bluuebunny/crossref_metadata_2025 huggingface dataset:
Catabolism of streptokinase and polyethylene glycol-streptokinase: evidence for transport of intact forms through the biliary system in the mouse
Authors: Brucato FH , Pizzo SV
Published: October 2019

The catabolism of streptokinase (SK) and polyethylene glycol derivatives of SK (PEG-SK) were studied in mice. The clearance and catabolism of SK:plasmin (SK:Pm) and PEG-SK:Pm activator complexes were also investigated. Native 125I-SK cleared rapidly (t1/2 = 15 minutes) from the circulation, with the majority of the ligand accumulating in the liver and gastrointestinal (GI) tract and a substantial fraction also localizing in the kidneys. SK, which was removed from the plasma by the liver, was secreted into bile and then the GI tract. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that 125I-SK recovered from liver and bile was homogeneous and of the same molecular weight (mol wt approximately 50,200) as native SK. PEG-125I-SK cleared slowly (t1/2 greater than 200 minutes), with more than 80% of the preparation localizing in liver and GI tract. The PEG-125I-SK secreted into the bile was also intact. The bile containing 125I-SK was incubated with stoichiometric amounts of plasminogen and electrophoresed under nondenaturing conditions. This study demonstrated that the secreted SK was able to form SK:Pg complexes. SDS-PAGE also showed activation of 125I-Pg that was incubated with recovered bile containing the SK. 125I-SK:Pm catabolism was also studied. In these experiments, the mol wt approximately 42,000 fragment obtained when SK is cleaved by plasmin was found in the bile. This fragment of 125I-SK was not recovered as part of a complex with plasmin, consistent with our previous observations that catabolism of SK:Pm involves transfer of the plasmin to plasma proteinase inhibitors while SK is catabolized independently. By contrast, when PEG-125I-SK:Pm was injected into mice, only intact PEG-125I-SK was found in the bile, consistent with our previous observations that the PEG derivatization blocks its degradation by plasmin.
Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region specific oligonucleotide probes
Authors: Jonsson OG , Kitchens RL , Scott FC , Smith RG
Published: October 2019

To develop a sensitive and specific assay for minimal residual disease in acute lymphoblastic leukemia (ALL), we exploited the enormous diversity of genomic sequences created by immune receptor gene rearrangements. To isolate clone-specific sequences, we first synthesized oligonucleotides that match conserved variable (VH) and joining (JH) sequences flanking the third hypervariable region (HVR3) in the rearranged immunoglobulin heavy chain (IgH) locus. In polymerase chain reactions (PCR), these primers were then used to amplify the intervening HVR3 segments from leukemic DNA samples. Of 12 B-lineage ALLs studied, ten generated one or more fragments of the size expected for HVR3 gene segments. Thus, this single pair of amplimers was sufficient to isolate HVR3 sequences from a majority of acute lymphoblastic leukemias. To verify that the amplified fragments originated from HVR3 alleles and to assess their diversity, we sequenced 7 PCR products derived from 6 leukemias. In addition to elements of recognized D segments, each of the 7 fragments contained novel VH-D and D-JH junctional sequences, including N nucleotides, not known to be present in the germline. Each sequence was unique, and allele-specific oligonucleotide probes hybridized only to HVR3 segments from which the probes were derived. Therefore, as anticipated, these HVR3 segments appeared to possess the diversity required to serve as clonal markers for leukemic populations. To demonstrate that these amplified HVR3 alleles could serve as the basis for a sensitive and specific assay to detect rare leukemic cells, we analyzed in detail one pre-B leukemia that had rearranged 2 IgH alleles. The HVR3 sequences were shown to be linked to rearranged JH-containing restriction fragments in digests of genomic DNA, establishing their origin in the leukemic cells. We synthesized oligonucleotides corresponding to the unique junctional sequences in the HVR3 segments. Using these novel amplimers in an allele-specific amplification and hybridization procedure, we showed that this assay can detect 10 leukemic cells in a background of 10(6) normal blood mononuclear cells. In contrast, the leukemic HVR3 sequences were not detected in extracts of normal or unrelated remission leukemic leukocytes. We conclude that the assay for specific IgH HVR3 sequences is a realistic strategy for detection of minimal residual disease in B-lineage ALL.
Response patterns of hairy cell leukemia to B-cell mitogens and growth factors
Authors: Barut BA , Cochran MK , O'Hara C , Anderson KC
Published: October 2019

The effect of mitogens and/or recombinant B-cell growth factors (M/GFs) on the in vitro growth of hairy cells was examined. Tumor cells were isolated from the spleens of four patients with hairy cell leukemia (HCL) by Ficoll-Hypaque sedimentation and E-rosetting. Enrichment for tumor cells was confirmed with intracytoplasmic immunoglobulin (Ig) staining, tartrate resistant acid phosphatase (TRAP) staining, and staining using monoclonal antibodies (MoAbs) directed at B, T, myeloid, and monocytoid antigens (Ags) in indirect immunofluorescence assays. Tumor cells were B1(CD20)+ B2(CD21)- B4(CD19)+ IL-2R(CD25)+ PCA-1 +/- TRAP+. HCLs neither synthesized DNA nor secreted Ig in response to culture with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-3, IL-4, IL-5, or IL-6. However, a proliferative response (stimulation index greater than or equal to 3.0) without Ig secretion was triggered in HCLs by mitogens or combinations of GFs. Specifically, DNA synthesis was induced at 3 days in three of four HCL samples cultured with Staphylococcus aureus Cowan A (SAC) or the combination of phorbol ester (TPA) and the calcium ionophore A 23187 (Ca2+); DNA synthesis was triggered later (day 7) by tumor necrosis factor (TNF) or by IL-4 and IL-5. In contrast, the fourth patient, a nonresponder to SAC or TPA/Ca2+, demonstrated increased DNA synthesis at day 3 when cocultured with IL-4 and IL-5. Both autoradiography and staining with antibromodeoxyuridine (BrdU) MoAb conjugated to fluorescein confirmed DNA synthesis by only a minority (5% to 23%) of tumor cells within each patient. Dual staining confirmed that responsive cells were both BrdU+ and TRAP+. DNA synthesis induced by TPA/Ca2+ was blocked specifically by anti-IL-6 Ab; in contrast, the HCL proliferative response to SAC, TNF, or IL-4 and IL- 5 was not inhibited by anti-IL-6 Ab. alpha-Interferon inhibited the response to TPA/Ca2+, TNF, or IL-4 and IL-5 without any effect on response to SAC. Finally, peroxidase-antiperoxidase staining demonstrated that HCLs are induced by TPA/Ca2+, but not by SAC, to produce intracytoplasmic IL-6. These data demonstrate IL-4, IL-5, and IL-6 mediated DNA synthesis by HCLs in vitro and suggest a possible in vivo role for these growth factors in the pathophysiology of HCL.
Benzylacyclouridine reverses azidothymidine-induced marrow suppression without impairment of anti-human immunodeficiency virus activity
Authors: Calabresi P , Falcone A , St Clair MH , Wiemann MC , Chu SH , Darnowski JW
Published: October 2019

Increased extracellular concentrations of uridine (Urd) have been reported to reduce, in vitro, azidothymidine (AZT)-induced inhibition of human granulocyte-macrophage progenitor cells without impairment of its antihuman immunodeficiency virus (HIV) activity. Because of the clinical toxicities associated with chronic Urd administration, the ability of benzylacyclouridine (BAU) to effect, in vivo, AZT-induced anemia and leukopenia was assessed. This agent inhibits Urd catabolism and, in vivo, increases the plasma concentration of Urd in a dose- dependent manner, without Urd-related toxicity. In mice rendered anemic and leukopenic by the administration of AZT for 28 days in drinking water (1.5 mg/mL), the continued administration of AZT plus daily BAU (300 mg/kg, orally) partially reversed AZT-induced anemia and leukopenia (P less than .05), increased peripheral reticulocytes (to 4.9%, P less than .01), increased cellularity in the marrow, and improved megaloblastosis. When coadministered with AZT from the onset of drug administration, BAU reduced AZT-induced marrow toxicity. In vitro, at a concentration of 100 mumol/L, BAU possesses minimal anti- HIV activity and has no effect on the ability of AZT to reverse the HIV- induced cytopathic effect in MT4 cells. The clinical and biochemical implications of these findings are discussed.
Detection of breakpoint cluster region- negative and nonclonal hematopoiesis in vitro and in vivo after transplantation of cells selected in cultures of chronic myeloid leukemia marrow
Authors: Turhan AG , Humphries RK , Eaves CJ , Barnett MJ , Phillips GL , Kalousek DK , Klingemann HG , Lansdorp PL , Reece DE , Shepherd JD
Published: October 2019

Philadelphia (Ph1) chromosome-positive clonogenic progenitors usually disappear within 4 to 6 weeks in long-term cultures established from the marrow of patients with chronic myeloid leukemia (CML). In contrast, coexisting chromosomally normal hematopoietic cells are relatively well maintained. Thus, even though normal cells are initially undetectable, they may become the predominant population. Recently, we have begun to explore the potential of such cultures as a strategy for preparing CML marrow for autografting, and based on cytogenetic studies of the differential kinetics of Ph1-positive and Ph1-negative clonogenic cells, have chosen a 10-day period in culture to obtain maximal numbers of selectively enriched normal stem cells. Here we present the results of molecular analyses of the cells regenerated in vivo for the initial three CML patients to be treated using this approach by comparison with the differentiated cells generated by continued maintenance of an aliquot of the autograft in vitro (using a slightly modified culture feeding procedure to enhance the production and release of cells into the nonadherent fraction after 4 weeks) for the one patient whose genotype made molecular analysis of clonality status also possible. These analyses showed that cells with a rearranged breakpoint cluster region (BCR) gene were not detectable by Southern blotting in either in vitro or in vivo populations of mature cells that might be assumed to represent the progeny of primitive cells present at the end of the initial 10 days in culture. Production of BCR- negative cells was also shown to be temporally correlated with the appearance of nonclonal hematopoietic cells both in culture and in vivo. These findings provide support for the view that prolonged maintenance of CML marrow cells in long-term culture may allow molecular characterization of both the BCR-genotype and clonality status of cells with in vivo regenerative potential.
Temporal association of CD40 antigen expression with discrete stages of human B-cell ontogeny and the efficacy of anti-CD40 immunotoxins against clonogenic B-lineage acute lymphoblastic leukemia as well as B- lineage non-Hodgkin's lymphoma cells
Authors: Uckun FM , Gajl-Peczalska K , Myers DE , Jaszcz W , Haissig S , Ledbetter JA
Published: October 2019

Detailed immunophenotypic analyses of immunologically classified leukemias and lymphomas showed that CD40 displays an exquisite B- lineage specificity within the human lymphopoietic system. Notably, 82% of B-lineage chronic lymphocytic leukemias (CLLs), 82% of B-lineage hairy cell leukemias (HCLs), 86% of B-lineage non-Hodgkin's lymphomas (NHLs), and 29% of B-lineage acute lymphoblastic leukemias (ALLs) were CD40+. Quantitative analyses of the correlated expression of CD40 and other B-lineage differentiation antigens on fetal lymphoid precursor cells by multiparameter two-color/three-color flow cytometry, combined with analyses of sequential antigen expression on fluorescence- activated cell fluorescence activated cell sorter (FACS) isolated immunologically distinct fetal B-cell precursor subpopulations during in vitro proliferation and differentiation, provided evidence that the acquisition of CD40 antigen in human B-cell ontogeny occurs subsequent to the expression of CD10 and CD19 antigens but before the surface expression of CD20, CD21, CD22, CD24, and surface immunoglobulin M (sIgM). Some leukemic pro-B cells from ALL patients as well as normal pro-B cell clones from fetal livers displaying germline Ig heavy chain genes were CD40+, indicating that the acquisition of CD40 antigen likely precedes the rearrangement of Ig heavy chain genes. CD40+ FACS- sorted malignant cells from B-lineage ALL as well as B-lineage NHL patients were capable of in vitro clonogenic growth, indicating the CD40 antigen is expressed on clonogenic leukemia and lymphoma cells. This hypothesis was confirmed by the ability of an anti-CD40 immunotoxin that we used as an antigen-specific cytotoxic probe to effectively kill clonogenic B-lineage ALL and NHL cells.
Thrombin generation is not increased in the blood of hemophilia B patients after the infusion of a purified factor IX concentrate
Authors: Mannucci PM , Bauer KA , Gringeri A , Barzegar S , Bottasso B , Simoni L , Rosenberg RD
Published: October 2019

Prothrombin complex concentrates (PCC), licensed for the treatment of hemophilia B, are known to carry a significant risk of thromboembolic complications. Although the reasons for thrombogenicity are not completely understood, several manufacturers have developed purified factor IX concentrates that contain negligible amounts of the other vitamin K-dependent factors. To evaluate whether or not the infusion of such a factor IX concentrate is followed by lesser activation of the hemostatic system than by the infusion of a PCC, we performed a series of coagulation assays on 11 hemophilia B patients before and after the administration of these two types of concentrate using a randomized cross-over design. The levels of prothrombin fragment F1 + 2, a sensitive measure of the in vivo cleavage of prothrombin by factor Xa, was significantly increased in plasma after PCC, but not after factor IX concentrate. Plasma fibrinopeptide A, a sensitive index of the enzymatic activity of thrombin on fibrinogen, also increased significantly after PCC but not after factor IX concentrate. The fragment B beta 15–42, a sensitive index of the enzymatic action of plasmin on fibrin II, did not change after either concentrate. There were also no differences in less sensitive coagulation measurements, such as plasma fibrinogen, antithrombin III, and fibrin monomers, nor in indices of platelet activation, such as beta-thromboglobulin and platelet factor 4. These findings show that the infusion of a purified factor IX concentrate can result in substantially less activation of the coagulation cascade than may be seen with PCC.
Expression of leukocyte alkaline phosphatase gene in normal and leukemic cells: regulation of the transcript by granulocyte colony- stimulating factor
Authors: Rambaldi A , Terao M , Bettoni S , Tini ML , Bassan R , Barbui T , Garattini E
Published: October 2019

The levels of leukocyte alkaline phosphatase (LAP) messenger RNA (mRNA) are evaluated in B and T lymphocytes, monocytes, and polymorphonuclear cells (PMNs), and this transcript is found to be present only in PMNs. Precursors of the myelomonocytic pathway, represented by leukemic cells isolated from several cases of chronic myelogenous leukemia (CML) in its stable and blastic phase and acute myelogenous leukemia (AML), are devoid of LAP transcript. These data support the notion that LAP is a marker of the granulocyte terminal differentiation. Despite the absence of LAP mRNA in both the myeloid and the lymphoid precursors, nuclear run-on experiments show constitutive transcription of the LAP gene in leukemic cells obtained from AML, CML, as well as acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia (B-CLL). In CML and in chronic myelo-monocytic leukemia (CMML) PMNs, granulocyte colony- stimulating factor (G-CSF) specifically accumulates LAP mRNA without showing a substantial increase in the rate of transcription of the LAP gene. Once increased by G-CSF, LAP mRNA is very stable, showing a half- life of more than 4 hours in the presence of actinomycin-D. G-CSF is suggested to play a pivotal role in the modulation of LAP transcript in PMNs.
Activation of the interleukin-3 gene by chromosome translocation in acute lymphocytic leukemia with eosinophilia [see comments]
Authors: Meeker TC , Hardy D , Willman C , Hogan T , Abrams J
Published: October 2019

The t(5;14)(q31;q32) translocation from B-lineage acute lymphocytic leukemia with eosinophilia has been cloned from two leukemia samples. In both cases, this translocation joined the IgH gene and the interleukin-3 (IL-3) gene. In one patient, excess IL-3 mRNA was produced by the leukemic cells. In the second patient, serum IL-3 levels were measured and shown to correlate with disease activity. There was no evidence of excess granulocyte/macrophage colony stimulating factor (GM-CSF) or IL-5 expression. Our data support the formulation that this subtype of leukemia may arise in part because of a chromosome translocation that activates the IL-3 gene, resulting in autocrine and paracrine growth effects.
Philadelphia chromosome positive childhood acute lymphoblastic leukemia: clinical and cytogenetic characteristics and treatment outcome. A Pediatric Oncology Group study
Authors: Crist W , Carroll A , Shuster J , Jackson J , Head D , Borowitz M , Behm F , Link M , Steuber P , Ragab A
Published: October 2019

Among 3,638 children with acute lymphoblastic leukemia (ALL) entered on Pediatric Oncology Group (POG) protocols between June 1981 and April 1989, successful cytogenetic studies were available for 2,519, 58 (2.3%) of which had the Philadelphia (Ph) chromosome detected. Features associated with the presence of the Ph chromosome were high leukocyte count (median, 33 x 10(9)/L), older age median, 9.6 years), a higher proportion of French-American-British L2 morphology, and a lower frequency of mediastinal mass. Immunologic marker studies at diagnosis in 56 Ph+ cases identified early pre-B ALL in 42 cases (75%), pre-B- cell in 9 (16%), and T-cell in 5 (9%). This distribution is similar to that found in Ph+ ALL. Intensive multiagent chemotherapy induced complete remissions in only 78% of eligible Ph+ patients compared with 96% of those without an identified Ph chromosome (P less than .001). Of 44 eligible Ph+ patients treated on POG frontline protocols for children with non-T, non-B-cell ALL, 27 have failed therapy, compared with 520 of 1,892 without an identified Ph chromosome (logrank P less than .001). Ph+ ALL is an aggressive form of acute leukemia that frequently presents in older children with a high leukocyte count, FAB L2 morphology, and a pseudodiploid karyotype, and becomes multidrug- resistant early. Thus, Ph+ cases require early identification to permit treatment with intensive induction regimens and experimental approaches such as bone marrow transplantation.
Increased blood clearance rate of indium-111 oxine-labeled autologous CD4+ blood cells in untreated patients with Hodgkin's disease
Authors: Grimfors G , Holm G , Mellstedt H , Schnell PO , Tullgren O , Bjorkholm M
Published: October 2019

Untreated patients with Hodgkin's disease (HD) have a blood T- lymphocytopenia mainly caused by a reduction of the CD4+ subset. Indirect support for a sequestration of T cells in the spleen and tumor- involved lymphoid tissue has accumulated. To test the hypothesis that the blood CD4 T-lymphocytopenia in patients with HD is caused by an altered lymphocyte traffic, 12 untreated HD patients and five in complete clinical remission (CCR) were studied. Blood lymphocytes were collected by leukapheresis and gradient centrifugation, and were further purified by an adherence step. The cells were labeled with indium-111 oxine and reinfused intravenously into the patient. The radioactivity of CD4+ and CD8+ blood lymphocytes separated by immunoabsorption was measured from serial blood samples. CD4+ cells were eliminated more rapidly in untreated patients than patients in CCR. Repeated gamma camera imaging after autotransfusion of indium-111 oxine labeled cells demonstrated an accumulation of radioactivity in tumor-involved tissue of untreated patients. These findings support the concept of an enhanced elimination of CD4+ cells in patients with active HD that may contribute to the observed blood T-lymphocytopenia and may reflect a biologic response to the tumor.
Near-triploid and near-tetraploid acute lymphoblastic leukemia of childhood
Authors: Pui CH , Carroll AJ , Head D , Raimondi SC , Shuster JJ , Crist WM , Link MP , Borowitz MJ , Behm FG , Land VJ
Published: October 2019

Cytogenetic and DNA flow cytometric analyses of leukemic cells from 1,971 children with newly diagnosed acute lymphoblastic leukemia (ALL) identified stem lines with modal chromosome numbers greater than 65 in 26 patients (1.3%). Near-triploidy (66 to 73 chromosomes) was found in six cases and near-tetraploidy (82 to 94 chromosomes) in 20. A striking morphologic finding was the presence of clumped chromatin with grooved nuclei or Rieder cell formation in 20 cases. Other than a slight excess of the pre-B immunophenotype, the near-triploid cases did not appear to differ substantially from the general ALL population in clinical features. In contrast, near-tetraploid cases were more often associated with a T-cell immunophenotype (47% v 14%, P less than .001) and L2 morphology (30% v 22%, P less than .01), and were older at diagnosis (median age, 8.6 v 4.8 years, P = .01) than cases with other ploidies. Moreover, an unusually high proportion of near-tetraploid cases tested (6 of 15) expressed one or more of the myeloid-associated antigens CD13, CD15, and CD33. Despite the use of contemporary intensive chemotherapy and short follow-up for most patients, 6 of the 20 near- tetraploid cases have relapsed or died. This study suggests that the near-tetraploid subtype differs from other cases of hyperdiploid greater than 50 ALL, which have been associated with a favorable prognosis.
Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping
Authors: Embury SH , Kropp GL , Stanton TS , Warren TC , Cornett PA , Chehab FF
Published: October 2019

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish beta A and beta E alleles. Competing 15mer primers were a fluorescein-labeled complement to beta A and a rhodamine-labeled complement to beta E, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine- labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha 3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.
Human immunodeficiency virus DNA amplification and serology in blood donors
Authors: Perrin LH , Yerly S , Adami N , Bachmann P , Butler-Brunner E , Burckhardt J , Kawashima E
Published: October 2019

The significance of indeterminate screening antibody test for human immunodeficiency virus (HIV) serology is still difficult to evaluate, especially in low-risk populations. One hundred twenty-seven blood donors with an initially reactive screening test for HIV antibodies were enrolled in this study. The sera of 95 of these blood donors were reactive on repetition of the test, and none had detectable circulating p24 antigen. Western blot (WB) analysis of the repeatedly reactive sera was as follows: 9 positive, 31 indeterminate, and 55 negative. One of the blood donors with indeterminate WB later presented a seroconversion. On subsequent control 3 to 12 months later, the sera from donors with indeterminate or negative WB did not present any parameters that may indicate a seroconversion. DNA was purified from citrated blood collected from the 127 blood donors at the time of the initial antibody screening. Five micrograms of each DNA sample corresponding to 7 x 10(5) nucleated white blood cells was amplified by polymerase chain reaction (PCR) in the presence of oligonucleotides (primers) corresponding to a highly conserved segment of the pol gene. The detection of amplified DNA was achieved by dot blot and Southern blot using appropriate 32P-labeled oligonucleotides. Ten DNA samples were positive, 9 corresponded to blood donors with a positive HIV serology, and 1 to the blood donor who later presented a seroconversion. These results confirm the sensitivity of the PCR for the diagnosis of HIV infection; they also suggest that repetition of the serology at 3- to 12-month intervals is a valuable procedure for the control of HIV infection status in blood donors.
A new treatment for polycythemia vera: recombinant interferon alfa [see comments]
Authors: Silver RT
Published: October 2019

Recombinant interferon alfa, a natural product with growth inhibitory capabilities, has been demonstrated for the first time to have significant therapeutic efficacy in controlling the red cell mass in patients with the myeloproliferative disease polycythemia vera. The starting dose was 3.0 X 10(6) U three times a week, subcutaneously (SC). In three patients the dose required was 5.0 X 10(6), U five times a week, SC. Side effects were easily tolerated. The striking advantage in the use of this drug is its presumed absence of antileukemic effect. Further evaluation is necessary, but the initial responses are encouraging.
Subcellular distribution and characterization of acetylcholinesterase activities from sheep platelets: relationships between temperature- dependence and environment
Authors: Sanchez-Yague J , Cabezas JA , Llanillo M
Published: October 2019

Acetylcholinesterase is a key enzyme in cholinergic neurotransmission for hydrolyzing acetylcholine and has been shown to possess arylacylamidase activity in addition to esterase activity. The enzyme is found at various loci, where its functional significance remains to be clarified, and it exists in multiple molecular forms. Sheep platelets have been shown to exhibit acetylcholinesterase activity associated with plasma membrane (Bp), endoplasmic reticulum (Cp), mitochondria granules (Dp), and soluble (As) fractions. These activities show differences in some physicochemical and kinetic properties. The soluble acetylcholinesterase is the most thermostable, and the enzyme from the Cp fractions shows the lowest affinity for the acetylthiocholine substrate and the strongest inhibition by fluoride. In all cases a noncompetitive inhibition of the enzyme by this ion is found. When membrane-bound acetylcholinesterases were assayed at temperatures between 12 degrees C and 33 degrees C, the Arrhenius plots of all activities exhibited a break point at about 17 degrees C. This discontinuity was abolished by addition of detergent to the assay medium (0.02% Triton X-100, final concentration). Their Hill coefficients were calculated in the presence of fluoride, showing unitary values in all cases, which points to a noncooperative effect and nonallosteric behavior in the particulate enzyme. These results suggest that the sheep platelet acetylcholinesterase associated with membrane-bound systems is modulated by the physical state of its environment, despite the fact that the enzyme might be lipid- or nonlipid-dependent.
Heritable severe combined anemia and thrombocytopenia in the mouse: description of the disease and successful therapy
Authors: Peters LL , McFarland-Starr EC , Wood BG , Barker JE
Published: October 2019

A new autosomal recessive mouse mutation, scat (severe combined anemia and thrombocytopenia), causes intermittent episodes of severe bleeding in the homozygote. At birth, affected mice are pale with intradermal petechiae and bruises on exposed surfaces. Central nervous system (CNS) bleeding occurs in 22% of the mice. Gastrointestinal (GI) hemorrhaging and splenomegaly are noted in moribund mice at autopsy. Of the 291 mice studied, 113 mice survived the initial crisis and entered a spontaneous remission period lasting from day 16 to day 27. A second crisis period ensued, and all but 22 mice died by 45 days. Mice in crisis show significantly decreased platelets, erythrocytes, and leukocytes and increased reticulocytes when compared to normal littermates. During remission all parameters are significantly improved or revert to normal values. Neither splenomegaly nor internal bleeding are observed during remission. A platelet-specific antibody is present in the plasma of mutant mice during crisis. The symptoms (severe bleeding, anemia, low- platelet counts) and platelet-specific antibody production are transferred to lethally irradiated normal mice through spleen cell transplantation. Splenectomy of mice in remission significantly increases survival. Exploitation of the features common to both scat/scat mice and patients with some forms of autoimmune thrombocytopenic purpura will undoubtedly prove useful in defining common pathways of disease development and in testing potential therapeutic measures.
Flow cytometric analysis of expression of interleukin-2 receptor beta chain (p70-75) on various leukemic cells
Authors: Hoshino S , Oshimi K , Tsudo M , Miyasaka M , Teramura M , Masuda M , Motoji T , Mizoguchi H
Published: October 2019

We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70–75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody (MoAb), designated Mik-beta 1, which has been recently developed. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T- cell leukemia (ATL) cells from all three patients with ATL, and on T- cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis. These results indicate that flow cytometric analysis using MoAbs, anti-Tac, and Mik-beta 1 is useful for detecting the expression of the IL-2R chains.
L428 cells derived from Hodgkin's disease produce E rosette-inhibiting factor
Authors: Katay I , Wirnitzer U , Burrichter H , von Kalle C , Schell-Frederick E , Diehl V , Schaadt M
Published: October 2019

Diminished rosetting capacity of T cells is a well-known phenomenon in Hodgkin's disease, and inhibitors of E rosette formation have been reported to be present in the plasma of patients with Hodgkin's disease. The cell line L428, representing an in vitro counterpart of Hodgkin and Sternberg-Reed cells, could be shown to release a factor capable of suppressing the binding of sheep red blood cells (SRBC) to normal peripheral-blood T lymphocytes or to a T-cell line (L735). At maximally effective concentrations, RIF (rosette inhibiting factor) inhibited T lymphocyte rosetting by approximately 40% (mean from 184 healthy controls). The diminished E rosetting of T lymphocytes from Hodgkin's patients was not further suppressed by added RIF. This factor inhibited binding of SRBC to their target cells at 37 degrees C but not at 4 degrees C. The factor could be stored lyophilized at -20 degrees C and was stable at 56 degrees C (30 minutes). RIF was inactive below pH 6 and above pH 9 or after trypsin digestion. Purification by affinity, ion exchange, and molecular sieve chromatography showed activity peaks at 12.5 Kd, 25 Kd, 50 Kd, and 100 Kd.
The ontogeny of a 57-Kd cationic antimicrobial protein of human polymorphonuclear leukocytes: localization to a novel granule population
Authors: Pereira HA , Spitznagel JK , Winton EF , Shafer WM , Martin LE , Guzman GS , Pohl J , Scott RW , Marra MN , Kinkade JM Jr
Published: October 2019

The ontogeny of a 57-Kd cationic antimicrobial protein (CAP57) that has substantial similarities to bactericidal permeability increasing protein (BPI) has been determined immunocytochemically. CAP57 was detected in the granules of mature peripheral blood neutrophils. However, it was absent from other cells of the peripheral blood: eosinophils, red blood cells (RBCs), and mononuclear cells. In human bone marrow, CAP57 was confined to the neutrophilic series. The earliest stage of development of the myeloid cells at which CAP57 was demonstrated was the promyelocyte. Double immunofluorescent labeling showed that CAP57 was detected in cells positive for myeloperoxidase. The absence of lactoferrin in certain cells (promyelocytes) containing CAP57 indicated that CAP57 was synthesized and packaged in a population of granules prior to the development of granules that contain lactoferrin. CAP57 could not be demonstrated in HL60 cells either by enzyme-linked immunosorbent assay (ELISA) or by immunocytochemistry. However, the presence of another granule-associated cationic antimicrobial protein of molecular weight 37 Kd (CAP37) was readily detected in undifferentiated HL60 cells. Amino acid sequence analysis showed that CAP57 and BPI were identical. Further indication of the identity between CAP57 and BPI was that monoclonal anti-CAP57 antibodies cross reacted with BPI. Sucrose density-gradient centrifugations showed CAP57 was confined to a granule population that exhibited a buoyant density intermediate of the previously described light and heavy azurophil granules. Further resolution of the individual azurophil granule populations by Percoll density-gradient centrifugation revealed that CAP57 was most concentrated in the density range of 1.093 to 1.100 g/cc. These results strongly suggest the unique finding that CAP57 may be associated with a heretofore unreported granule type.
What role for prednisone in prevention of acute graft-versus-host disease in patients undergoing marrow transplants?
Authors: Storb R , Pepe M , Anasetti C , Appelbaum FR , Beatty P , Doney K , Martin P , Stewart P , Sullivan KM , Witherspoon R
Published: October 2019

One hundred forty-seven consecutive patients with leukemia, myelodysplastic syndrome, or aplastic anemia were treated by marrow grafts from genotypically HLA-identical siblings (n = 122) or HLA- haploidentical family members (n = 25). Haploidentical recipients differed from their donors for no more than one HLA locus on the nonshared haplotype. All were given postgrafting immunosuppression with a combination of methotrexate and cyclosporine. In a randomized study we explored whether prednisone administered from day 0 through 35 along with methotrexate/cyclosporine could improve prevention of acute graft- versus-host disease (GVHD). The GVHD incidence in patients not given prednisone was comparable with that previously reported with methotrexate/cyclosporine. Unexpectedly, significant increases in acute and also chronic GVHD were seen in HLA-identical recipients administered prednisone, but not in the small number of patients administered HLA-nonidentical grafts. However, the resultant increase in transplant-related mortality in patients administered prednisone was offset by an increase in leukemic relapse in patients not administered prednisone, presumably related to the absence of a graft-versus- leukemia effect. Therefore, overall disease-free survival of the two groups of patients was comparable, with slightly more than 50% of the patients being alive at more than 2 years after transplantation. We speculated that prednisone adversely affected GVHD prophylaxis, interfering with methotrexate's cell cycle-dependent suppression of donor lymphocyte proliferation in response to host antigens. In a pilot study we explored whether beginning prednisone on day 15, after completion of methotrexate administration, would avoid this adverse effect. The GVHD incidence in patients administered methotrexate/cyclosporine along with “late” prednisone was comparable with that in patients not administered prednisone. We conclude that methotrexate/cyclosporine is effective in decreasing the incidence of grade II through IV GVHD, and that the addition of prednisone to this regimen is not beneficial in recipients of HLA-identical marrow grafts.
Synthesis of proteins from [35S]methionine by guinea pig megakaryocytes in vivo and time course of appearance of newly synthesized proteins in platelets
Authors: Schick BP
Published: October 2019

The relationship of protein synthesis to megakaryocyte maturation has been studied in guinea pigs in vivo. Guinea pigs were injected with a single dose of [35S]methionine. Megakaryocytes and platelets were isolated daily for 4 days, and proteins from both cells were isolated by DEAE-Sephacel chromatography and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. All proteins in megakaryocytes corresponding to stained bands on the SDS- PAGE gels were radiolabeled at 3 hours after injection. The greatest loss of radioactivity from the megakaryocytes occurred between 1 and 3 days after injection. Only trace labeling of platelet proteins was seen at 3 hours, representing almost entirely three bands at molecular weights 47,000, 52,000, and 66,000. At 24 hours only about 13% of the maximal labeling was present, but not all proteins were labeled. The maximal labeling was at 3 days. The pattern of labeling of platelets at 3 days was identical to that of megakaryocytes at 3 hours. The protein pattern of nonmegakaryocytic marrow cells was different from that of the platelets and megakaryocytes. Data presented here suggest that most protein synthesis in megakaryocytes is completed at least 24 hours before release of the platelets to the circulation, and suggest some specificity in the proteins that are synthesized at the terminal stages of maturation.
Chromosome aberrations and prognostic factors in therapy-related myelodysplasia and acute nonlymphocytic leukemia
Authors: Pedersen-Bjergaard J , Philip P , Larsen SO , Jensen G , Byrsting K
Published: October 2019

Cytogenetic studies of 91 consecutive patients with therapy-related myelodysplasia or overt acute nonlymphocytic leukemia disclosed characteristic defects of chromosome 7 in 48 cases and of chromosome 5 in 21 cases. The chromosome 5 abnormalities were consistently present in all abnormal mitoses at the time of diagnosis, as were the chromosome 7 abnormalities in 45 of the 48 patients. Various abnormalities, primarily of the short arm of chromosome 17, were observed in 13 cases, abnormalities of the long arm of chromosome 21 were observed in 12 cases, and rearrangements of 11q23 were seen in nine cases. Thirteen patients presented a normal karyotype. Previous therapy with alkylating agents, the presence of an initial myelodysplastic phase, and abnormalities of chromosome 7 or 5 were interdependent. Patients with 11q23 rearrangement typically developed overt leukemia of FAB types M4 or M5a without myelodysplasia and with a short latent period. Evaluated by Cox regression analysis, complete remission of the primary malignancy and a malignant lymphoma as primary tumor were the two most important and independent prognostic factors indicating a longer survival (P = .008). In addition, the platelet count at diagnosis was a significant prognostic factor (P = .01). For the subgroup of 62 patients with myelodysplasia, the number of chromosome aberrations, the percentage of blasts in the bone marrow, and the hemoglobin level were other significant and independent prognostic factors (P = .05, .05, and .004, respectively). The most important predictive factor for a favorable response to intensive antileukemic chemotherapy in overt leukemia was the absence of a preceding myelodysplastic phase (P = .0014).
Murine leukemia inhibitory factor enhances retroviral-vector infection efficiency of hematopoietic progenitors
Authors: Fletcher FA , Williams DE , Maliszewski C , Anderson D , Rives M , Belmont JW
Published: October 2019

We have investigated the in vitro effects of the cytokine leukemia inhibitory factor (LIF) on normal murine hematopoietic progenitors by measuring recovery and retroviral vector infection efficiency of 13-day posttransplant, spleen-colony-forming cell (CFU-S 13) in short-term culture. Up to a twofold increase in CFU-S13 recovery was observed, from 9.7 x 10(-5) cells in untreated controls to 17.8 to 19.5 x 10(-5) cells, depending on the concentration of LIF. Histologic analysis of spleen colonies from control and LIF-treated marrows demonstrated that there was no detectable alteration in the differentiative potential of CFU-S13. The efficiency of CFU-S13 infection was increased from 15% in untreated controls to 84% to 91% in LIF-treated marrows. Analysis of proviral integration sites in spleen colonies indicated that some CFU- S13 precursors were infected in the LIF-treated marrows.
Analysis of immunoglobulin and T-cell receptor gene rearrangements in human fetal bone marrow B lineage cells
Authors: LeBien TW , Elstrom RL , Moseley M , Kersey JH , Griesinger F
Published: October 2019

Fetal bone marrow B lineage cells representing multiple stages of B cell development were isolated by two-color cell sorting and analyzed for immunoglobulin H and T-cell receptor (TCR) gamma and delta gene rearrangements. Analysis of CD10+/surface mu- cells using a JH probe revealed a high frequency of rearrangements; some of these rearrangements used the 3′ D region gene DQ52. Analysis of CD10+/surface mu- cells revealed no detectable TCR-gamma or -delta rearrangements, nor were TCR-delta rearrangements detected in CD10+/surface mu+ cells, despite the limited repertoire of these genes. These observations are surprising given the high frequency of TCR delta/gamma rearrangements in B cell precursor acute lymphoblastic leukemia, and identify a potential difference in patterns of gene rearrangement that distinguish normal and leukemic B cell precursors.
Activation mechanisms of adherent human neutrophils
Authors: Ginis I , Tauber AI
Published: October 2019

The mechanism by which unstimulated human neutrophils initiate a respiratory burst on adherence to a surface has been examined. When neutrophils adhere to a plastic surface, they immediately generate a sustained burst of superoxide (O2-). However, this respiratory burst is not initiated by adherence alone, since neutrophils attached to fibronectin fail to mount a response. Adhesion to plastic is calcium (Ca2+) independent, but O2- production requires Ca2(+)-containing buffer in the initiation phase, that is, during adhesion and the early phase of O2- production. The Ca2(+)-dependent step was shown to involve protein kinase C (PK-C) in that the O2- production, but not adherence, was blocked with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and PK-C was found to translocate from the cytosol to the membrane on adhesion. Furthermore, it may be inferred that this translocation results in the generation of a Ca2+ independent form of PK-C, PK-M, since leupeptin, which inhibits the generation of PK-M, also blocked O2- production. This finding was corroborated by showing that after 5 minutes in a Ca2(+)-containing buffer, enough time to initiate O2- production and PK-C translocation, Ca2+ is no longer required for sustained O2- release. These results, in aggregate, demonstrate that neutrophils are activated by adhesion to plastic to generate O2-, a PK- C-dependent process that appears to involve a Ca2(+)-independent form of the kinase, PK-M. Why adherent neutrophils generate a respiratory burst on plastic and not fibronectin surfaces probably reflects activation of distinct receptors, whose nature must still be defined. Another issue to address is the priming effect of adhesion, since cells adherent to plastic- or fibronectin-coated surfaces have an enhanced O2- response to formylmethionyl-leucine-phenylalanine (FMLP) compared with neutrophils stimulated in suspension. This may relate to increased Ca2+ mobilization, an important mediator of priming for FMLP responses. Thus, adhesion as a priming event does not necessarily initiate cell effector function, and the further elucidation of the plastic and fibronectin models suggests a means of characterizing the crucial event that control neutrophil activation.
The influence of oxygen tension, temperature, and hemoglobin concentration on the rheologic properties of sickle erythrocytes
Authors: Mackie LH , Hochmuth RM
Published: October 2019

With the use of micromanipulation techniques, the shear modulus or “rigidity” mu, the recovery time tc, and the unfolding time tf for individual sickle cells have been measured at different oxygen tensions, temperatures, and cell densities. In these experiments, the partial pressure of oxygen was varied from 156 to 40 mm Hg and the temperature was controlled at 25 degrees C or 37 degrees C. Three mean cellular hemoglobin concentrations were studied: 29 g/dL, 33 g/dL, and 46 g/dL. The lighter cells (29 and 33 g/dL) exhibited at most a threefold increase in rigidity as the pO2 was decreased from 156 to 40 mm Hg. At 25 degrees C, the densest cells (46 g/dL) also exhibited a threefold increase. However, at 37 degrees C, the rigidity of these cells increased eightfold between 156 to 40 mm Hg. Compared with normal cells, this gives a rigidity that is 18 times larger. In contrast to the values for mu, the values for tc and tf remained essentially unchanged (within the accuracy of the experiments) for the lighter cells and could not be measured for the densest cells.