T , Ohno K , Morimoto M , Yamazaki M , Dohmae K , Nishimune Y , Kondo K , Kitamura Y
Published: October 2019

Mice possessing two mutant alleles at the W or Sl locus are anemic and deficient in mast cells. These mouse mutants have black eyes and white hair. Because homozygous mutant rats at the newly found white spotting (Ws) locus were also black-eyed whites, the numbers of erythrocytes and mast cells were examined. Suckling Ws/Ws rats showed a severe macrocytic anemia and were deficient in mast cells. When bone marrow cells of normal (+/+) control or Ws/Ws rats were injected into C3H/He mice that had received cyclophosphamide injection and whole-body irradiation, remarkable erythropoiesis occurred in the spleen of +/+ marrow recipients but not in the spleen of Ws/Ws marrow recipients. When skin pieces of Ws/Ws embryos were grafted under the kidney capsule of nude athymic rats, mast cells did develop in the grafted skin tissues. Therefore, the anemia and mast cell deficiency of Ws/Ws rats were attributed to a defect of precursors of erythrocytes and mast cells. Because the magnitude of the anemia decreased and that of the mast cell deficiency increased in adult Ws/Ws rats, this mutant is potentially useful for investigations about differentiation and function of mast cells.
T-cell malignancies with mature phenotypes: altered cell cycle regulation by HLA class I molecules
Authors: Turco MC , Alfinito F , De Felice M , Lamberti A , Ferrone S , Venuta S
Published: October 2019

Soluble anti-HLA class I monoclonal antibodies (MoAbs) modulate normal T-lymphocyte proliferation induced via the CD3/Ti and the CD2 pathway, but do not induce proliferation of normal T lymphocytes in the absence of additional mitogenic stimuli. In this report, we show that anti-HLA class I MoAbs induce DNA synthesis in peripheral blood mononuclear cells from a patient with a CD4+CD8+T-prolymphocytic leukemia (T-PLL) and from a patient with a CD4-CD8+ T-chronic lymphocytic leukemia (T- CLL), in the absence of detectable additional mitogenic stimuli. Proliferation of leukemic T cells is induced by both whole Igs and Fab' fragments of anti-HLA class I MoAbs, arguing in favor of their direct interactions with the proliferating cells as the mechanism underlying the mitogenic effect. This interpretation is also supported by the ability of anti-HLA class I MoAbs to induce proliferation of leukemic T- cell preparations, depleted of accessory cells. DNA synthesis in T-CLL and T-PLL cells is preceded by expression of G1-specific messenger RNAs, ie. c-myc, 2F1, Tac, and interferon-gamma, in activated cells. Cell proliferation is inhibited by the protein kinase C inhibitor H7, indicating that activation of this enzyme is required for the mitogenic effect of anti-HLA class I MoAbs. The latter inhibit the proliferation of T-CLL cells as well as that of normal T cells stimulated with anti- CD3 MoAbs and enhance that of both types of cells stimulated with anti- CD2 MoAbs. In addition, anti-HLA class I MoAb Q6/64 in combination with anti-CD2 MoAb 9.6 or MoAb 9–1 induces proliferation of leukemic T cells to a greater extent than the individual MoAbs, but is not mitogenic for normal T cells. Anti-HLA class I MoAbs restore the cytolytic activity of T-CLL cells that is lost after 5 days of incubation of control medium, suggesting that HLA class I antigens may mediate a signal contributing to the activation state. The present results indicate that leukemic T-cell proliferation can be triggered via HLA class I molecules and suggest a potential role for these antigens in the in vivo growth of malignant clones.
Semiquantitative analysis of integrated genomes of human T-lymphotropic virus type I in asymptomatic virus carriers
Authors: Shinzato O , Ikeda S , Momita S , Nagata Y , Kamihira S , Nakayama E , Shiku H
Published: October 2019

A semiquantitative estimation of human T-lymphotropic virus type I (HTLV-I) integration by peripheral blood mononuclear cells (PBMC) was performed. Genomic DNA samples derived from 134 HTLV-I carriers were subjected to 40 or 60 cycles of the polymerase chain reaction to amplify the pol region of HTLV-I. The HTLV-I genome was detected by dot hybridization using a 32P-labeled oligonucleotide probe for the pol region. The radioactivity of hybridized dot membranes was then counted with an RI Imaging System (Ambis Inc, San Diego, CA) and the HTLV-I genome dose was determined by comparison with standard curve for serially diluted HTLV-I genome-positive DNA. A wide range of variation of HTLV-I genome integration was observed. When the integrated genome dose was calculated as the number of HTLV-I copies per 100 PBMC, 7 carriers (5%) had more than 10 copies, 56 (42%) had 1 to 10 copies, 46 (34%) had 0.1 to 1 copy, and 24 (18%) had less than 0.1 copy. In one sample, the HTLV-I genome was undetectable, which may indicate that the integrated genome was present at less than 0.01 copies per 100 PBMC. Age- or sex-related variations in the distribution of individuals with different HTLV-I genome were rather limited. However, carriers with a high level of the HTLV-I genome were always more than 30 years old and were predominantly male (six of seven).
Mast cell growth factor (c-kit ligand) supports the growth of human multipotential progenitor cells with a high replating potential
Authors: Carow CE , Hangoc G , Cooper SH , Williams DE , Broxmeyer HE
Published: October 2019

The replating capability of human multipotential (colony-forming unit- granulocyte-erythrocyte-macrophage-megakaryocyte [CFU-GEMM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitors was assessed in vitro as a potential measure of self-renewal using purified, recombinant (r) human (hu) or murine (mu) mast cell growth factor (MGF), a ligand for the c-kit proto-oncogene receptor. Primary cultures of human umbilical cord blood or adult human bone marrow cells were initiated in methylcellulose with erythropoietin (Epo) alone or in combination with rhu interleukin-3 (IL-3) or MGF. Individual day 14 to 18 CFU-GEMM or BFU-E colonies were removed from primary cultures and reseeded into secondary methylcellulose cultures containing a combination of Epo, MGF, and rhu granulocyte-macrophage colony- stimulating factor (GM-CSF). The data showed a high replating efficiency of cord blood and bone marrow CFU-GEMM in response to Epo + MGF in terms of the percentage of colonies that could be replated and the number of secondary colonies formed per replated primary colony. The average number of hematopoietic colonies and clusters apparent from replated cultures of cord blood or bone marrow CFU-GEMM stimulated by Epo + MGF was greater than with Epo + rhuIL-3 or Epo alone. Replated cord blood CFU-GEMM gave rise to CFU-GEMM, BFU-E, and GM colony-forming units (CFU-GM) in secondary cultures. Replated bone marrow CFU-GEMM gave rise mainly to CFU-GM in secondary cultures. A more limited capacity for replating of cord blood and bone marrow BFU-E was observed. These studies show that CFU-GEMM responding to MGF have an enhanced replating potential, which may be promoted by MGF. These studies also support the concept that MGF acts on more primitive progenitors than IL-3.
Identification of novel protein tyrosine phosphatases of hematopoietic cells by polymerase chain reaction amplification
Authors: Yi T , Cleveland JL , Ihle JN
Published: October 2019

Polymerase chain reaction (PCR) conditions were used to amplify cDNAs that encode putative protein tyrosine phosphatases (PTPs) from a murine interleukin-3-dependent myeloid cell line. Primers for the reactions were based on conserved sequences of the catalytic domain that are shared among all known PTPs. Sequencing of 100 PCR-amplified cDNA clones identified seven different cDNA sequences. Two of these sequences were identical to the murine PTP genes Ly5/CD45/LCA and LRP/R- PTP-alpha. Two of the cDNA sequences were 95% identical to human PTP epsilon (HPTP epsilon) and rat brain PTP (PTP1B), respectively, and are likely to represent their murine homologs. Three of the cDNA sequences encoded novel potential PTPs. One of the putative PTPs was ubiquitously expressed while a second was predominantly expressed in brain, kidney, and liver and at much lower levels in a variety of other cell tkpes and tissues. The third novel putative phosphatase was expressed primarily in hematopoietic cells and tissues in a pattern that was comparable with Ly5/CD45/LCA. Further characterization of these novel PTPs will provide insights into the growth regulation of hematopoietic cells.
Erythroid-restricted expression of homeobox genes of the human HOX 2 locus
Authors: Mathews CH , Detmer K , Boncinelli E , Lawrence HJ , Largman C
Published: October 2019

We have previously reported that certain members of the HOX 1 and HOX 2 clusters of class 1 homeobox-containing genes showed lineage-restricted patterns of expression in a small series of human hematopoietic cell lines. We now report on the expression patterns of the entire HOX 2 cluster, consisting of nine homeobox genes, in a broad survey of leukemic cell lines of different phenotypes. The most striking observation is that all but one of the HOX 2 genes are consistently expressed in cells with erythroid character and/or potential, but, with rare exception, not in cells with myelomonocytic or T- or B-lymphoid phenotype. By contrast, several genes of the HOX 1 and 3 loci are not expressed in erythroid lines. Within erythroid cell lines, many of the HOX 2 genes are expressed as multiple transcripts. Expression of some HOX 2 genes is detectable in normal human marrow. These data show that in human hematopoietic cell lines HOX 2 homeobox gene expression is largely restricted to cells of erythroid phenotype and suggest that these genes play a role in erythropoiesis.
An autocrine role for erythropoietin in mouse hematopoietic cell differentiation [published erratum appears in Blood 1992 Jun 15;79(12):3397]
Authors: Hermine O , Beru N , Pech N , Goldwasser E
Published: October 2019

Erythropoietin (epo) is the primary regulator of the rate of red blood cell formation in mammals. Because it is formed in the kidney and acts on the bone marrow, its action is classically endocrine. We have shown by PCR that marrow cells contain epo mRNA and that antisense oligodeoxynucleotides, to both epo and its receptor, act on multipotent hematopoietic cells to cause a decrease in mixed erythroid:nonerythroid colonies. The antisense oligonucleotides also cause an increase in mixed nonerythroid colonies with no effect on erythroid burst formation. Sense oligonucleotides have no effect. The antisense suppression is not due to adherent cells, cycling late differentiated cells or lymphocytes, and not reversed by exogenous epo. We conclude that normal erythroid differentiation may have an early phase that is dependent on an internal autocrine mechanism involving epo and its receptor.
Establishment and erythroid differentiation of a cytokine-dependent human leukemic cell line F-36: a parental line requiring granulocyte- macrophage colony-stimulating factor or interleukin-3, and a subline requiring erythropoietin
Authors: Chiba S , Takaku F , Tange T , Shibuya K , Misawa C , Sasaki K , Miyagawa K , Yazaki Y , Hirai H
Published: October 2019

We have established a new nonlymphoid leukemic cell ine from a patient with myelodysplastic syndrome (MDS), which progressed to overt leukemia. The parental cell line and a subline derived from this line have absolute dependency on several cytokines for their long-term survival and growth. The parental line designated F-36P requires granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) for continuous growth, while a subline designated F-36E can be maintained in the presence of erythropoietin (Epo) alone. When these cytokines are depleted, both the parental and the subline cells die within several days, even in medium supplemented with fetal calf serum (FCS). F-36E, maintained in the presence of Epo, constitutively synthesizes hemoglobin at a significant level. F-36P, which is usually maintained in the presence of GM-CSF or IL-3, can be induced to synthesize hemoglobin when GM-CSF or IL-3 is substituted by Epo. The surface marker profile shows that the F-36P cells are positive for the leukocyte common antigen (CD45) and some common multilineage markers such as CD13, CD33, and CD34, and negative for T- and B-cell antigens and mature myelomonocytic antigens. However, some monoclonal antibodies recognizing erythroid and platelet glycoproteins react with these cells. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in a multipotent stem cell. It is also evident that the F-36 cells can be induced to differentiate into the erythroid lineage in the presence of Epo. This, to our knowledge, is the first description of a human leukemic cell line that can be stimulated to synthesize hemoglobin by Epo.
Isolation from commercial aurintricarboxylic acid of the most effective polymeric inhibitors of von Willebrand factor interaction with platelet glycoprotein Ib. Comparison with other polyanionic and polyaromatic polymers
Authors: Weinstein M , Vosburgh E , Phillips M , Turner N , Chute-Rose L , Moake J
Published: October 2019

Solutions of commercial aurintricarboxylic acid (ATA) inhibit ristocetin- or shear stress-induced, von Willebrand factor (vWF)- mediated platelet aggregation by interacting with vWF and blocking its attachment to platelet membrane glycoprotein Ib. ATA has also been shown to prevent cyclic platelet clumping in a dog model of coronary artery thrombosis. Because these ATA solutions are actually a heterogeneous mixture of polyanionic, polycarboxylic polyaromatic polymers of molecular weight (Mr) 200 to greater than 6,000, we separated the most effective inhibitory components of commercial ATA using exclusion chromatography. ATA polymers larger than Mr 700 inhibited ristocetin-induced, vWF-mediated platelet aggregation more effectively than smaller ATA polymers, whereas shear-induced, vWF- mediated platelet aggregation was optimally inhibited by ATA polymers of Mr greater than or equal to 2,500. Platelet aggregation mediated by vWF was not inhibited by a nonphenolic, polyanionic polymer (polyglutamic acid) or by a polyphenolic ATA-like polymer (aurin) devoid of carboxyl groups. Polyanionic, polysulfonated aromatic polymers (polystyrene sulfonate) of Mr 35, 17.4, 8, and 4.6 x 10(3) inhibited ristocetin- and shear-induced, vWF-mediated aggregation with less potency on a mass/volume basis than large polymers of ATA. We conclude that a polyanionic, polycarboxylated, polyphenolic ATA polymer of Mr 2,500 is optimally potent as an inhibitor of shear- and ristocetin-induced, vWF-mediated platelet aggregation and is likely to be more effective than solutions of commercial ATA as an anti-arterial thrombotic agent.
Characterization of prostaglandin and thromboxane receptors expressed on a megakaryoblastic leukemia cell line, MEG-01s
Authors: Watanabe T , Yatomi Y , Sunaga S , Miki I , Ishii A , Nakao A , Higashihara M , Seyama Y , Ogura M , Saito H
Published: October 2019

MEG-01s, an established human megakaryoblastic leukemia cell line, exhibited specific high-affinity binding sites for [3H]iloprost, a stable prostaglandin (PG) I2 analogue, for [3H]SQ-29548, a stable thromboxane (TX) A2 antagonist and, for [3H]PGE2/PGE1, but not for [3H]PGD2. In the MEG-01s cells, iloprost/PGI2, or PGE1 stimulated cAMP production with ED50 values practically identical to the IC50 values for the [3H] iloprost binding. STA2 and U46619, TXA2/PGH2 agonists, PGE2/PGE1, iloprost/PGI2, and thrombin elevated the intracellular concentrations of Ca2+ ([Ca2+]i), as determined by Fura-2 fluorescence signals. Elevation of [Ca2+]i by PGE2/PGE1 and iloprost, but not that by TX-agonists or thrombin, was totally dependent on the presence of extracellular Ca2+. This effect by PGE2/PGE1 was partially inhibited by prior treatment of the cells with islet-activating protein (IAP), while that by TX-agonists or by PGI2/iloprost was not affected. We tentatively conclude from these results that: (1) MEG-01s cells express (a) PGI2/PGE1 receptor(s) coupled to adenylate cyclase and Ca2+ influx, a TXA2/PGH2 receptor coupled to the phosphatidylinositol-turnover-Ca2+ system, and the PGE2/PGE1 receptor coupled to Ca2+ influx; (2) the receptors for TXA2/PGH2 and iloprost and those for PGE2/PGE1 and thrombin are coupled to IAP-insensitive and IAP-sensitive GTP-binding proteins, respectively, and function in a different manner to elevate [Ca2+]i. Thus, the MEG-01s cell line is a pertinent model for studying eicosanoid receptor-mediated signal transduction in platelet/megakaryocyte systems.
BCR-ABL rearrangements in children with Philadelphia chromosome- positive chronic myelogenous leukemia
Authors: Aurer I , Butturini A , Gale RP
Published: October 2019

Leukemia cells from adults with Philadelphia (Ph1)-chromosome positive chronic myelogenous leukemia (CML) have a characteristic molecular rearrangement between the BCR and ABL genes whereby major breakpoint cluster region (Mbcr) exons 2 or 3 are joined to ABL exon II. Ph1- chromosome positive CML is uncommon in children and it is unknown whether these children have similar rearrangements. We studied 17 children with Ph1-chromosome positive CML. Five were studied for Mbcr rearrangement using Southern blotting, nine for the presence of chimeric BCR-ABL mRNA using reverse transcription and polymerase chain reaction, and three for both. All eight children studied by Southern blotting had BCR rearrangement. Of 12 children in whom BCR-ABL mRNA was studied, 10 had Mbcr exon 2 joined to ABL exon II, one had Mbcr exon 3 joined to ABL II, and one had both Mbcr-ABL junctions. These data indicate a similarity to adult CML. However, mRNA processing in children may preferentially splice Mbcr exon 2 to ABL exon II. No child had BCR exon 1 joined to ABL exon II, the rearrangement typical of childhood Ph1-chromosome positive acute lymphoblastic leukemia.
Significance of the P210 versus P190 molecular abnormalities in adults with Philadelphia chromosome-positive acute leukemia
Authors: Kantarjian HM , Talpaz M , Dhingra K , Estey E , Keating MJ , Ku S , Trujillo J , Huh Y , Stass S , Kurzrock R
Published: October 2019

We investigated the significance of p210 and p190 molecular abnormalities in 32 adults with Philadelphia chromosome (Ph)-positive acute leukemia. p210 was detected in 15 patients (47%), p190 in 16 (50%), and both in one (3%). p210 was noted in 11 of 24 patients (46%) with acute lymphocytic leukemia, and in four of eight patients (50%) with acute myelogenous or undifferentiated leukemia. Among 29 patients with untreated disease (p210, 14 patients; p190, 15 patients), no significant differences in the two molecularly distinct groups were observed by pretreatment characteristics including age, degree of organomegaly, anemia, leukocytosis, thrombocytopenia, occurrence of karyotypic abnormalities in addition to Ph, or residual diploid metaphases. Complete response (CR) rates were also similar. Although the remission duration tended to be longer with p190 (P = .08), the differences were minor (median duration 29 v 20 weeks) and not paralleled by differences in survival rate. In 10 patients studied by karyotypic analysis in remission, two of four patients with p190 and two of six patients with p210 showed 100% normal metaphases. One of the seven patients (14%) with p210 who achieved CR manifested a morphologic picture of second chronic-phase chronic myelogenous leukemia lasting for 1 month. We conclude that the molecular studies in Ph-positive acute leukemia are not associated with significantly different clinico- laboratory, karyotypic, or prognostic implications.
Transcriptional regulation of two myeloid-specific genes, myeloperoxidase and lactoferrin, during differentiation of the murine cell line 32D C13
Authors: Friedman AD , Krieder BL , Venturelli D , Rovera G
Published: October 2019

The transcriptional regulation of myeloperoxidase (MPO) and lactoferrin (LF) was examined during terminal myeloid differentiation of the murine cell line 32D C13. The rates of transcription initiation for MPO and LF, determined by an in vitro nuclear run-on assay, increased approximately ninefold. The accumulation of MPO mRNA in 32D C13 cells, determined by Northern blot analysis, correlated temporally with the observed increase in MPO transcription initiation. On the other hand, accumulation of LF mRNA lagged behind the observed increase in LF transcription initiation. In mouse L cells, the LF gene was transcribed more frequently than the MPO gene, though neither mRNA accumulated. Finally, murine MPO transcription is shown, by Northern blot and primer extension analysis, to initiate at multiple sites. These results indicate that whereas transcription induction may largely account for the accumulation of MPO mRNA during terminal myeloid differentiation, both transcriptional and posttranscriptional mechanisms operate to allow accumulation of LF mRNA. The 32D C13 cell system will be a useful model for elucidating these mechanisms.